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Key points The electrogenic Na+/HCO3 −cotransporter NBCe1‐B is widely expressed in many tissues, including pancreas, submandibular gland, brain, heart, etc. NBCe1‐B has very low activity under basal condition due to auto‐inhibition, but can be fully activated by protein interaction with the IP3R‐binding protein released with inositol 1,4,5‐trisphosphate (IRBIT).The structural components of the auto‐inhibition domain and the IRBIT‐binding domain of NBCe1‐B are finely characterized based on systematic mutations in the present study and data from previous studies.Reducing negative charges on the cytosol side of the transmembrane domain greatly decreases the magnitude of the auto‐inhibition of NBCe1‐B.We propose that the auto‐inhibition domain functions as a brake module that inactivates NBCe1‐B by binding to, via electrostatic attraction, the transmembrane domain; IRBIT activates NBCe1‐B by releasing the brake from the transmembrane domain via competitive binding to the auto‐inhibition domain. Abstract The electrogenic Na+/HCO3 − cotransporter NBCe1‐B is widely expressed in many tissues in the body. NBCe1‐B exhibits only basal activity due to the action of the auto‐inhibition domain (AID) in its unique amino‐terminus. However, NBCe1‐B can be activated by interaction with the IP3R‐binding protein released with inositol 1,4,5‐trisphosphate (IRBIT). Here, we investigate the molecular mechanism underlying the auto‐inhibition of NBCe1‐B and its activation by IRBIT. The IRBIT‐binding domain (IBD) of NBCe1‐B spans residues 1−52, essentially consisting of two arms, one negatively charged (residues 1−24) and the other positively charged (residues 40−52). The AID mainly spans residues 40−85, overlapping with the IBD in the positively charged arm. The magnitude of auto‐inhibition of NBCe1‐B is greatly decreased by manipulating the positively charged residues in the AID or by replacing a set of negatively charged residues with neutral ones in the transmembrane domain. The interaction between IRBIT and NBCe1‐B is abolished by mutating a set of negatively charged Asp/Glu residues (to Asn/Gln) plus a set of Ser/Thr residues (to Ala) in the PEST domain of IRBIT. However, this interaction is not affected by replacing the same set of Ser/Thr residues in the PEST domain with Asp. We propose that: (1) the AID, acting as a brake, binds to the transmembrane domain via electrostatic interaction to slow down NBCe1‐B; (2) IRBIT activates NBCe1‐B by releasing the brake from the transmembrane domain., Key points The electrogenic Na+/HCO3 −cotransporter NBCe1‐B is widely expressed in many tissues, including pancreas, submandibular gland, brain, heart, etc. NBCe1‐B has very low activity under basal condition due to auto‐inhibition, but can be fully activated by protein interaction with the IP3R‐binding protein released with inositol 1,4,5‐trisphosphate (IRBIT).The structural components of the auto‐inhibition domain and the IRBIT‐binding domain of NBCe1‐B are finely characterized based on systematic mutations in the present study and data from previous studies.Reducing negative charges on the cytosol side of the transmembrane domain greatly decreases the magnitude of the auto‐inhibition of NBCe1‐B.We propose that the auto‐inhibition domain functions as a brake module that inactivates NBCe1‐B by binding to, via electrostatic attraction, the transmembrane domain; IRBIT activates NBCe1‐B by releasing the brake from the transmembrane domain via competitive binding to the auto‐inhibition domain.

Key points Mechanoelectrical transduction at auditory hair cells requires highly specialized stereociliary bundles that project from their apical surface, forming a characteristic graded ‘staircase’ structure.The morphogenesis and maintenance of these stereociliary bundles is a tightly regulated process requiring the involvement of several actin‐binding proteins, many of which are still unidentified.We identify a new stereociliary protein, the I‐BAR protein BAIAP2L2, which localizes to the tips of the shorter transducing stereocilia in both inner and outer hair cells (IHCs and OHCs).We find that Baiap2l2 deficient mice lose their second and third rows of stereocilia, their mechanoelectrical transducer current, and develop progressive hearing loss, becoming deaf by 8 months of age.We demonstrate that BAIAP2L2 localization to stereocilia tips is dependent on the motor protein MYO15A and its cargo EPS8.We propose that BAIAP2L2 is a new key protein required for the maintenance of the transducing stereocilia in mature cochlear hair cells. Abstract The transduction of sound waves into electrical signals depends upon mechanosensitive stereociliary bundles that project from the apical surface of hair cells within the cochlea. The height and width of these actin‐based stereocilia is tightly regulated throughout life to establish and maintain their characteristic staircase‐like structure, which is essential for normal mechanoelectrical transduction. Here, we show that BAIAP2L2, a member of the I‐BAR protein family, is a newly identified hair bundle protein that is localized to the tips of the shorter rows of transducing stereocilia in mouse cochlear hair cells. BAIAP2L2 was detected by immunohistochemistry from postnatal day 2.5 (P2.5) throughout adulthood. In Baiap2l2 deficient mice, outer hair cells (OHCs), but not inner hair cells (IHCs), began to lose their third row of stereocilia and showed a reduction in the size of the mechanoelectrical transducer current from just after P9. Over the following post‐hearing weeks, the ordered staircase structure of the bundle progressively deteriorates, such that, by 8 months of age, both OHCs and IHCs of Baiap2l2 deficient mice have lost most of the second and third rows of stereocilia and become deaf. We also found that BAIAP2L2 interacts with other key stereociliary proteins involved in normal hair bundle morphogenesis, such as CDC42, RAC1, EPS8 and ESPNL. Furthermore, we show that BAIAP2L2 localization to the stereocilia tips depends on the motor protein MYO15A and its cargo EPS8. We propose that BAIAP2L2 is key to maintenance of the normal actin structure of the transducing stereocilia in mature mouse cochlear hair cells., Key points Mechanoelectrical transduction at auditory hair cells requires highly specialized stereociliary bundles that project from their apical surface, forming a characteristic graded ‘staircase’ structure.The morphogenesis and maintenance of these stereociliary bundles is a tightly regulated process requiring the involvement of several actin‐binding proteins, many of which are still unidentified.We identify a new stereociliary protein, the I‐BAR protein BAIAP2L2, which localizes to the tips of the shorter transducing stereocilia in both inner and outer hair cells (IHCs and OHCs).We find that Baiap2l2 deficient mice lose their second and third rows of stereocilia, their mechanoelectrical transducer current, and develop progressive hearing loss, becoming deaf by 8 months of age.We demonstrate that BAIAP2L2 localization to stereocilia tips is dependent on the motor protein MYO15A and its cargo EPS8.We propose that BAIAP2L2 is a new key protein required for the maintenance of the transducing stereocilia in mature cochlear hair cells.

Key points Few reports have explored the possibility of involvement of non‐inflammatory factors in lacrimal hyposecretion in Sjögren's syndrome (SS).RNA‐sequencing analysis revealed that only four genes, including arginase 1, were downregulated in the lacrimal gland of SS model male mice (NOD mice) after onset of lacrimal hyposecretion and dacryoadenitis.Even in non‐dacryoadenitis‐type NOD mice, tear secretion and arginase 1 expression remained low.An arginase 1 inhibitor reduced tear secretion and partially reduced saliva secretion in BALB/c mice.The results indicate that a non‐inflammatory factor, arginase 1, is involved in lacrimal hyposecretion in male NOD mice, regardless of dacryoadenitis status. Abstract Lacrimal fluid (tears) is important for preservation of the ocular surface, and thus lacrimal hyposecretion in Sjögren's syndrome (SS) leads to reduced quality of life. However, the cause(s) of lacrimal hyposecretion remains unknown, even though many studies have been conducted from the perspective of inflammation. Here, we hypothesized that a non‐inflammatory factor induces lacrimal hyposecretion in SS pathology, and to elucidate such a factor, we conducted transcriptome analysis of the lacrimal glands in male non‐obese diabetic (NOD) mice as an SS model. The NOD mice showed inflammatory cell infiltration and decreased pilocarpine‐induced tear secretion at and after 6 weeks of age compared to age‐matched BALB/c mice. RNA‐sequencing analysis revealed that only four genes, including arginase 1, were downregulated, whereas many genes relating to inflammation were upregulated, in the lacrimal glands of male NOD mice after onset of lacrimal hyposecretion and dacryoadenitis (lacrimal gland inflammation). Changes in the level of arginase 1 expression were confirmed by real‐time RT‐PCR and western blot analysis. Furthermore, non‐dacryoadenitis‐type NOD mice were used to investigate the relationships among arginase 1 expression, lacrimal hyposecretion and dacryoadenitis. Interestingly, these NOD mice retained the phenotype of dacryoadenitis with regard to tear secretion and arginase 1 expression level. An arginase 1 inhibitor reduced tear secretion and partially reduced saliva secretion in BALB/c mice. In conclusion, a non‐inflammatory factor, arginase 1, is involved in lacrimal hyposecretion in male NOD mice, regardless of dacryoadenitis status. These results shed light on the pathophysiological role of arginase 1 in SS (dry eye)., Key points Few reports have explored the possibility of involvement of non‐inflammatory factors in lacrimal hyposecretion in Sjögren's syndrome (SS).RNA‐sequencing analysis revealed that only four genes, including arginase 1, were downregulated in the lacrimal gland of SS model male mice (NOD mice) after onset of lacrimal hyposecretion and dacryoadenitis.Even in non‐dacryoadenitis‐type NOD mice, tear secretion and arginase 1 expression remained low.An arginase 1 inhibitor reduced tear secretion and partially reduced saliva secretion in BALB/c mice.The results indicate that a non‐inflammatory factor, arginase 1, is involved in lacrimal hyposecretion in male NOD mice, regardless of dacryoadenitis status.

Key points Loss‐of‐function mutations in proteins found at glycinergic synapses, most commonly in the α1 subunit of the glycine receptor (GlyR), cause the startle disease/hyperekplexia channelopathy in man.It was recently proposed that the receptors responsible are presynaptic homomeric GlyRs, rather than postsynaptic heteromeric GlyRs (which mediate glycinergic synaptic transmission), because heteromeric GlyRs are less affected by many startle mutations than homomers.We examined the α1 startle mutation S270T, at the extracellular end of the M2 transmembrane helix.Recombinant heteromeric GlyRs were less impaired than homomers by this mutation when we measured their response to equilibrium applications of glycine.However, currents elicited by synaptic‐like millisecond applications of glycine to outside‐out patches were much shorter (7‐ to 10‐fold) in all mutant receptors, both homomeric and heteromeric. Thus, the synaptic function of heteromeric receptors is likely to be impaired by the mutation. Abstract Human startle disease is caused by mutations in glycine receptor (GlyR) subunits or in other proteins associated with glycinergic synapses. Many startle mutations are known, but it is hard to correlate the degree of impairment at molecular level with the severity of symptoms in patients. It was recently proposed that the disease is caused by disruption in the function of presynaptic homomeric GlyRs (rather than postsynaptic heteromeric GlyRs), because homomeric GlyRs are more sensitive to loss‐of‐function mutations than heteromers. Our patch‐clamp recordings from heterologously expressed GlyRs characterised in detail the functional consequences of the α1S270T startle mutation, which is located at the extracellular end of the pore lining M2 transmembrane segment (18ʹ). This mutation profoundly decreased the maximum single‐channel open probability of homomeric GlyRs (to 0.16; cf. 0.99 for wild type) but reduced only marginally that of heteromeric GlyRs (0.96; cf. 0.99 for wild type). However, both heteromeric and homomeric mutant GlyRs became less sensitive to the neurotransmitter glycine. Responses evoked by brief, quasi‐synaptic pulses of glycine onto outside‐out patches were impaired in mutant receptors, as deactivation was approximately 10‐ and 7‐fold faster for homomeric and heteromeric GlyRs, respectively. Our data suggest that the α1S270T mutation is likely to affect the opening step in GlyR activation. The faster decay of synaptic currents mediated by mutant heteromeric GlyRs is expected to reduce charge transfer at the synapse, despite the high equilibrium open probability of these mutant channels., Key points Loss‐of‐function mutations in proteins found at glycinergic synapses, most commonly in the α1 subunit of the glycine receptor (GlyR), cause the startle disease/hyperekplexia channelopathy in man.It was recently proposed that the receptors responsible are presynaptic homomeric GlyRs, rather than postsynaptic heteromeric GlyRs (which mediate glycinergic synaptic transmission), because heteromeric GlyRs are less affected by many startle mutations than homomers.We examined the α1 startle mutation S270T, at the extracellular end of the M2 transmembrane helix.Recombinant heteromeric GlyRs were less impaired than homomers by this mutation when we measured their response to equilibrium applications of glycine.However, currents elicited by synaptic‐like millisecond applications of glycine to outside‐out patches were much shorter (7‐ to 10‐fold) in all mutant receptors, both homomeric and heteromeric. Thus, the synaptic function of heteromeric receptors is likely to be impaired by the mutation.

Key points Taste transduction occurs in taste buds in the tongue epithelium.The Ca2+‐activated Cl– channels TMEM16A and TMEM16B play relevant physiological roles in several sensory systems.Here, we report that TMEM16A, but not TMEM16B, is expressed in the apical part of taste buds.Large Ca2+‐activated Cl− currents blocked by Ani‐9, a selective inhibitor of TMEM16A, are measured in type I taste cells but not in type II or III taste cells.ATP indirectly activates Ca2+‐activated Cl– currents in type I cells through TMEM16A channels.These results indicate that TMEM16A is functional in type I taste cells and contribute to understanding the largely unknown physiological roles of these cells. Abstract The Ca2+‐activated Cl– channels TMEM16A and TMEM16B have relevant roles in many physiological processes including neuronal excitability and regulation of Cl– homeostasis. Here, we examined the presence of Ca2+‐activated Cl– channels in taste cells of mouse vallate papillae by using immunohistochemistry and electrophysiological recordings. By using immunohistochemistry we showed that only TMEM16A, and not TMEM16B, was expressed in taste bud cells where it largely co‐localized with the inwardly rectifying K+ channel KNCJ1 in the apical part of type I cells. By using whole‐cell patch‐clamp recordings in isolated cells from taste buds, we measured an average current of −1083 pA at −100 mV in 1.5 μm Ca2+ and symmetrical Cl– in type I cells. Ion substitution experiments and blockage by Ani‐9, a specific TMEM16A channel blocker, indicated that Ca2+ activated anionic currents through TMEM16A channels. We did not detect any Ca2+‐activated Cl– currents in type II or III taste cells. ATP is released by type II cells in response to various tastants and reaches type I cells where it is hydrolysed by ecto‐ATPases. Type I cells also express P2Y purinergic receptors and stimulation of type I cells with extracellular ATP produced large Ca2+‐activated Cl− currents blocked by Ani‐9, indicating a possible role of TMEM16A in ATP‐mediated signalling. These results provide a definitive demonstration that TMEM16A‐mediated currents are functional in type I taste cells and provide a foundation for future studies investigating physiological roles for these often‐neglected taste cells., Key points Taste transduction occurs in taste buds in the tongue epithelium.The Ca2+‐activated Cl– channels TMEM16A and TMEM16B play relevant physiological roles in several sensory systems.Here, we report that TMEM16A, but not TMEM16B, is expressed in the apical part of taste buds.Large Ca2+‐activated Cl− currents blocked by Ani‐9, a selective inhibitor of TMEM16A, are measured in type I taste cells but not in type II or III taste cells.ATP indirectly activates Ca2+‐activated Cl– currents in type I cells through TMEM16A channels.These results indicate that TMEM16A is functional in type I taste cells and contribute to understanding the largely unknown physiological roles of these cells.

Key points 25‐Hydroxyvitamin D (25OHD) is a partial agonist of TRPV1 whereby 25OHD can weakly activate TRPV1 yet antagonize the stimulatory effects of the full TRPV1 agonists capsaicin and oleoyl dopamine.25OHD binds to TRPV1 within the same vanilloid binding pocket as capsaicin.25OHD inhibits the potentiating effects of PKC‐mediated TRPV1 activity.25OHD reduces T‐cell activation and trigeminal neuron calcium signalling mediated by TRPV1 activity.These results provide evidence that TRPV1 is a novel receptor for the biological actions of vitamin D in addition to the well‐documented effects of vitamin D upon the nuclear vitamin D receptor.The results may have important implications for our current understanding of certain diseases where TRPV1 and vitamin D deficiency have been implicated, such as chronic pain and autoimmune diseases, such as type 1 diabetes. Abstract The capsaicin receptor TRPV1 plays an important role in nociception, inflammation and immunity and its activity is regulated by exogenous and endogenous lipophilic ligands. As vitamin D is lipophilic and involved in similar biological processes as TRPV1, we hypothesized that it directly regulates TRPV1 activity and function. Our calcium imaging and electrophysiological data demonstrate that vitamin D (25‐hydroxyvitamin D (25OHD) and 1,25‐hydroxyvitamin D (1,25OHD)) can weakly activate TRPV1 at physiologically relevant concentrations (100 nM). Furthermore, both 25OHD and 1,25OHD can inhibit capsaicin‐induced TRPV1 activity (IC50 = 34.3 ± 0.2 and 11.5 ± 0.9 nM, respectively), but not pH‐induced TRPV1 activity, suggesting that vitamin D interacts with TRPV1 in the same region as the TRPV1 agonist capsaicin. This hypothesis is supported by our in silico TRPV1 structural modelling studies, which place 25OHD in the same binding region as capsaicin. 25OHD also attenuates PKC‐dependent TRPV1 potentiation via interactions with a known PKC phospho‐acceptor residue in TRPV1. To provide evidence for a physiological role for the interaction of vitamin D with TRPV1, we employed two different cellular models known to express TRPV1: mouse CD4+ T‐cells and trigeminal neurons. Our results indicate that 25OHD reduces TRPV1‐induced cytokine release from T‐cells and capsaicin‐induced calcium activity in trigeminal neurons. In summary, we provide evidence that vitamin D is a novel endogenous regulator of TRPV1 channel activity that may play an important physiological role in addition to its known effects through the canonical nuclear vitamin D receptor pathway., Key points 25‐Hydroxyvitamin D (25OHD) is a partial agonist of TRPV1 whereby 25OHD can weakly activate TRPV1 yet antagonize the stimulatory effects of the full TRPV1 agonists capsaicin and oleoyl dopamine.25OHD binds to TRPV1 within the same vanilloid binding pocket as capsaicin.25OHD inhibits the potentiating effects of PKC‐mediated TRPV1 activity.25OHD reduces T‐cell activation and trigeminal neuron calcium signalling mediated by TRPV1 activity.These results provide evidence that TRPV1 is a novel receptor for the biological actions of vitamin D in addition to the well‐documented effects of vitamin D upon the nuclear vitamin D receptor.The results may have important implications for our current understanding of certain diseases where TRPV1 and vitamin D deficiency have been implicated, such as chronic pain and autoimmune diseases, such as type 1 diabetes.

Key points We report a novel method for the transient expression of SARS‐CoV‐2 envelope (E) protein in intracellular organelles and the plasma membrane of mammalian cells and Xenopus oocytes.Intracellular expression of SARS‐CoV‐2 E protein increases intra‐Golgi pH.By targeting the SARS‐CoV‐2 E protein to the plasma membrane, we show that it forms a cation channel, viroporin, that is modulated by changes of pH.This method for studying the activity of viroporins may facilitate screening for new antiviral drugs to identify novel treatments for COVID‐19. Abstract The envelope (E) protein of coronaviruses such as SARS‐CoV‐1 is proposed to form an ion channel or viroporin that participates in viral propagation and pathogenesis. Here we developed a technique to study the E protein of SARS‐CoV‐2 in mammalian cells by directed targeting using a carboxyl‐terminal fluorescent protein tag, mKate2. The wild‐type SARS‐CoV‐2 E protein can be trafficked to intracellular organelles, notably the endoplasmic reticulum–Golgi intermediate complex, where its expression increases pH inside the organelle. We also succeeded in targeting SARS‐CoV‐2 E to the plasma membrane, which enabled biophysical analysis using whole‐cell patch clamp recording in a mammalian cell line, HEK 293 cells, and two‐electrode voltage clamp electrophysiology in Xenopus oocytes. The results suggest that the E protein forms an ion channel that is permeable to monovalent cations such as Na+, Cs+ and K+. The E current is nearly time‐ and voltage‐independent when E protein is expressed in mammalian cells, and is modulated by changes of pH. At pH 6.0 and 7.4, the E protein current is activated, whereas at pH 8.0 and 9.0, the amplitude of E protein current is reduced, and in oocytes the inward E current fades at pH 9 in a time‐ and voltage‐dependent manner. Using this directed targeting method and electrophysiological recordings, potential inhibitors of the E protein can be screened and subsequently investigated for antiviral activity against SARS‐CoV‐2 in vitro and possible efficacy in treating COVID‐19., Key points We report a novel method for the transient expression of SARS‐CoV‐2 envelope (E) protein in intracellular organelles and the plasma membrane of mammalian cells and Xenopus oocytes.Intracellular expression of SARS‐CoV‐2 E protein increases intra‐Golgi pH.By targeting the SARS‐CoV‐2 E protein to the plasma membrane, we show that it forms a cation channel, viroporin, that is modulated by changes of pH.This method for studying the activity of viroporins may facilitate screening for new antiviral drugs to identify novel treatments for COVID‐19.

Key points In vascular smooth muscle cells (VSMCs), activation of Ca2+‐permeable store‐operated channels (SOCs) composed of canonical transient receptor potential channel 1 (TRPC1) subunits mediates Ca2+ entry pathways that regulate contraction, proliferation and migration, which are processes associated with vascular disease.Activation of TRPC1‐based SOCs requires protein kinase C (PKC) activity, which is proposed to phosphorylate TRPC1 proteins to promote channel opening by phosphatidylinositol 4,5‐bisphosphate (PIP2). We investigated the identity of the PKC isoform involved in activating TRPC1‐based SOCs in rat mesenteric artery VSMCs.TRPC1‐based SOCs were reduced by PKCδ inhibitors and knockdown of PKCδ expression. Store depletion induced interactions between TRPC1 and PKCδ and PKCδ‐dependent phosphorylation of TRPC1. Furthermore, generation of store‐operated interactions between PIP2 and TRPC1 and activation of TRPC1‐based SOCs by PIP2 required PKCδ.These findings reveal that PKCδ activity has an obligatory role in activating TRPC1‐based SOCs, through regulating PIP2‐mediated channel opening. Abstract In vascular smooth muscle cells (VMSCs), stimulation of Ca2+‐permeable canonical transient receptor potential channel 1 (TRPC1)‐based store‐operated channels (SOCs) mediates Ca2+ entry pathways that regulate cell contraction, proliferation and migration, which are processes associated with vascular disease. It is therefore important to understand how TRPC1‐based SOCs are activated. Stimulation of TRPC1‐based SOCs requires protein kinase C (PKC) activity, with store‐operated PKC‐dependent phosphorylation of TRPC1 essential for channel opening by phosphatidylinositol 4,5‐bisphosphate (PIP2). Experimental protocols used to activate TRPC1‐based SOCs suggest that the PKC isoform involved requires diacylglycerol (DAG) but is Ca2+‐insensitive, which are characteristics of the novel group of PKC isoforms (δ, ε, η, θ). Hence, the present study examined whether a novel PKC isoform(s) is involved in activating TRPC1‐based SOCs in contractile rat mesenteric artery VSMCs. Store‐operated whole‐cell cation currents were blocked by Pico145, a highly selective and potent TRPC1/4/5 channel blocker and T1E3, a TRPC1 blocking antibody. PKCδ was expressed in VSMCs, and selective PKCδ inhibitory peptides and knockdown of PKCδ expression with morpholinos oligomers inhibited TRPC1‐based SOCs. TRPC1 and PKCδ interactions and phosphorylation of TRPC1 induced by store depletion were both reduced by pharmacological inhibition and PKCδ knockdown. In addition, store‐operated PIP2 and TRPC1 interactions were blocked by PKCδ inhibition, and PKCδ was required for PIP2‐mediated activation of TRPC1 currents. These results identify the involvement of PKCδ in stimulation of TRPC1‐based SOCs and highlight that store‐operated PKCδ activity is obligatory for channel opening by PIP2, the probable activating ligand., Key points In vascular smooth muscle cells (VSMCs), activation of Ca2+‐permeable store‐operated channels (SOCs) composed of canonical transient receptor potential channel 1 (TRPC1) subunits mediates Ca2+ entry pathways that regulate contraction, proliferation and migration, which are processes associated with vascular disease.Activation of TRPC1‐based SOCs requires protein kinase C (PKC) activity, which is proposed to phosphorylate TRPC1 proteins to promote channel opening by phosphatidylinositol 4,5‐bisphosphate (PIP2). We investigated the identity of the PKC isoform involved in activating TRPC1‐based SOCs in rat mesenteric artery VSMCs.TRPC1‐based SOCs were reduced by PKCδ inhibitors and knockdown of PKCδ expression. Store depletion induced interactions between TRPC1 and PKCδ and PKCδ‐dependent phosphorylation of TRPC1. Furthermore, generation of store‐operated interactions between PIP2 and TRPC1 and activation of TRPC1‐based SOCs by PIP2 required PKCδ.These findings reveal that PKCδ activity has an obligatory role in activating TRPC1‐based SOCs, through regulating PIP2‐mediated channel opening.

Key points Cl− and HCO3 − had similar paracellular permeabilities in human airway epithelia.PCl/PNa of airway epithelia was unaltered by pH 7.4 vs. pH 6.0 solutions.Under basal conditions, calculated paracellular HCO3 − flux was secretory.Cytokines that increased airway surface liquid pH decreased or reversed paracellular HCO3 − flux.HCO3 − flux through the paracellular pathway may counterbalance effects of cellular H+ and HCO3 − secretion. Abstract Airway epithelia control the pH of airway surface liquid (ASL), thereby optimizing respiratory defences. Active H+ and HCO3 − secretion by airway epithelial cells produce an ASL that is acidic compared with the interstitial space. The paracellular pathway could provide a route for passive HCO3 − flux that also modifies ASL pH. However, there is limited information about paracellular HCO3 − flux, and it remains uncertain whether an acidic pH produced by loss of cystic fibrosis transmembrane conductance regulator anion channels or proinflammatory cytokines might alter the paracellular pathway function. To investigate paracellular HCO3 − transport, we studied differentiated primary cultures of human cystic fibrosis (CF) and non‐CF airway epithelia. The paracellular pathway was pH‐insensitive at pH 6.0 vs. pH 7.4 and was equally permeable to Cl− and HCO3 −. Under basal conditions at pH ∼6.6, calculated paracellular HCO3 − flux was weakly secretory. Treating epithelia with IL‐17 plus TNFα alkalinized ASL pH to ∼7.0, increased paracellular HCO3 − permeability, and paracellular HCO3 − flux was negligible. Applying IL‐13 increased ASL pH to ∼7.4 without altering paracellular HCO3 − permeability, and calculated paracellular HCO3 − flux was absorptive. These results suggest that HCO3 − flux through the paracellular pathway counterbalances, in part, changes in the ASL pH produced via cellular mechanisms. As the pH of ASL increases towards that of basolateral liquid, paracellular HCO3 − flux becomes absorptive, tempering the alkaline pH generated by transcellular HCO3 − secretion., Key points Cl− and HCO3 − had similar paracellular permeabilities in human airway epithelia.PCl/PNa of airway epithelia was unaltered by pH 7.4 vs. pH 6.0 solutions.Under basal conditions, calculated paracellular HCO3 − flux was secretory.Cytokines that increased airway surface liquid pH decreased or reversed paracellular HCO3 − flux.HCO3 − flux through the paracellular pathway may counterbalance effects of cellular H+ and HCO3 − secretion.

Key points Some ion channels are known to behave as inductors and make up the parallel resonant circuit in the plasma membrane of neurons, which enables neurons to respond to current inputs with a specific frequency (so‐called ‘resonant properties’).Here, we report that heterologous expression of mouse Kv11 voltage‐dependent K+ channels generate resonance and oscillation at depolarized membrane potentials in HEK293 cells; expressions of individual Kv11 subtypes generate resonance and oscillation with different frequency properties.Kv11.3‐expressing HEK293 cells exhibited transient conductance changes that opposed the current changes induced by voltage steps; this probably enables Kv11 channels to behave like an inductor.The resonance and oscillation of inferior olivary neurons were impaired at the resting membrane potential in Kv11.3 knockout mice.This study helps to elucidate basic ion channel properties that are crucial for the frequency responses of neurons. Abstract The plasma membranes of some neurons preferentially respond to current inputs with a specific frequency, and output as large voltage changes. This property is called resonance, and is thought to be mediated by ion channels that show inductor‐like behaviour. However, details of the candidate ion channels remain unclear. In this study, we mainly focused on the functional roles of Kv11 potassium (K+) channels, encoded by ether‐á‐go‐go‐related genes, in resonance in mouse inferior olivary (IO) neurons. We transfected HEK293 cells with long or short splice variants of Kv11.1 (Merg1a and Merg1b) or Kv11.3, and examined membrane properties using whole‐cell recording. Transfection with Kv11 channels reproduced resonance at membrane potentials depolarized from the resting state. Frequency ranges of Kv11.3‐, Kv11.1(Merg1b)‐ and Kv11.1(Merg1a)‐expressing cells were 2–6 Hz, 2–4 Hz, and 0.6–0.8 Hz, respectively. Responses of Kv11.3 currents to step voltage changes were essentially similar to those of inductor currents in the resistor–inductor–capacitor circuit. Furthermore, Kv11 transfections generated membrane potential oscillations. We also confirmed the contribution of HCN1 channels as a major mediator of resonance at more hyperpolarized potentials by transfection into HEK293 cells. The Kv11 current kinetics and properties of Kv11‐dependent resonance suggested that Kv11.3 mediated resonance in IO neurons. This finding was confirmed by the impairment of resonance and oscillation at –30 to –60 mV in Kcnh7 (Kv11.3) knockout mice. These results suggest that Kv11 channels have important roles in inducing frequency‐dependent responses in a subtype‐dependent manner from resting to depolarized membrane potentials., Key points Some ion channels are known to behave as inductors and make up the parallel resonant circuit in the plasma membrane of neurons, which enables neurons to respond to current inputs with a specific frequency (so‐called ‘resonant properties’).Here, we report that heterologous expression of mouse Kv11 voltage‐dependent K+ channels generate resonance and oscillation at depolarized membrane potentials in HEK293 cells; expressions of individual Kv11 subtypes generate resonance and oscillation with different frequency properties.Kv11.3‐expressing HEK293 cells exhibited transient conductance changes that opposed the current changes induced by voltage steps; this probably enables Kv11 channels to behave like an inductor.The resonance and oscillation of inferior olivary neurons were impaired at the resting membrane potential in Kv11.3 knockout mice.This study helps to elucidate basic ion channel properties that are crucial for the frequency responses of neurons.

KEY POINTS: Slc4a4 (mouse) encodes at least five variants of the electrogenic sodium/bicarbonate transporter NBCe1. The initial 41 cytosolic amino acids of NBCe1‐A and ‐D are unique; NBCe1‐A has high activity. The initial 85 amino acids of NBCe1‐B, ‐C and ‐E are unique; NBCe1‐B and ‐C have low activity. Previous work showed that deleting residues 1–85 or 40–62 of NBCe1‐B, or 1–87 of NBCe1‐C, eliminates autoinhibition. These regions also include binding determinants for IRBIT (inositol trisphosphate (IP(3))‐receptor binding protein released with IP(3)), which relieves autoinhibition. Here, systematically replacing/deleting residues 28–62, we find that only the nine amino acid cationic cluster (residues 40–48) of NBCe1‐B is essential for autoinhibition. IRBIT stimulates all but one low‐activity construct. We suggest that electrostatic interactions – which IRBIT presumably interrupts – between the cationic cluster and the membrane or other domains of NBCe1 play a central role in tempering the activity of NBCe1‐B in the pancreas, brain and other organs. ABSTRACT: Variant B of the electrogenic Na(+)/HCO(3) (−) cotransporter (NBCe1‐B) contributes to the vectorial transport of HCO(3) (−) in epithelia (e.g. pancreatic ducts) and to the maintenance of intracellular pH in the central nervous systems (e.g. astrocytes). NBCe1‐B has very low basal activity due to an autoinhibitory domain (AID) located, at least in part, in the unique portion (residues 1–85) of the cytosolic NH(2)‐terminus. Previous work has shown that removing 23 amino acids (residues 40–62) stimulates NBCe1‐B. Here, we test the hypothesis that a cationic cluster of nine consecutive positively charged amino acids (residues 40−48) is a necessary part of the AID. Using two‐electrode voltage clamping of Xenopus oocytes, we assess the activity of human NBCe1‐B constructs in which we systematically replace or delete residues 28–62, which includes the cationic cluster. We find that replacing or deleting all residues within the cationic cluster markedly increases NBCe1‐B activity (i.e. eliminates autoinhibition). On the background of a cationic clusterless construct, systematically restoring Arg residues restores autoinhibition in two distinct quanta, with one to three Arg residues restoring ∼50%, and four or more Arg residues restoring virtually all autoinhibition. Systematically deleting residues before the cluster reduces autoinhibition by, at most, a small amount. Replacing or deleting residues after the cluster has no effect. For constructs with low NBCe1 activity (but good surface expression, as assessed by biotinylation), co‐expression with super‐IRBIT (lacking PP1‐binding site) restores full activity (i.e. relieves autoinhibition). In summary, the cationic cluster is a necessary component of the AID of NBCe1‐B.

Key points Na+ current inactivation is biphasic in insulin‐secreting cells, proceeding with two voltage dependences that are half‐maximal at ∼−100 mV and −60 mV.Inactivation of voltage‐gated Na+ (NaV) channels occurs at ∼30 mV more negative voltages in insulin‐secreting Ins1 and primary β‐cells than in HEK, CHO or glucagon‐secreting αTC1‐6 cells.The difference in inactivation between Ins1 and non‐β‐cells persists in the inside‐out patch configuration, discounting an involvement of a diffusible factor.In Ins1 cells and primary β‐cells, but not in HEK cells, inactivation of a single NaV subtype is biphasic and follows two voltage dependences separated by 30–40 mV.We propose that NaV channels adopt different inactivation behaviours depending on the local membrane environment. Abstract Pancreatic β‐cells are equipped with voltage‐gated Na+ channels that undergo biphasic voltage‐dependent steady‐state inactivation. A small Na+ current component (10–15%) inactivates over physiological membrane potentials and contributes to action potential firing. However, the major Na+ channel component is completely inactivated at −90 to −80 mV and is therefore inactive in the β‐cell. It has been proposed that the biphasic inactivation reflects the contribution of different NaV α‐subunits. We tested this possibility by expression of TTX‐resistant variants of the NaV subunits found in β‐cells (NaV1.3, NaV1.6 and NaV1.7) in insulin‐secreting Ins1 cells and in non‐β‐cells (including HEK and CHO cells). We found that all NaV subunits inactivated at 20–30 mV more negative membrane potentials in Ins1 cells than in HEK or CHO cells. The more negative inactivation in Ins1 cells does not involve a diffusible intracellular factor because the difference between Ins1 and CHO persisted after excision of the membrane. NaV1.7 inactivated at 15‐­20 mV more negative membrane potentials than NaV1.3 and NaV1.6 in Ins1 cells but this small difference is insufficient to solely explain the biphasic inactivation in Ins1 cells. In Ins1 cells, but never in the other cell types, widely different components of NaV inactivation (separated by 30 mV) were also observed following expression of a single type of NaV α‐subunit. The more positive component exhibited a voltage dependence of inactivation similar to that found in HEK and CHO cells. We propose that biphasic NaV inactivation in insulin‐secreting cells reflects insertion of channels in membrane domains that differ with regard to lipid and/or membrane protein composition., Key points Na+ current inactivation is biphasic in insulin‐secreting cells, proceeding with two voltage dependences that are half‐maximal at ∼−100 mV and −60 mV.Inactivation of voltage‐gated Na+ (NaV) channels occurs at ∼30 mV more negative voltages in insulin‐secreting Ins1 and primary β‐cells than in HEK, CHO or glucagon‐secreting αTC1‐6 cells.The difference in inactivation between Ins1 and non‐β‐cells persists in the inside‐out patch configuration, discounting an involvement of a diffusible factor.In Ins1 cells and primary β‐cells, but not in HEK cells, inactivation of a single NaV subtype is biphasic and follows two voltage dependences separated by 30–40 mV.We propose that NaV channels adopt different inactivation behaviours depending on the local membrane environment.

Key points Cav1.2 channels maintain activity through interactions with calmodulin (CaM). In this study, activities of the Cav1.2 channel (α1C) and of mutant‐derivatives, C‐terminal deleted (α1CΔ) and α1CΔ linked with CaM (α1CΔCaM), were compared in the inside‐out mode.α1CΔ with CaM, but not without CaM, and α1CΔCaM were active, suggesting that CaM induced channel activity through a dynamic interaction with the channel, even without the distal C‐tail.ATP induced α1C activity with CaM and enhanced activity of the mutant channels. Okadaic acid mimicked the effect of ATP on the wildtype but not mutant channels.These results supported the hypothesis that CaM and ATP maintain activity of Cav1.2 channels through their dynamic interactions. ATP effects involve mechanisms both related and unrelated to channel phosphorylation.CaM‐linked channels are useful tools for investigating Cav1.2 channels in the inside‐out mode; the fast run‐down is prevented by only ATP and the slow run‐down is nearly absent. Abstract Calmodulin (CaM) plays a critical role in regulation of Cav1.2 Ca2+ channels. CaM binds to the channel directly, maintaining channel activity and regulating it in a Ca2+‐dependent manner. To explore the molecular mechanisms involved, we compared the activity of the wildtype channel (α1C) and mutant derivatives, C‐terminal deleted (α1C∆) and α1C∆ linked to CaM (α1C∆CaM). These were co‐expressed with β2a and α2δ subunits in HEK293 cells. In the inside‐out mode, α1C and α1C∆ showed minimal open‐probabilities in a basic internal solution (run‐down), whereas α1C∆ with CaM and α1C∆CaM maintained detectable channel activity, confirming that CaM was necessary, but not sufficient, for channel activity. Previously, we reported that ATP was required to maintain channel activity of α1C. Unlike α1C, the mutant channels did not require ATP for activation in the early phase (3–5 min). However, α1C∆ with CaM + ATP and α1C∆CaM with ATP maintained activity, even in the late phase (after 7–9 min). These results suggested that CaM and ATP interacted dynamically with the proximal C‐terminal tail of the channel and, thereby, produced channel activity. In addition, okadaic acid, a protein phosphatase inhibitor, could substitute for the effects of ATP on α1C but not on the mutant channels. These results supported the hypothesis that CaM and ATP maintain activity of Cav1.2 channels, further indicating that ATP has dual effects. One maintains phosphorylation of the channel and the other becomes apparent when the distal carboxyl‐terminal tail is removed., Key points Cav1.2 channels maintain activity through interactions with calmodulin (CaM). In this study, activities of the Cav1.2 channel (α1C) and of mutant‐derivatives, C‐terminal deleted (α1CΔ) and α1CΔ linked with CaM (α1CΔCaM), were compared in the inside‐out mode.α1CΔ with CaM, but not without CaM, and α1CΔCaM were active, suggesting that CaM induced channel activity through a dynamic interaction with the channel, even without the distal C‐tail.ATP induced α1C activity with CaM and enhanced activity of the mutant channels. Okadaic acid mimicked the effect of ATP on the wildtype but not mutant channels.These results supported the hypothesis that CaM and ATP maintain activity of Cav1.2 channels through their dynamic interactions. ATP effects involve mechanisms both related and unrelated to channel phosphorylation.CaM‐linked channels are useful tools for investigating Cav1.2 channels in the inside‐out mode; the fast run‐down is prevented by only ATP and the slow run‐down is nearly absent.

Key points The physiological significance of the developmental switch from fetal to adult acetylcholine receptors in muscle (AChRs) and the functional impact of AChR clustering by rapsyn are not well studied.Using patch clamp experiments, we show that recovery from desensitization is faster in the adult AChR isoform.Recovery from desensitization is determined by the AChR isoform‐specific cytoplasmic M3–M4 domain.The co‐expression of rapsyn in muscle cells induced AChR clustering and facilitated recovery from desensitization in both fetal and adult AChRs. In fetal AChRs, facilitation of recovery kinetics by rapsyn was independent of AChR clustering.These effects could be crucial adaptations to motor neuron firing rates, which, in rodents, have been shown to increase around the time of birth when AChRs cluster at the developing neuromuscular junctions. Abstract The neuromuscular junction (NMJ) is the site of a number of autoimmune and genetic disorders, many involving the muscle‐type nicotinic acetylcholine receptor (AChR), although there are aspects of normal NMJ development and function that need to be better understood. In particular, there are still questions regarding the implications of the developmental switch from fetal to adult AChRs, as well as how their functions might be modified by rapsyn that clusters the AChRs. Desensitization of human muscle AChRs was investigated using the patch clamp technique to measure whole‐cell currents in muscle‐type (TE671/CN21) and non‐muscle (HEK293) cell lines expressing either fetal or adult AChRs. Desensitization time constants were similar with both AChR isoforms but recovery time constants were shorter in cells expressing adult compared to fetal AChRs (P, Key points The physiological significance of the developmental switch from fetal to adult acetylcholine receptors in muscle (AChRs) and the functional impact of AChR clustering by rapsyn are not well studied.Using patch clamp experiments, we show that recovery from desensitization is faster in the adult AChR isoform.Recovery from desensitization is determined by the AChR isoform‐specific cytoplasmic M3–M4 domain.The co‐expression of rapsyn in muscle cells induced AChR clustering and facilitated recovery from desensitization in both fetal and adult AChRs. In fetal AChRs, facilitation of recovery kinetics by rapsyn was independent of AChR clustering.These effects could be crucial adaptations to motor neuron firing rates, which, in rodents, have been shown to increase around the time of birth when AChRs cluster at the developing neuromuscular junctions.

Key points In the epididymis, elaborate communication networks between epithelial cells are important with respect to establishing an optimal acidic luminal environment for the maturation and storage of spermatozoa, which is essential for male fertility. Proton secretion by epididymal clear cells is achieved via the proton pumping V-ATPase located in their apical membrane. In the present study, we dissect the molecular mechanisms by which clear cells respond to luminal ATP and adenosine to modulate their acidifying activity via the adenosine receptor ADORA2B and the pH-sensitive ATP receptor P2X4. We demonstrate that the hydrolysis of ATP to produce adenosine by ectonucleotidases plays a key role in V-ATPase-dependent proton secretion, and is part of a feedback loop that ensures acidification of the luminal compartment These results help us better understand how professional proton-secreting cells respond to extracellular cues to modulate their functions, and how they communicate with neighbouring cells. Abstract Cell-cell cross-talk is crucial for the dynamic function of epithelia, although how epithelial cells detect and respond to variations in extracellular stimuli to modulate their environment remains incompletely understood. In the present study, we used the epididymis as a model system to investigate epithelial cell regulation by luminal factors. In the epididymis, elaborate communication networks between the different epithelial cell types are important for establishing an optimal acidic luminal environment for the maturation and storage of spermatozoa. In particular, clear cells (CCs) secrete protons into the lumen via the proton pumping V-ATPase located in their apical membrane, a process that is activated by luminal alkalinization. However, how CCs detect luminal pH variations to modulate their function remains uncharacterized. Purinergic regulation of epithelial transport is modulated by extracellular pH in other tissues. In the present study, functional analysis of the mouse cauda epididymis perfused in vivo showed that luminal ATP and adenosine modulate the acidifying activity of CCs via the purinergic ADORA2B and P2X4 receptors, and that luminal adenosine content is itself regulated by luminal pH. Altogether, our observations illustrate mechanisms by which CCs are activated by pH sensitive P2X4 receptor and ectonucleotidases, providing a feedback mechanism for the maintenance of luminal pH. These novel mechanisms by which professional proton-secreting cells respond to extracellular cues to modulate their functions, as well as how they communicate with neighbouring cells, might be translatable to other acidifying epithelia.

KEY POINTS : Amino acid absorption requires luminal uptake into and subsequent basolateral efflux out of epithelial cells, with the latter step being critical to regulate the intracellular concentration of the amino acids. The basolateral essential neutral amino acid uniporter LAT4 (SLC43A2) has been suggested to drive the net efflux of non-essential and cationic amino acids via parallel amino acid antiporters by recycling some of their substrates; its deletion has been shown to cause defective postnatal growth and death in mice. Here we test the regulatory function of LAT4 phosphorylation sites by mimicking their phosphorylated and dephosphorylated states in Xenopus laevis oocytes and show that dephosphorylation of S274 and phosphorylation of S297 increase LAT4 membrane localization and function. Using new phosphorylation site-specific antibodies, we observe changes in LAT4 phosphorylation in mouse small intestine that correspond to its upregulation at the expected feeding time. These results strongly suggest that LAT4 phosphorylation participates in the regulation of transepithelial amino acid absorption. ABSTRACT : The essential amino acid uniporters LAT4 and TAT1 are located at the basolateral side of intestinal and kidney epithelial cells and their transport function has been suggested to control the transepithelial (re)absorption of neutral and possibly also cationic amino acids. Uniporter LAT4 selectively transports the branched chain amino acids leucine, isoleucine and valine, and additionally methionine and phenylalanine. Its deletion leads to a postnatal growth failure and early death in mice. Since LAT4 has been reported to be phosphorylated in vivo, we hypothesized that phosphorylation regulates its function. Using Xenopus laevis oocytes, we tested the impact of LAT4 phosphorylation at Ser274 and Ser297 by expressing mutant constructs mimicking phosphorylated and dephosphorylated states. We then investigated the in vivo regulation of LAT4 in mouse small intestine using new phosphorylation site-specific antibodies and a time-restricted diet. In Xenopus oocytes, mimicking non-phosphorylation of Ser274 led to an increase in affinity and apparent surface membrane localization of LAT4, stimulating its transport activity, while the same mutation of Ser297 decreased LAT4's apparent surface expression and transport rate. In wild-type mice, LAT4 phosphorylation on Ser274 was uniform at the beginning of the inactive phase (ZT0). In contrast, at the beginning of the active phase (ZT12), corresponding to the anticipated feeding time, Ser274 phosphorylation was decreased and restricted to relatively large patches of cells, while Ser297 phosphorylation was increased. We conclude that phosphorylation of small intestinal LAT4 is under food-entrained circadian control, leading presumably to an upregulation of LAT4 function at the anticipated feeding time.

KEY POINTS: Exogenous Na(+)/H(+) exchanger 1 (NHE1) expression stimulated the collective migration of epithelial cell sheets. Stimulation with epidermal growth factor, a key morphogen, primarily increased migration of the front row of cells, whereas NHE1 increased that of submarginal cell rows, and the two stimuli were additive. Accordingly, NHE1 localized not only to the leading edges of leader cells, but also in cryptic lamellipodia in submarginal cell rows. NHE1 expression disrupted the morphology of epithelial cell sheets and three‐dimensional cysts. ABSTRACT: Collective cell migration plays essential roles in embryonic development, in normal epithelial repair processes, and in many diseases including cancer. The Na(+)/H(+) exchanger 1 (NHE1, SLC9A1) is an important regulator of motility in many cells and has been widely studied for its roles in cancer, although its possible role in collective migration of normal epithelial cells has remained unresolved. In the present study, we show that NHE1 expression in MDCK‐II kidney epithelial cells accelerated collective cell migration. NHE1 localized to the leading edges of leader cells, as well as to cryptic lamellipodia in submarginal cell rows. Epidermal growth factor, a kidney morphogen, increased displacement of the front row of collectively migrating cells and reduced the number of migration fingers. NHE1 expression increased the number of migration fingers and increased displacement of submarginal cell rows, resulting in additive effects of NHE1 and epidermal growth factor. Finally, NHE1 expression resulted in disorganized development of MDCK‐II cell cysts. Thus, NHE1 contributes to collective migration and epithelial morphogenesis, suggesting roles for the transporter in embryonic and early postnatal development.

KEY POINTS: Electrophysiological characterization of Q1412X‐CFTR, a C‐terminal truncation mutation of cystic fibrosis transmembrane conductance regulator (CFTR) associated with the severe form of cystic fibrosis (CF), reveals a gating defect that has not been reported previously. Mechanistic investigations of the gating deficit in Q1412X‐CFTR suggest that the reduced open probability in Q1412X‐CFTR is the result of a disruption of the function of the second ATP binding site (or site 2) in the nucleotide binding domains (NBDs). Detailed comparisons of several mutations with different degrees of truncation in the C‐terminal region of NBD2 reveal the importance of the last two beta‐strands in NBD2 for maintaining proper gating functions. The results of the present study also show that the application of clinically‐approved drugs (VX‐770 and VX‐809) can greatly enhance the function of Q1412X, providing in vitro evidence for a therapeutic strategy employing both reagents for patients bearing Q1412X or similar truncation mutations. ABSTRACT: Cystic fibrosis (CF) is caused by loss‐of‐function mutations of cystic fibrosis transmembrane conductance regulator (CFTR), a phosphorylation‐activated but ATP‐gated chloride channel. Based on the molecular mechanism of CF pathogenesis, disease‐associated mutations are categorized into six classes. Among them, Class VI, whose members include some of the C‐terminal truncation mutations such as Q1412X, is defined as decreased membrane expression because of a faster turnover rate. In the present study, we characterized the functional properties of Q1412X‐CFTR, a severe‐form premature stop codon mutation. We confirmed previous findings of a ∼90% decrease in membrane expression but found a ∼95% reduction in the open probability (P (o)). Detailed kinetic studies support the idea that the gating defect is the result of a dysfunctional ATP‐binding site 2 in the nucleotide binding domains (NBDs). Because the Q1412X mutation results in a deletion of the last two beta‐strands in NBD2 and the whole C‐terminal region, we further characterized truncation mutations with different degrees of deletion in this segment. Mutations that completely or partially remove the C‐terminus of CFTR at the same time as keeping an intact NBD2 (i.e. D1425X and S1455X) assume gating function almost identical to that of wild‐type channels. However, the deletion of the last beta‐strand in the NBD2 (i.e. N1419X) causes gating dysfunction that is milder than that of Q1412X. Thus, normal CFTR gating requires structural integrity of NBD2. Moreover, our observation that clinically‐approved VX‐809 (Lumacaftor, Vertex Pharmaceuticals, Boston, MA, USA) and VX‐770 (Ivacaftor, Vertex Pharmaceuticals, Boston, MA, USA) significantly enhance the overall function of Q1412X‐CFTR provides the conceptual basis for the treatment of patients carrying this mutation.

Key points Arginine vasopressin (AVP) stimulates the release of enteroendocrine L‐cell derived hormones glucagon‐like peptide‐1 (GLP‐1) and peptide YY (PYY) in vitro from mouse and human colons.This is mediated by the AVP receptor 1B, which is highly enriched in colonic L‐cells and linked to the elevation of L‐cell calcium and cAMP concentrations.By means of Ussing chambers, we show that AVP reduced colonic anion secretion, although this was blocked by a specific neuropeptide Y receptor Y1 receptor antagonist, suggesting that L‐cell‐released PYY acts locally on the epithelium to modulate fluid balance.In human serum samples, PYY concentrations were higher in samples with raised osmolality and copeptin (surrogate marker for AVP).These findings describe, for the first time, the role of L‐cells in AVP regulated intestinal fluid secretion, potentially linking together hormonal control of blood volume and blood glucose levels, and thus adding to our understanding of the complex pathways involved in the gut hormonal response to different stimuli. Abstract Arginine vasopressin (AVP) regulates fluid balance and blood pressure via AVP receptor (AVPR)2 in the kidney and AVP receptor 1A in vascular smooth muscle. Its role in intestinal function has received less attention. We hypothesized that enteroendocrine L‐cells producing glucagon‐like peptide 1 (GLP‐1) and peptide YY (PYY) may be a target of AVP and contribute to the control of fluid balance. Avpr1b expression was assessed by quantitative RT‐PCR on flourescence‐activated cell sorting‐isolated L‐ and control cells and was enriched in colonic L‐cells. AVP stimulated GLP‐1 and PYY release from primary cultured murine and human colonic cells and was associated with elevated calcium and cAMP concentrations in L‐cells as measured in cultures from GLU‐Cre/ROSA26‐GCaMP3 and GLU‐Epac2camps mice. An antagonist of AVPR1B reduced AVP‐triggered hormone secretion from murine and human cells. In Ussing chambers, basolaterally applied AVP reduced colonic anion secretion and this effect was blocked by a specific neuropeptide Y receptor Y1 (NPY1R) antagonist. In human serum, PYY concentrations were higher in samples with raised osmolality or copeptin (a surrogate marker for AVP). In conclusion, we propose that AVP activates L‐cell AVPR1B, causing GLP‐1 and PYY secretion. PYY in turn reduces colonic anion secretion via epithelial NPY1R. Our data suggest L‐cells are active players in the hypothalamic control of intestinal fluid homeostasis, providing a potential link between the regulation of blood volume/pressure/osmolality and blood glucose., Key points Arginine vasopressin (AVP) stimulates the release of enteroendocrine L‐cell derived hormones glucagon‐like peptide‐1 (GLP‐1) and peptide YY (PYY) in vitro from mouse and human colons.This is mediated by the AVP receptor 1B, which is highly enriched in colonic L‐cells and linked to the elevation of L‐cell calcium and cAMP concentrations.By means of Ussing chambers, we show that AVP reduced colonic anion secretion, although this was blocked by a specific neuropeptide Y receptor Y1 receptor antagonist, suggesting that L‐cell‐released PYY acts locally on the epithelium to modulate fluid balance.In human serum samples, PYY concentrations were higher in samples with raised osmolality and copeptin (surrogate marker for AVP).These findings describe, for the first time, the role of L‐cells in AVP regulated intestinal fluid secretion, potentially linking together hormonal control of blood volume and blood glucose levels, and thus adding to our understanding of the complex pathways involved in the gut hormonal response to different stimuli.

Key points Raised arterial blood CO2 (hypercapnia) is a feature of many lung diseases.CO2 has been shown to act as a cell signalling molecule in human cells, notably by influencing the levels of cell signalling second messengers: cAMP and Ca2+.Hypercapnia reduced cAMP‐stimulated cystic fibrosis transmembrane conductance regulator‐dependent anion and fluid transport in Calu‐3 cells and primary human airway epithelia but did not affect cAMP‐regulated HCO3 − transport via pendrin or Na+/HCO3 − cotransporters.These results further support the role of CO2 as a cell signalling molecule and suggests CO2‐induced reductions in airway anion and fluid transport may impair innate defence mechanisms of the lungs. Abstract Hypercapnia is clinically defined as an arterial blood partial pressure of CO2 of above 40 mmHg and is a feature of chronic lung disease. In previous studies we have demonstrated that hypercapnia modulates agonist‐stimulated cAMP levels through effects on transmembrane adenylyl cyclase activity. In the airways, cAMP is known to regulate cystic fibrosis transmembrane conductance regulator (CFTR)‐mediated anion and fluid secretion, which contributes to airway surface liquid homeostasis. The aim of the current work was to investigate if hypercapnia could modulate cAMP‐regulated ion and fluid transport in human airway epithelial cells. We found that acute exposure to hypercapnia significantly reduced forskolin‐stimulated elevations in intracellular cAMP as well as both adenosine‐ and forskolin‐stimulated increases in CFTR‐dependent transepithelial short‐circuit current, in polarised cultures of Calu‐3 human airway cells. This CO2‐induced reduction in anion secretion was not due to a decrease in HCO3 − transport given that neither a change in CFTR‐dependent HCO3 − efflux nor Na+/HCO3 − cotransporter‐dependent HCO3 − influx were CO2‐sensitive. Hypercapnia also reduced the volume of forskolin‐stimulated fluid secretion over 24 h, yet had no effect on the HCO3 − content of the secreted fluid. Our data reveal that hypercapnia reduces CFTR‐dependent, electrogenic Cl− and fluid secretion, but not CFTR‐dependent HCO3 − secretion, which highlights a differential sensitivity of Cl− and HCO3 − transporters to raised CO2 in Calu‐3 cells. Hypercapnia also reduced forskolin‐stimulated CFTR‐dependent anion secretion in primary human airway epithelia. Based on current models of airways biology, a reduction in fluid secretion, associated with hypercapnia, would be predicted to have important consequences for airways hydration and the innate defence mechanisms of the lungs., Key points Raised arterial blood CO2 (hypercapnia) is a feature of many lung diseases.CO2 has been shown to act as a cell signalling molecule in human cells, notably by influencing the levels of cell signalling second messengers: cAMP and Ca2+.Hypercapnia reduced cAMP‐stimulated cystic fibrosis transmembrane conductance regulator‐dependent anion and fluid transport in Calu‐3 cells and primary human airway epithelia but did not affect cAMP‐regulated HCO3 − transport via pendrin or Na+/HCO3 − cotransporters.These results further support the role of CO2 as a cell signalling molecule and suggests CO2‐induced reductions in airway anion and fluid transport may impair innate defence mechanisms of the lungs.

KEY POINTS: Re‐sensitization of P2X(4) receptors depends on a protonation/de‐protonation cycle. Protonation and de‐protonation of the receptors is achieved by internalization and recycling of P2X(4) receptors via acidic compartments. Protonation and de‐protonation occurs at critical histidine residues within the extracellular loop of P2X(4) receptors. Re‐sensitization is blocked in the presence of the receptor agonist ATP. ABSTRACT: P2X(4) receptors are members of the P2X receptor family of cation‐permeable, ligand‐gated ion channels that open in response to the binding of extracellular ATP. P2X(4) receptors are implicated in a variety of biological processes, including cardiac function, cell death, pain sensation and immune responses. These physiological functions depend on receptor activation on the cell surface. Receptor activation is followed by receptor desensitization and deactivation upon removal of ATP. Subsequent re‐sensitization is required to return the receptor into its resting state. Desensitization and re‐sensitization are therefore crucial determinants of P2X receptor signal transduction and responsiveness to ATP. However, the molecular mechanisms controlling desensitization and re‐sensitization are not fully understood. In the present study, we provide evidence that internalization and recycling via acidic compartments is essential for P2X(4) receptor re‐sensitization. Re‐sensitization depends on a protonation/de‐protonation cycle of critical histidine residues within the extracellular loop of P2X(4) receptors that is mediated by receptor internalization and recycling. Interestingly, re‐sensitization under acidic conditions is completely revoked by receptor agonist ATP. Our data support the physiological importance of the unique subcellular distribution of P2X(4) receptors that is predominantly found within acidic compartments. Based on these findings, we suggest that recycling of P2X(4) receptors regulates the cellular responsiveness in the sustained presence of ATP.

KEY POINTS: Maternal high‐fat diet consumption predisposes to metabolic dysfunction in male and female offspring at young adulthood. Maternal obesity programs non‐alcoholic fatty liver disease (NAFLD) in a sex‐dependent manner. We demonstrate sex‐dependent liver transcriptome profiles in rat offspring of obese mothers. In this study, we focused on pathways related to insulin, glucose and lipid signalling. These results improve understanding of the mechanisms by which a maternal high‐fat diet affects the offspring. ABSTRACT: Maternal obesity (MO) predisposes offspring (F1) to obesity, insulin resistance (IR) and non‐alcoholic fatty liver disease (NAFLD). MO's effects on the F1 liver transcriptome are poorly understood. We used RNA‐seq to determine the liver transcriptome of male and female F1 of MO and control‐fed mothers. We hypothesized that MO‐F1 are predisposed to sex‐dependent adult liver dysfunction. Female Wistar rat mothers ate a control (C) or obesogenic (MO) diet from the time they were weaned through breeding at postnatal day (PND) 120, delivery and lactation. After weaning, all male and female F1 ate a control diet. At PND 110, F1 serum, liver and fat were collected to analyse metabolites, histology and liver differentially expressed genes. Male and female MO‐F1 showed increased adiposity index, triglycerides, insulin and homeostatic model assessment vs. C‐F1 with similar body weight and glucose serum concentrations. MO‐F1 males presented greater physiological and histological NAFLD characteristics than MO‐F1 females. RNA‐seq revealed 1365 genes significantly changed in male MO‐F1 liver and only 70 genes in female MO‐F1 compared with controls. GO and KEGG analysis identified differentially expressed genes related to metabolic processes. Male MO‐F1 liver showed the following altered pathways: insulin signalling (22 genes), phospholipase D signalling (14 genes), NAFLD (13 genes) and glycolysis/gluconeogenesis (7 genes). In contrast, few genes were altered in these pathways in MO‐F1 females. In summary, MO programs sex‐dependent F1 changes in insulin, glucose and lipid signalling pathways, leading to liver dysfunction and insulin resistance.

Key points The kinetics of NMDA receptor (NMDAR) signalling are a critical aspect of the physiology of excitatory synaptic transmission in the brain.Here we develop a mechanistic description of NMDAR function based on the receptor tetrameric structure and the principle that each agonist‐bound subunit must undergo some rate‐limiting conformational change after agonist binding, prior to channel opening.By fitting this mechanism to single channel data using a new MATLAB‐based software implementation of maximum likelihood fitting with correction for limited time resolution, rate constants were derived for this mechanism that reflect distinct structural changes and predict the properties of macroscopic and synaptic NMDAR currents.The principles applied here to develop a mechanistic description of the heterotetrameric NMDAR, and the software used in this analysis, can be equally applied to other heterotetrameric glutamate receptors, providing a unifying mechanistic framework to understanding the physiology of glutamate receptor signalling in the brain. Abstract NMDA receptors (NMDARs) are tetrameric complexes comprising two glycine‐binding GluN1 and two glutamate‐binding GluN2 subunits. Four GluN2 subunits encoded by different genes can produce up to 10 different di‐ and triheteromeric receptors. In addition, some neurological patients contain a de novo mutation or inherited rare variant in only one subunit. There is currently no mechanistic framework to describe tetrameric receptor function that can be extended to receptors with two different GluN1 or GluN2 subunits. Here we use the structural features of glutamate receptors to develop a mechanism describing both single channel and macroscopic NMDAR currents. We propose that each agonist‐bound subunit undergoes some rate‐limiting conformational change after agonist binding, prior to channel opening. We hypothesize that this conformational change occurs within a triad of interactions between a short helix preceding the M1 transmembrane helix, the highly conserved M3 motif encoded by the residues SYTANLAAF, and the linker preceding the M4 transmembrane helix of the adjacent subunit. Molecular dynamics simulations suggest that pre‐M1 helix motion is uncorrelated between subunits, which we interpret to suggest independent subunit‐specific conformational changes may influence these pre‐gating steps. According to this interpretation, these conformational changes are the main determinants of the key kinetic properties of NMDA receptor activation following agonist binding, and so these steps sculpt their physiological role. We show that this structurally derived tetrameric model describes both single channel and macroscopic data, giving a new approach to interpreting functional properties of synaptic NMDARs that provides a logical framework to understanding receptors with non‐identical subunits., Key points The kinetics of NMDA receptor (NMDAR) signalling are a critical aspect of the physiology of excitatory synaptic transmission in the brain.Here we develop a mechanistic description of NMDAR function based on the receptor tetrameric structure and the principle that each agonist‐bound subunit must undergo some rate‐limiting conformational change after agonist binding, prior to channel opening.By fitting this mechanism to single channel data using a new MATLAB‐based software implementation of maximum likelihood fitting with correction for limited time resolution, rate constants were derived for this mechanism that reflect distinct structural changes and predict the properties of macroscopic and synaptic NMDAR currents.The principles applied here to develop a mechanistic description of the heterotetrameric NMDAR, and the software used in this analysis, can be equally applied to other heterotetrameric glutamate receptors, providing a unifying mechanistic framework to understanding the physiology of glutamate receptor signalling in the brain.

KEY POINTS: The ClC‐3 2Cl(−)/1H(+) exchanger modulates endosome pH and Cl(−) concentration. We investigated the relationships between ClC‐3‐mediated ion transport (steady‐state transport current, I (SS)), gating charge (Q) and cytoplasmic alkalization. ClC‐3 transport is functionally unidirectional. ClC‐5 and ClC‐3 display indistinguishable exchange ratios, but ClC‐3 cycling is less “efficient”, as reflected by a large Q/I (SS). An M531A mutation predicted to increase water‐wire stability and cytoplasmic proton supply improves efficiency. Protonation (pH 5.0) of the outer glutamate gate (Glu(ext); E224) reduces Q, inhibits transport, and weakens coupling. Removal of the central tyrosine anion gate (Y572S) greatly increases uncoupled anion current. Tyrosine –OH removal (Y572F) alters anion selectivity and impairs coupling. E224 and Y572 act as anion barriers, and contribute to gating. The Y572 side chain and –OH regulate Q movement kinetics and voltage dependence. E224 and Y572 interact to create a “closed” inner gate conformation that maintains coupling during cycling. ABSTRACT: We utilized plasma membrane‐localized ClC‐3 to investigate relationships between steady‐state transport current (I (SS)), gating charge (Q) movement, and cytoplasmic alkalization rate. ClC‐3 exhibited lower transport efficiency than ClC‐5, as reflected by a larger Q/I (SS) ratio, but an indistinguishable Cl(−)/H(+) coupling ratio. External SCN(−) reduced H(+) transport rate and uncoupled anion/H(+) exchange by 80–90%. Removal of the external gating glutamate (“Glu(ext)”) (E224A mutation) reduced Q and abolished H(+) transport. We hypothesized that Methionine 531 (M531) impedes “water wire” H(+) transfer from the cytoplasm to E224. Accordingly, an M531A mutation decreased the Q/I (SS) ratio by 50% and enhanced H(+) transport. External protons (pH 5.0) inhibited I (SS) and markedly reduced Q while shifting the Q–voltage (V) relationship positively. The Cl(−)/H(+) coupling ratio at pH 5.0 was significantly increased, consistent with externally protonated Glu(ext) adopting an outward/open position. Internal “anion gate” removal (Y572S) dramatically increased I (SS) and impaired coupling, without slowing H(+) transport rate. Loss of both gates (Y572S/E224A) resulted in a large “open pore” conductance. Y572F (removing only the phenolic hydroxide) and Y572S shortened Q duration similarly, resulting in faster Q kinetics at all voltages. These data reveal a complex relationship between Q and ion transport. Q/I (SS) must be assessed together with coupling ratio to properly interpret efficiency. Coupling and transport rate are influenced by the anion, internal proton supply and external protons. Y572 regulates H(+) coupling as well as anion selectivity, and interacts directly with E224. Disruption of this “closed gate” conformation by internal protons may represent a critical step in the ClC‐3 transport cycle.

KEY POINTS: The mechanisms by which Rho kinase (ROCK) regulates airway smooth muscle contraction were determined in tracheal smooth muscle tissues. ROCK may mediate smooth muscle contraction by inhibiting myosin regulatory light chain (RLC) phosphatase. ROCK can also regulate F‐actin dynamics during cell migration, and actin polymerization is critical for airway smooth muscle contraction. Our results show that ROCK does not regulate airway smooth muscle contraction by inhibiting myosin RLC phosphatase or by stimulating myosin RLC phosphorylation. We find that ROCK regulates airway smooth muscle contraction by activating the serine–threonine kinase Pak, which mediates the activation of Cdc42 and neuronal Wiskott–Aldrich syndrome protein (N‐WASp). N‐WASP transmits signals from Cdc42 to the Arp2/3 complex for the nucleation of actin filaments. These results demonstrate a novel molecular function for ROCK in the regulation of Pak and Cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle. ABSTRACT: Rho kinase (ROCK), a RhoA GTPase effector, can regulate the contraction of airway and other smooth muscle tissues. In some tissues, ROCK can inhibit myosin regulatory light chain (RLC) phosphatase, which increases the phosphorylation of myosin RLC and promotes smooth muscle contraction. ROCK can also regulate cell motility and migration by affecting F‐actin dynamics. Actin polymerization is stimulated by contractile agonists in airway smooth muscle tissues and is required for contractile tension development in addition to myosin RLC phosphorylation. We investigated the mechanisms by which ROCK regulates the contractility of tracheal smooth muscle tissues by expressing a kinase‐inactive mutant of ROCK, ROCK‐K121G, in the tissues or by treating them with the ROCK inhibitor H‐1152P. Our results show no role for ROCK in the regulation of non‐muscle or smooth muscle myosin RLC phosphorylation during contractile stimulation in this tissue. We found that ROCK regulates airway smooth muscle contraction by mediating activation of p21‐activated kinase (Pak), a serine–threonine kinase, to promote actin polymerization. Pak catalyses paxillin phosphorylation on Ser273 and coupling of the GIT1–βPIX–Pak signalling module to paxillin, which activates the guanine nucleotide exchange factor (GEF) activity of βPIX towards Cdc42. Cdc42 is required for the activation of neuronal Wiskott–Aldrich syndrome protein (N‐WASp), which transmits signals from Cdc42 to the Arp2/3 complex for the nucleation of actin filaments. Our results demonstrate a novel molecular function for ROCK in the regulation of Pak and Cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle.

KEY POINTS: Ex vivo proliferated c‐Kit(+) endogenous cardiac progenitor cells (eCPCs) obtained from mouse and human cardiac tissues have been reported to express a wide range of functional ion channels. In contrast to previous reports in cultured c‐Kit(+) eCPCs, we found that ion currents were minimal in freshly isolated cells. However, inclusion of free Ca(2+) intracellularly revealed a prominent inwardly rectifying current identified as the intermediate conductance Ca(2+)‐activated K(+) current (KCa3.1). Electrical function of both c‐Kit(+) eCPCs and bone marrow‐derived mesenchymal stem cells is critically governed by KCa3.1 calcium‐dependent potassium channels. Ca(2+)‐induced increases in KCa3.1 conductance are necessary to optimize membrane potential during Ca(2+) entry. Membrane hyperpolarization due to KCa3.1 activation maintains the driving force for Ca(2+) entry that activates stem cell proliferation. Cardiac disease downregulates KCa3.1 channels in resident cardiac progenitor cells. Alterations in KCa3.1 may have pathophysiological and therapeutic significance in regenerative medicine. ABSTRACT: Endogenous c‐Kit(+) cardiac progenitor cells (eCPCs) and bone marrow (BM)‐derived mesenchymal stem cells (MSCs) are being developed for cardiac regenerative therapy, but a better understanding of their physiology is needed. Here, we addressed the unknown functional role of ion channels in freshly isolated eCPCs and expanded BM‐MSCs using patch‐clamp, microfluorometry and confocal microscopy. Isolated c‐Kit(+) eCPCs were purified from dog hearts by immunomagnetic selection. Ion currents were barely detectable in freshly isolated c‐Kit(+) eCPCs with buffering of intracellular calcium (Ca(2+) (i)). Under conditions allowing free intracellular Ca(2+), freshly isolated c‐Kit(+) eCPCs and ex vivo proliferated BM‐MSCs showed prominent voltage‐independent conductances that were sensitive to intermediate‐conductance K(+)‐channel (KCa3.1 current, I (KCa3.1)) blockers and corresponding gene (KCNN4)‐expression knockdown. Depletion of Ca(2+) (i) induced membrane‐potential (V (mem)) depolarization, while store‐operated Ca(2+) entry (SOCE) hyperpolarized V (mem) in both cell types. The hyperpolarizing SOCE effect was substantially reduced by I (KCa3.1) or SOCE blockade (TRAM‐34, 2‐APB), and I (KCa3.1) blockade (TRAM‐34) or KCNN4‐knockdown decreased the Ca(2+) entry resulting from SOCE. I (KCa3.1) suppression reduced c‐Kit(+) eCPC and BM‐MSC proliferation, while significantly altering the profile of cyclin expression. I (KCa3.1) was reduced in c‐Kit(+) eCPCs isolated from dogs with congestive heart failure (CHF), along with corresponding KCNN4 mRNA. Under perforated‐patch conditions to maintain physiological [Ca(2+)](i), c‐Kit(+) eCPCs from CHF dogs had less negative resting membrane potentials (−58 ± 7 mV) versus c‐Kit(+) eCPCs from control dogs (−73 ± 3 mV, P

Key points The post‐translational modification of target proteins by SUMOylation occurs in response to stressful stimuli in a variety of organ systems.Small ubiquitin‐like modifier (SUMO) isoforms 1–4 have recently been identified in the human placenta, and are upregulated in the major obstetrical complication of pre‐eclampsia.This is the first study to characterize the spatiotemporal distribution of SUMO isoforms and their targets during placental development across gestation and in response to stress induced by pre‐eclampsia and chorioamnionitis.Keratins were identified as major targets of placental SUMOylation. The interaction with SUMOs and cytoskeletal filaments provides evidence for SUMOylation possibly contributing to underlying dysfunctional trophoblast turnover, which is a hallmark feature of pre‐eclampsia.Further understanding the role of individual SUMO isoforms and SUMOylation underlying placental dysfunction may provide a target for a novel therapeutic candidate as an approach for treating pre‐eclampsia complicated with placental pathology. Abstract SUMOylation is a dynamic, reversible post‐translational modification that regulates cellular protein stability and localization. SUMOylation occurs in response to various stressors, including hypoxia and inflammation, features common in the obstetrical condition of pre‐eclampsia. SUMO isoforms 1–4 have recently been identified in the human placenta, but less is known about their role in response to pre‐eclamptic stress. We hypothesized that SUMOylation components have a unique spatiotemporal distribution during placental development and that their subcellular localization can be further modulated by extra‐cellular stressors. Placental SUMO expression was examined across gestation. First‐trimester human placental explants and JAR cells were subjected to hypoxia or TNF‐α cytokine, and subcellular translocation of SUMOs was monitored. SUMOylation target proteins were elucidated using mass spectrometry and proximity ligation assay. Placental SUMO‐1 and SUMO‐4 were restricted to villous cytotrophoblast cells in first trimester and syncytium by term, while SUMO‐2/3 staining was evenly distributed throughout the trophoblast across gestation. In placental villous explants, oxidative stress induced hyperSUMOylation of SUMO‐1 and SUMO‐4 in the syncytial cytoplasm, whereas SUMO‐2/3 nuclear expression increased. Oxidative stress also upregulated cytoplasmic SUMO‐1 and SUMO‐4 protein expression (P, Key points The post‐translational modification of target proteins by SUMOylation occurs in response to stressful stimuli in a variety of organ systems.Small ubiquitin‐like modifier (SUMO) isoforms 1–4 have recently been identified in the human placenta, and are upregulated in the major obstetrical complication of pre‐eclampsia.This is the first study to characterize the spatiotemporal distribution of SUMO isoforms and their targets during placental development across gestation and in response to stress induced by pre‐eclampsia and chorioamnionitis.Keratins were identified as major targets of placental SUMOylation. The interaction with SUMOs and cytoskeletal filaments provides evidence for SUMOylation possibly contributing to underlying dysfunctional trophoblast turnover, which is a hallmark feature of pre‐eclampsia.Further understanding the role of individual SUMO isoforms and SUMOylation underlying placental dysfunction may provide a target for a novel therapeutic candidate as an approach for treating pre‐eclampsia complicated with placental pathology.

KEY POINTS: Activation of the shoulder abductor muscles in the arm opposite a unilateral brain injury causes involuntary increases in elbow, wrist and finger flexion in the same arm, a phenomenon referred to as the flexion synergy. It has been proposed that flexion synergy expression is related to reduced output from ipsilesional motor cortex and corticospinal pathways. In this human subjects study, we provide evidence that the magnitude of flexion synergy expression is instead related to a progressive, task‐dependent recruitment of contralesional cortex. We also provide evidence that recruitment of contralesional cortex may induce excessive activation of ipsilateral reticulospinal descending motor pathways that cannot produce discrete movements, leading to flexion synergy expression. We interpret these findings as an adaptive strategy that preserves low‐level motor control at the cost of fine motor control. ABSTRACT: A hallmark of hemiparetic stroke is the loss of fine motor control in the contralesional arm and hand and the constraint to a grouped movement pattern known as the flexion synergy. In the flexion synergy, increasing shoulder abductor activation drives progressive, involuntary increases in elbow, wrist and finger flexion. The neural mechanisms underlying this phenomenon remain unclear. Here, across 25 adults with moderate to severe hemiparesis following chronic stroke and 18 adults without neurological injury, we test the overall hypothesis that two inter‐related mechanisms are necessary for flexion synergy expression: increased task‐dependent activation of the intact, contralesional cortex and recruitment of contralesional motor pathways via ipsilateral reticulospinal projections. First, we imaged brain activation in real time during reaching motions progressively constrained by flexion synergy expression. Using this approach, we found that cortical activity indeed shifts towards the contralesional hemisphere in direct proportion to the degree of shoulder abduction loading in the contralesional arm. We then leveraged the post‐stroke reemergence of a developmental brainstem reflex to show that anatomically diffuse reticulospinal motor pathways are active during synergy expression. We interpret this progressive recruitment of contralesional cortico‐reticulospinal pathways as an adaptive strategy that preserves low‐level motor control at the cost of fine motor control.

Key points Giant trypsin‐containing endocytic vacuoles are formed in pancreatic acinar cells stimulated with inducers of acute pancreatitis.F‐actin envelops endocytic vacuoles and regulates their properties.Endocytic vacuoles can rupture and release their content into the cytosol of acinar cells.Endocytic vacuoles can fuse with the plasma membrane of acinar cells and exocytose their content. Abstract Intrapancreatic activation of trypsinogen is an early event in and hallmark of the development of acute pancreatitis. Endocytic vacuoles, which form by disconnection and transport of large post‐exocytic structures, are the only resolvable sites of the trypsin activity in live pancreatic acinar cells. In the present study, we characterized the dynamics of endocytic vacuole formation induced by physiological and pathophysiological stimuli and visualized a prominent actin coat that completely or partially surrounded endocytic vacuoles. An inducer of acute pancreatitis taurolithocholic acid 3‐sulphate and supramaximal concentrations of cholecystokinin triggered the formation of giant (more than 2.5 μm in diameter) endocytic vacuoles. We discovered and characterized the intracellular rupture of endocytic vacuoles and the fusion of endocytic vacuoles with basal and apical regions of the plasma membrane. Experiments with specific protease inhibitors suggest that the rupture of endocytic vacuoles is probably not induced by trypsin or cathepsin B. Perivacuolar filamentous actin (observed on the surface of ∼30% of endocytic vacuoles) may play a stabilizing role by preventing rupture of the vacuoles and fusion of the vacuoles with the plasma membrane. The rupture and fusion of endocytic vacuoles allow trypsin to escape the confinement of a membrane‐limited organelle, gain access to intracellular and extracellular targets, and initiate autodigestion of the pancreas, comprising a crucial pathophysiological event., Key points Giant trypsin‐containing endocytic vacuoles are formed in pancreatic acinar cells stimulated with inducers of acute pancreatitis.F‐actin envelops endocytic vacuoles and regulates their properties.Endocytic vacuoles can rupture and release their content into the cytosol of acinar cells.Endocytic vacuoles can fuse with the plasma membrane of acinar cells and exocytose their content.

KEY POINTS: Na(+) conducting hypertonicity‐induced cation channels (HICCs) are key players in the volume restoration of osmotically shrunken cells and, under isotonic conditions, considered as mediators of proliferation – thereby opposing apoptosis. In an siRNA screen of ion channels and transporters in HepG2 cells, with the regulatory volume increase (RVI) as read‐out, δENaC, TRPM2 and TRPM5 were identified as HICCs. Subsequently, all permutations of these channels were tested in RVI and patch‐clamp recordings and, at first sight, HICCs were found to operate in an independent mode. However, there was synergy in the siRNA perturbations of HICC currents. Accordingly, proximity ligation assays showed that δENaC was located in proximity to TRPM2 and TRPM5 suggesting a physical interaction. Furthermore, δENaC, TRPM2 and TRPM5 were identified as mediators of HepG2 proliferation – their silencing enhanced apoptosis. Our study defines the architecture of HICCs in human hepatocytes as well as their molecular functions. ABSTRACT: Hypertonicity‐induced cation channels (HICCs) are a substantial element in the regulatory volume increase (RVI) of osmotically shrunken cells. Under isotonic conditions, they are key effectors in the volume gain preceding proliferation; HICC repression, in turn, significantly increases apoptosis rates. Despite these fundamental roles of HICCs in cell physiology, very little is known concerning the actual molecular architecture of these channels. Here, an siRNA screening of putative ion channels and transporters was performed, in HepG2 cells, with the velocity of RVI as the read‐out; in this first run, δENaC, TRPM2 and TRPM5 could be identified as HICCs. In the second run, all permutations of these channels were tested in RVI and patch‐clamp recordings, with special emphasis on the non‐additivity and additivity of siRNAs – which would indicate molecular interactions or independent ways of channel functioning. At first sight, the HICCs in HepG2 cells appeared to operate rather independently. However, a proximity ligation assay revealed that δENaC was located in proximity to both TRPM2 and TRPM5. Furthermore, a clear synergy of HICC current knock‐downs (KDs) was observed. δENaC, TRPM2 and TRPM5 were defined as mediators of HepG2 cell proliferation and their silencing increased the rates of apoptosis. This study provides a molecular characterization of the HICCs in human hepatocytes and of their role in RVI, cell proliferation and apoptosis.

Missense mutations in the gene encoding the α1 subunit of the skeletal muscle voltage‐gated Ca2+ channel induce type 1 hypokalaemic periodic paralysis, a poorly understood neuromuscular disease characterized by episodic attacks of paralysis associated with low serum K+.Acute expression of human wild‐type and R1239H HypoPP1 mutant α1 subunits in mature mouse muscles showed that R1239H fibres displayed Ca2+ currents of reduced amplitude and larger resting leak inward current increased by external acidification.External acidification also produced intracellular acidification at a higher rate in R1239H fibres and inhibited inward rectifier K+ currents.These data suggest that the R1239H mutation induces an elevated leak H+ current at rest flowing through a gating pore and could explain why paralytic attacks preferentially occur during the recovery period following muscle exercise.

Endothelial cell function in resistance arteries integrates Ca2+ signalling with hyperpolarization to promote relaxation of smooth muscle cells and increase tissue blood flow. Whether complementary signalling occurs in lymphatic endothelium is unknown.Intracellular calcium and membrane potential were evaluated in endothelial cell tubes freshly isolated from mouse collecting lymphatic vessels of the popliteal fossa. Resting membrane potential measured using intracellular microelectrodes averaged ∼−70 mV.Stimulation of lymphatic endothelium by acetylcholine or a TRPV4 channel agonist increased intracellular Ca2+ with robust depolarization. Findings from Trpv4 −/− mice and with computational modelling suggest that the initial mobilization of intracellular Ca2+ leads to influx of Ca2+ and Na+ through TRPV4 channels to evoke depolarization.Lymphatic endothelial cells lack the Ca2+‐activated K+ channels present in arterial endothelium to generate endothelium‐derived hyperpolarization. Absence of this signalling pathway with effective depolarization may promote rapid conduction of contraction along lymphatic muscle during lymph propulsion.

Key points The G-protein coupled inwardly rectifying potassium (GIRK) channel is an important mediator of neurotransmission via Gβγ subunit of the heterotrimeric Gi/o protein released by G-protein coupled receptor (GPCR) activation. Channels containing the GIRK1 subunit exhibit high basal currents, whereas channels that are formed by the GIRK2 subunit have very low basal currents. GIRK1-containing channels, but not channels consisting of GIRK2 only, recruit Gβγ to the plasma membrane. The Gα subunit of the G protein is not recruited by either GIRK1/2 or GIRK2. The unique distal C terminus of GIRK1 (G1-dCT) endows the channel with strong interaction with Gβγ, and deletion of G1-dCT abolishes the Gβγ recruitment and reduces the basal currents. These findings suggest that the basal activity of GIRK channels depends on channel-induced recruitment of Gβγ. The unique C terminus of GIRK1 subunit plays an important role in Gβγ recruitment. Abstract The G-protein coupled inwardly rectifying potassium (GIRK, or Kir3) channels are important mediators of inhibitory neurotransmission via activation of G-protein coupled receptors (GPCRs). GIRK channels are tetramers comprising combinations of subunits (GIRK1–4), activated by direct binding of the Gβγ subunit of Gi/o proteins. Heterologously expressed GIRK1/2 exhibit high, Gβγ-dependent basal currents (Ibasal) and a modest activation by GPCR or coexpressed Gβγ. Inversely, the GIRK2 homotetramers exhibit low Ibasal and strong activation by Gβγ. The high Ibasal of GIRK1 seems to be associated with its unique distal C terminus (G1-dCT), which is not present in the other subunits. We investigated the role of G1-dCT using electrophysiological and fluorescence assays in Xenopus laevis oocytes and protein interaction assays. We show that expression of GIRK1/2 increases the plasma membrane level of coexpressed Gβγ (a phenomenon we term ‘Gβγ recruitment’) but not of coexpressed Gαi3. All GIRK1-containing channels, but not GIRK2 homomers, recruited Gβγ to the plasma membrane. In biochemical assays, truncation of G1-dCT reduces the binding between the cytosolic parts of GIRK1 and Gβγ, but not Gαi3. Nevertheless, the truncation of G1-dCT does not impair activation by Gβγ. In fluorescently labelled homotetrameric GIRK1 channels and in the heterotetrameric GIRK1/2 channel, the truncation of G1-dCT abolishes Gβγ recruitment and decreases Ibasal. Thus, we conclude that G1-dCT carries an essential role in Gβγ recruitment by GIRK1 and, consequently, in determining its high basal activity. Our results indicate that G1-dCT is a crucial part of a Gβγ anchoring site of GIRK1-containing channels, spatially and functionally distinct from the site of channel activation by Gβγ.

The muscarinic acetylcholine receptor (mAChR)‐mediated increase in excitability in rat adrenal medullary cells is at least in part due to inhibition of TWIK (tandem of P domains in a weak inwardly rectifying K+ channel)‐related acid‐sensitive K+ (TASK)1 channels.In this study we focused on the molecular mechanism of mAChR‐mediated inhibition of TASK1 channels.Exposure to muscarine resulted in a clathrin‐dependent endocytosis of TASK1 channels following activation of the muscarinic M1 receptor (M1R).This muscarinic signal for the endocytosis was mediated in sequence by phospholipase C (PLC), protein kinase C (PKC), and then the non‐receptor tyrosine kinase Src with the consequent tyrosine phosphorylation of TASK1.The present results establish that TASK1 channels are tyrosine phosphorylated and internalized in a clathrin‐dependent manner in response to M1R stimulation and this translocation is at least in part responsible for muscarinic inhibition of TASK1 channels in rat AM cells.

Endothelial cells employ transient receptor potential isoform 4 (TRPV4) channels to sense ambient mechanical and chemical stimuli. In retinal microvascular endothelial cells, TRPV4 channels regulate calcium homeostasis, cytoskeletal signalling and the organization of adherens junctional contacts. Intracellular calcium increases induced by TRPV4 agonists include a significant contribution from calcium release from internal stores. Activation of TRPV4 channels regulates retinal endothelial barriers in vitro and in vivo. TRPV4 sensing may provide a feedback mechanism between sensing shear flow and eicosanoid modulators, vascular permeability and contractility at the inner retinal endothelial barrier.The identity of microvascular endothelial (MVE) mechanosensors that sense blood flow in response to mechanical and chemical stimuli and regulate vascular permeability in the retina is unknown. Using immunohistochemistry, calcium imaging, electrophysiology, impedance measurements and vascular permeability assays, we show that the transient receptor potential isoform 4 (TRPV4) plays a major role in Ca

Glomus cells in the carotid body (CB) and chromaffin cells in the adrenal medulla (AM) are essential for reflex cardiorespiratory adaptation to hypoxia. However, the mechanisms whereby these cells detect changes in O2 tension are poorly understood.The metabolic properties of acute O2‐sensing cells have been investigated by comparing the transcriptomes of CB and AM cells, which are O2‐sensitive, with superior cervical ganglion neurons, which are practically O2‐insensitive.In O2‐sensitive cells, we found a characteristic prolyl hydroxylase 3 down‐regulation and hypoxia inducible factor 2α up‐regulation, as well as overexpression of genes coding for three atypical mitochondrial electron transport subunits and pyruvate carboxylase, an enzyme that replenishes tricarboxylic acid cycle intermediates.In agreement with this observation, the inhibition of succinate dehydrogenase impairs CB acute O2 sensing. The responsiveness of peripheral chemoreceptor cells to acute hypoxia depends on a ‘signature metabolic profile’.

Dynamin 2 is a ubiquitously expressed protein involved in membrane trafficking processes.Mutations in the gene encoding dynamin 2 are responsible for a congenital myopathy associated with centrally located nuclei in the muscle fibres.Using muscle fibres from a mouse model of the most common mutation responsible for this disease in humans, we tested whether altered Ca2+ signalling and excitation–contraction coupling contribute to muscle weakness.The plasma membrane network that carries the electrical excitation is moderately perturbed in the diseased muscle fibres.The excitation‐activated Ca2+ input fluxes across both the plasma membrane and the membrane of the sarcoplasmic reticulum are defective in the diseased fibres, which probably contributes to muscle weakness in patients.

While several studies have investigated the effects of exercise training in human skeletal muscle and the chronic effect of β2‐agonist treatment in rodent muscle, their effects on muscle proteome signature with related functional measures in humans are still incompletely understood.Herein we show that daily β2‐agonist treatment attenuates training‐induced enhancements in exercise performance and maximal oxygen consumption, and alters muscle proteome signature and phenotype in trained young men.Daily β2‐agonist treatment abolished several of the training‐induced enhancements in muscle oxidative capacity and caused a repression of muscle metabolic pathways; furthermore, β2‐agonist treatment induced a slow‐to‐fast twitch muscle phenotype transition.The present study indicates that chronic β2‐agonist treatment confounds the positive effect of high intensity training on exercise performance and oxidative capacity, which is of interest for the large proportion of persons using inhaled β2‐agonists on a daily basis, including athletes.

Key points The proton sensing ovarian cancer G protein coupled receptor 1 (OGR1, aka GPR68) promotes expression of the canonical transient receptor potential channel subunit TRPC4 in normal and transformed cerebellar granule precursor (DAOY) cells.OGR1 and TRPC4 are prominently expressed in healthy cerebellar tissue throughout postnatal development and in primary cerebellar medulloblastoma tissues.Activation of TRPC4‐containing channels in DAOY cells, but not non‐transformed granule precursor cells, results in prominent increases in [Ca2+]i and promotes cell motility in wound healing and transwell migration assays.Medulloblastoma cells not arising from granule precursor cells show neither prominent rises in [Ca2+]i nor enhanced motility in response to TRPC4 activation unless they overexpressTRPC4.Our results suggest that OGR1 enhances expression of TRPC4‐containing channels that contribute to enhanced invasion and metastasis of granule precursor‐derived human medulloblastoma. Abstract Aberrant intracellular Ca2+ signalling contributes to the formation and progression of a range of distinct pathologies including cancers. Rises in intracellular Ca2+ concentration occur in response to Ca2+ influx through plasma membrane channels and Ca2+ release from intracellular Ca2+ stores, which can be mobilized in response to activation of cell surface receptors. Ovarian cancer G protein coupled receptor 1 (OGR1, aka GPR68) is a proton‐sensing Gq‐coupled receptor that is most highly expressed in cerebellum. Medulloblastoma (MB) is the most common paediatric brain tumour that arises from cerebellar precursor cells. We found that nine distinct human MB samples all expressed OGR1. In both normal granule cells and the transformed human cerebellar granule cell line DAOY, OGR1 promoted expression of the proton‐potentiated member of the canonical transient receptor potential (TRPC) channel family, TRPC4. Consistent with a role for TRPC4 in MB, we found that all MB samples also expressed TRPC4. In DAOY cells, activation of TRPC4‐containing channels resulted in large Ca2+ influx and enhanced migration, while in normal cerebellar granule (precursor) cells and MB cells not derived from granule precursors, only small levels of Ca2+ influx and no enhanced migration were observed. Our results suggest that OGR1‐dependent increases in TRPC4 expression may favour formation of highly Ca2+‐permeable TRPC4‐containing channels that promote transformed granule cell migration. Increased motility of cancer cells is a prerequisite for cancer invasion and metastasis, and our findings may point towards a key role for TRPC4 in progression of certain types of MB., Key points The proton sensing ovarian cancer G protein coupled receptor 1 (OGR1, aka GPR68) promotes expression of the canonical transient receptor potential channel subunit TRPC4 in normal and transformed cerebellar granule precursor (DAOY) cells.OGR1 and TRPC4 are prominently expressed in healthy cerebellar tissue throughout postnatal development and in primary cerebellar medulloblastoma tissues.Activation of TRPC4‐containing channels in DAOY cells, but not non‐transformed granule precursor cells, results in prominent increases in [Ca2+]i and promotes cell motility in wound healing and transwell migration assays.Medulloblastoma cells not arising from granule precursor cells show neither prominent rises in [Ca2+]i nor enhanced motility in response to TRPC4 activation unless they overexpressTRPC4.Our results suggest that OGR1 enhances expression of TRPC4‐containing channels that contribute to enhanced invasion and metastasis of granule precursor‐derived human medulloblastoma.

Paramyotonia congenita is a hereditary channelopathy caused by missense mutations in the SCN4A gene, which encodes the α subunit of the human skeletal muscle voltage-gated sodium channel NaV1.4. Affected individuals suffered from myotonia and paralysis of muscles, which were aggravated by exposure to cold. We report a three-generation Chinese family with patients presenting paramyotonia congenita and identify a novel N1366S mutation of NaV1.4. Whole-cell electrophysiological recordings of the N1366S channel reveal a gain-of-function change of gating in response to cold. Modelling and molecular dynamic simulation data suggest that an arginine-to-serine substitution at position 1366 increases the distance from N1366 to R1454 and disrupts the hydrogen bond formed between them at low temperature. We demonstrate that N1366S is a disease-causing mutation and that the temperature-sensitive alteration of N1366S channel activity may be responsible for the pronounced paramyotonia congenita symptoms of these patients.Paramyotonia congenita is an autosomal dominant skeletal muscle channelopathy caused by missense mutations in SCN4A, the gene encoding the α subunit of the human skeletal muscle voltage-gated sodium channel NaV1.4. We report a three-generation family in which six members present clinical symptoms of paramyotonia congenita characterized by a marked worsening of myotonia by cold and by the presence of clear episodes of paralysis. We identified a novel mutation in SCN4A (Asn1366Ser, N1366S) in all patients in the family but not in healthy relatives or in 500 normal control subjects. Functional analysis of the channel protein expressed in HEK293 cells by whole-cell patch clamp recording revealed that the N1366S mutation led to significant alterations in the gating process of the NaV1.4 channel. The N1366S mutant displayed a cold-induced hyperpolarizing shift in the voltage dependence of activation and a depolarizing shift in fast inactivation, as well as a reduced rate of fast inactivation and accelerated recovery from fast inactivation. In addition, homology modelling and molecular dynamic simulation of N1366S and wild-type NaV1.4 channels indicated that the arginine-to-serine substitution disrupted the hydrogen bond formed between N1366 and R1454. Together, our results suggest that N1366S is a gain-of-function mutation of NaV1.4 at low temperature and the mutation may be responsible for the clinical symptoms of paramyotonia congenita in the affected family and constitute a basis for studies into its pathogenesis.

Calcium homeostasis modulator 1 (CALHM1), a new voltage-gated ATP- and CaEmerging roles of CALHM1, a recently discovered voltage-gated ion channel, include purinergic neurotransmission of tastes in taste buds and memory formation in the brain, highlighting its physiological importance. However, the regulatory mechanisms of the CALHM1 channel remain entirely unexplored, hindering full understanding of its contribution in vivo. The different gating properties of CALHM1 in vivo and in vitro suggest undiscovered regulatory mechanisms. Here, in searching for post-translational regulatory mechanisms, we discovered the regulation of CALHM1 gating and association with lipid microdomains via protein S-palmitoylation, the only reversible lipid modification of proteins on cysteine residues. CALHM1 is palmitoylated at two intracellular cysteines located in the juxtamembrane regions of the third and fourth transmembrane domains. Enzymes that catalyse CALHM1 palmitoylation were identified by screening 23 members of the DHHC protein acyltransferase family. Epitope tagging of endogenous CALHM1 proteins in mice revealed that CALHM1 is basally palmitoylated in taste buds in vivo. Functionally, palmitoylation downregulates CALHM1 without effects on its synthesis, degradation and cell surface expression. Mutation of the palmitoylation sites has a profound impact on CALHM1 gating, shifting the conductance-voltage relationship to more negative voltages and accelerating the activation kinetics. The same mutation also reduces CALHM1 association with detergent-resistant membranes. Our results comprehensively uncover a post-translational regulation of the voltage-dependent gating of CALHM1 by palmitoylation.

Smooth muscle myosin regulatory light chain (RLC) is phosphorylated by Ca2+/calmodulin‐dependent myosin light chain kinase and dephosphorylated by myosin light chain phosphatase (MLCP).Tracheal smooth muscle contains significant amounts of myosin binding subunit 85 (MBS85), another myosin phosphatase targeting subunit (MYPT) family member, in addition to MLCP regulatory subunit MYPT1.Concentration/temporal responses to carbachol demonstrated similar sensitivities for bovine tracheal force development and phosphorylation of RLC, MYPT1, MBS85 and paxillin.Electrical field stimulation releases ACh from nerves to increase RLC phosphorylation but not MYPT1 or MBS85 phosphorylation. Thus, nerve‐mediated muscarinic responses in signalling modules acting on RLC phosphorylation are different from pharmacological responses with bath added agonist.The conditional knockout of MYPT1 or the knock‐in mutation T853A in mice had no effect on muscarinic force responses in isolated tracheal tissues. MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction.

Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. The extent of cross-talk between fibroblasts, as the source of matrix protein, and satellite cells in humans is unknown. We studied this in human muscle biopsies and cell-culture studies. We observed a strong stimulation of myogenesis by human fibroblasts in cell culture. In biopsies collected 30 days after a muscle injury protocol, fibroblast number increased to four times control levels, where fibroblasts were found to be preferentially located immediately surrounding regenerating muscle fibres. These novel findings indicate an important role for fibroblasts in supporting the regeneration of muscle fibres, potentially through direct stimulation of satellite cell differentiation and fusion, and contribute to understanding of cell-cell cross-talk during physiological and pathological muscle remodelling.Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. In addition to the indispensable role satellite cells play in muscle regeneration, there is emerging evidence in rodents for a regulatory influence on fibroblast activity. However, the influence of fibroblasts on satellite cells and muscle regeneration in humans is unknown. The purpose of this study was to investigate this in vitro and during in vivo regeneration in humans. Following a muscle injury protocol in young healthy men (n = 7), the number of fibroblasts (TCF7L2+), satellite cells (Pax7+), differentiating myogenic cells (myogenin+) and regenerating fibres (neonatal/embryonic myosin+) was determined from biopsy cross-sections. Fibroblasts and myogenic precursor cells (MPCs) were also isolated from human skeletal muscle (n = 4) and co-cultured using different cell ratios, with the two cell populations either in direct contact with each other or separated by a permeable membrane. MPC proliferation, differentiation and fusion were assessed from cells stained for BrdU, desmin and myogenin. On biopsy cross-sections, fibroblast number was seen to increase, along with myogenic cell number, by d7 and increase further by d30, where fibroblasts were observed to be preferentially located immediately surrounding regenerating muscle fibres. In vitro, the presence of fibroblasts in direct contact with MPCs was found to moderately stimulate MPC proliferation and strongly stimulate both MPC differentiation and MPC fusion. It thus appears, in humans, that fibroblasts exert a strong positive regulatory influence on MPC activity, in line with observations during in vivo skeletal muscle regeneration.

β‐Adrenergic stimulation enhances Ca2+ entry via L‐type CaV1.2 channels, causing stronger contraction of cardiac muscle cells.The signalling pathway involves activation of protein kinase A (PKA), but the molecular details of PKA regulation of CaV1.2 remain controversial despite extensive research.We show that PKA regulation of CaV1.2 can be reconstituted in Xenopus oocytes when the distal C‐terminus (dCT) of the main subunit, α1C, is truncated.The PKA upregulation of CaV1.2 does not require key factors previously implicated in this mechanism: the clipped dCT, the A kinase‐anchoring protein 15 (AKAP15), the phosphorylation sites S1700, T1704 and S1928, or the β subunit of CaV1.2. The gating element within the initial segment of the N‐terminus of the cardiac isoform of α1C is essential for the PKA effect.We propose that the regulation described here is one of two or several mechanisms that jointly mediate the PKA regulation of CaV1.2 in the heart.

We found that extracellular CaTransient receptor potential ankyrin 1 (TRPA1) is a homotetrameric non-selective cation-permeable channel that has six transmembrane domains and cytoplasmic N- and C-termini. The N-terminus is characterized by an unusually large number of ankyrin repeats. Although the 3-dimensional structure of human TRPA1 has been determined, and TRPA1 channels from insects to birds are known to be activated by heat stimulus, the mechanism for temperature-dependent TRPA1 activation is unclear. We previously reported that extracellular Ca

Chronic alcohol consumption causes a spectrum of liver diseases, but the pathogenic mechanisms driving the onset and progression of disease are not clearly defined.We show that chronic alcohol feeding sensitizes rat hepatocytes to Ca2+‐mobilizing hormones resulting in a leftward shift in the concentration–response relationship and the transition from oscillatory to more sustained and prolonged Ca2+ increases.Our data demonstrate that alcohol‐dependent adaptation in the Ca2+ signalling pathway occurs at the level of hormone‐induced inositol 1,4,5 trisphosphate (IP3) production and does not involve changes in the sensitivity of the IP3 receptor or size of internal Ca2+ stores.We suggest that prolonged and aberrant hormone‐evoked Ca2+ increases may stimulate the production of mitochondrial reactive oxygen species and contribute to alcohol‐induced hepatocyte injury.

Maternal high-fat diet impairs brown adipocyte function and correlates with obesity in offspring. Maternal resveratrol administration recovers metabolic activity of offspring brown adipose tissue. Maternal resveratrol promotes beige adipocyte development in offspring white adipose tissue. Maternal resveratrol intervention protects offspring against high-fat diet-induced obesity.Promoting beige/brite adipogenesis and thermogenic activity is considered as a promising therapeutic approach to reduce obesity and metabolic syndrome. Maternal obesity impairs offspring brown adipocyte function and correlates with obesity in offspring. We previously found that dietary resveratrol (RES) induces beige adipocyte formation in adult mice. Here, we evaluated further the effect of resveratrol supplementation of pregnant mice on offspring thermogenesis and energy expenditure. Female C57BL/6 J mice were fed a control diet (CON) or a high-fat diet (HFD) with or without 0.2% (w/w) RES during pregnancy and lactation. Male offspring were weaned onto a HFD and maintained on this diet for 11 weeks. The offspring thermogenesis and related regulatory factors in adipose tissue were evaluated. At weaning, HFD offspring had lower thermogenesis in brown and white adipose tissues compared with CON offspring, which was recovered by maternal RES supplementation, along with the appearance of multilocular brown/beige adipocytes and elevated thermogenic gene expression. Adult offspring of RES-treated mothers showed increased energy expenditure and insulin sensitivity when on an obesogenic diet compared with HFD offspring. The elevated metabolic activity was correlated with enhanced brown adipose function and white adipose tissue browning in HFD+RES compared with HFD offspring. In conclusion, RES supplementation of HFD-fed dams during pregnancy and lactation promoted white adipose browning and thermogenesis in offspring at weaning accompanied by persistent beneficial effects in protecting against HFD-induced obesity and metabolic disorders.

Outward and inward background currents across the cell membrane balance, determining resting membrane potential. Inward rectifier K+ channel subfamily 2 (Kir2) channels primarily maintain the resting membrane potential of cardiomyocytes.Human cardiomyocytes exhibit two levels of resting membrane potential at subphysiological extracellular K+ concentrations or pathological hypokalaemia, however, the underlying mechanism is unclear.In the present study, we show that human cardiomyocytes derived from induced pluripotent stem cells with enhanced expression of isoform 1 of Kir2 (Kir2.1) channels and mouse HL‐1 cardiomyocytes with ectopic expression of two pore‐domain K+ channel isoform 1 (K2P1) recapitulate two levels of resting membrane potential, indicating the contributions of Kir2.1 and K2P1 channels to the phenomenon.In Chinese hamster ovary cells that express the channels, Kir2.1 currents non‐linearly counterbalance hypokalaemia‐induced K2P1 leak cation currents, reconstituting two levels of resting membrane potential.These findings support the hypothesis that Kir2 currents non‐linearly counterbalance inward background cation currents, such as K2P1 currents, accounting for two levels of resting membrane potential in human cardiomyocytes and demonstrating a novel mechanism that regulates excitability.

Key points: In human sperm, proton flux across the membrane is controlled by the voltage-gated proton channel Hv1. We show that sperm harbour both Hv1 and an N-terminally cleaved isoform termed Hv1Sper. The pH-control of Hv1Sper and Hv1 is distinctively different. Hv1Sper and Hv1 can form heterodimers that combine features of both constituents. Cleavage and heterodimerization of Hv1 might represent an adaptation to the specific requirements of pH control in sperm. Abstract: In human sperm, the voltage-gated proton channel Hv1 controls the flux of protons across the flagellar membrane. Here, we show that sperm harbour Hv1 and a shorter isoform, termed Hv1Sper. Hv1Sper is generated from Hv1 by removal of 68 amino acids from the N-terminus by post-translational proteolytic cleavage. The pH-dependent gating of the channel isoforms is distinctly different. In both Hv1 and Hv1Sper, the conductance-voltage relationship is determined by the pH difference across the membrane (∆pH). However, simultaneous changes in intracellular and extracellular pH that leave ΔpH constant strongly shift the activation curve of Hv1Sper but not that of Hv1, demonstrating that cleavage of the N-terminus tunes pH sensing in Hv1. Moreover, we show that Hv1 and Hv1Sper assemble as heterodimers that combine features of both constituents. We suggest that cleavage and heterodimerization of Hv1 represents an adaptation to the specific requirements of pH control in sperm.