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Aims Mutation type, location, dominant-negative I Ks reduction, and possibly loss of cyclic adenosine monophosphate (cAMP)-dependent I Ks stimulation via protein kinase A (PKA) influence the clinical severity of long QT syndrome type 1 (LQT1). Given the malignancy of KCNQ1-p.A341V, we assessed whether mutations neighbouring p.A341V in the S6 channel segment could also increase arrhythmic risk. Methods and results Clinical and genetic data were obtained from 1316 LQT1 patients [450 families, 166 unique KCNQ1 mutations, including 277 p.A341V-positive subjects, 139 patients with p.A341-neighbouring mutations (91 missense, 48 non-missense), and 900 other LQT1 subjects]. A first cardiac event represented the primary endpoint. S6 segment missense variant characteristics, particularly cAMP stimulation responses, were analysed by cellular electrophysiology. p.A341-neighbouring mutation carriers had a QTc shorter than p.A341V carriers (477 ± 33 vs. 490 ± 44 ms) but longer than the remaining LQT1 patient population (467 ± 41 ms) (P Conclusion KCNQ1 S6 segment mutations surrounding p.A341 increase arrhythmic risk. p.A341V-specific loss of PKA-dependent I Ks enhancement correlates with its phenotypic severity. Cellular studies providing further insights into I Ks-channel regulation and knowledge of structure-function relationships could improve risk stratification. These findings impact on clinical management.

Facilitation of cardiac function in response to signals from the sympathetic nervous system is initiated by the phosphorylation of L-type voltage-dependent Ca2+ channels (VDCCs) by protein kinase A (PKA), which in turn is activated by β-adrenoceptors. Among the five subunits (α1, β, α2/δ, and γ) of VDCCs, the α1 subunit and the family of β subunits are substrates for PKA-catalyzed phosphorylation; however, the subunit responsible for β-adrenergic augmentation of Ca2+ channel function has yet to be specifically identified. Here we show that the VDCC β2 subunit is required for PKA phosphorylation upon sympathetic acceleration. In mice with β2 subunit-null mutations, cardiac muscle contraction in response to isoproterenol was reduced and there was no significant increase in Ca2+ channel currents upon PKA-dependent phosphorylation. These findings indicate that within the sympathetic nervous system the β2 subunit of VDCCs is required for physiological PKA-dependent channel phosphorylation.

Parkinson's disease (PD) is a socially significant disease, during the development of which oxidative stress and inflammation play a significant role. Here, we studied the neuroprotective effects of four Kunitz-type peptides from Heteractis crispa and Heteractis magnifica sea anemones against PD inductors. The peptide HCIQ1c9, which was obtained for the first time, inhibited trypsin less than other peptides due to unfavorable interactions of Arg17 with Lys43 in the enzyme. Its activity was reduced by up to 70% over the temperature range of 60-100 °C, while HCIQ2c1, HCIQ4c7, and HMIQ3c1 retained their conformation and stayed active up to 90-100 °C. All studied peptides inhibited paraquat- and rotenone-induced intracellular ROS formation, in particular NO, and scavenged free radicals outside the cells. The peptides did not modulate the TRPV1 channels but they affected the P2X7R, both of which are considered therapeutic targets in Parkinson's disease. HMIQ3c1 and HCIQ4c7 almost completely inhibited the ATP-induced uptake of YO-PRO-1 dye in Neuro-2a cells through P2X7 ion channels and significantly reduced the stable calcium response in these cells. The complex formation of the peptides with the P2X7R extracellular domain was determined via SPR analysis. Thus, these peptides may be considered promising compounds to protect neuronal cells against PD inductors, which act as ROS production inhibitors and partially act as ATP-induced P2X7R activation inhibitors. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:23 issue:9 ispartof: location:Switzerland status: published

Arabidopsis thaliana contains five tandem-pore domain potassium channels, TPK1-TPK5 and the related one-pore domain potassium channel, KCO3. Although KCO3 is unlikely to be an active channel, it still has a physiological role in plant cells. TPK2 is most similar to KCO3 and both are localized to the tonoplast. However, their function remains poorly understood. Here, taking advantage of the similarities between TPK2 and KCO3, we evaluated Ca2+ binding to the EF hands in TPK2, and the elements of KCO3 required for K+ channel activity. Presence of both EF-hand motifs in TPK2 resulted in Ca2+ binding, but EF1 or EF2 alone failed to interact with Ca2+. The EF hands were not required for K+ transport activity. EF1 contains two cysteines separated by two amino acids. Replacement of both cysteines with serines in TPK2 increased Ca2+ binding. We generated a two-pore domain chimeric K+ channel by replacing the missing pore region in KCO3 with a pore domain of TPK2. Alternatively, we generated two versions of simple one-pore domain K+ channels by removal of an extra region from KCO3. The chimera and one of the simple one-pore variants were functional channels. This strongly suggests that KCO3 is not a pseudogene and KCO3 retains components required for the formation of a functional K+ channel and oligomerization. Our results contribute to our understanding of the structural properties required for K+ channel activity.

High-resolution structural information on membrane proteins is essential for understanding cell biology and for the structure-based design of new medical drugs and drug delivery strategies. X-ray diffraction (XRD) can provide angstrom-level information about the structure of membrane proteins, yet for XRD experiments, proteins are removed from their native membrane environment, chemically stabilized, and crystallized, all of which can compromise the conformation. Here, we describe how a combination of surface-sensitive vibrational spectroscopy and molecular dynamics simulations can account for the native membrane environment. We observe the structure of a glycerol facilitator channel (GlpF), an aquaporin membrane channel finely tuned to selectively transport water and glycerol molecules across the membrane barrier. We find subtle but significant differences between the XRD structure and the inferred in situ structure of GlpF.

Cystic fibrosis (CF) is a recessive genetic disease caused by mutations in a gene encoding a protein called Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). The CFTR protein is known to acts as a chloride (Cl− ) channel expressed in the exocrine glands of several body systems where it also regulates other ion channels, including the epithelial sodium (Na+ ) channel (ENaC) that plays a key role in salt absorption. This function is crucial to the osmotic balance of the mucus and its viscosity. However, the pathophysiology of CF is more challenging than a mere dysregulation of epithelial ion transport, mainly resulting in impaired mucociliary clearance (MCC) with consecutive bronchiectasis and in exocrine pancreatic insufficiency. This review shows that the CFTR protein is not just a chloride channel. For a long time, research in CF has focused on abnormal Cl− and Na+ transport. Yet, the CFTR protein also regulates numerous other pathways, such as the transport of HCO3−, glutathione and thiocyanate, immune cells, and the metabolism of lipids. It influences the pH homeostasis of airway surface liquid and thus the MCC as well as innate immunity leading to chronic infection and inflammation, all of which are considered as key pathophysiological characteristics of CF., SCOPUS: re.j, info:eu-repo/semantics/published

Asymmetrical flow field-flow (AF4) fractionation aims in separation of sample components to yield elution of homogenous fractions identified as well-defined peaks in the chromatograms. Separation that occurs in matrixfree open channel potentiates high recovery that can be close to 100%. However, sample properties and separation conditions may induce carryover of sample components during AF4 analysis and in sample sequences. This compromises the quality of the data collected from the online detectors and the downstream offline analytics of the collected fractions. In this study, we followed sample carryover in AF4 using model viruses and analyzed various cleaning solutions and rinse methods to reduce carryover. We introduce an SDS-NaOH -based rinsing and decontamination protocol for the AF4 instrument enabling high-quality data collection.

The expression of the channels-enzymes TRPM6 and TRPM7 in the human heart remains poorly defined, and TRPM6 is generally considered not to be expressed in cardiomyocytes. We examined their expression at protein and mRNA levels using right atrial samples resected from patients (n = 72) with or without ischemic heart disease (IHD) and samples from all chamber walls of explanted human hearts (n = 9). TRPM6 and TRPM7 proteins were detected using immunofluorescence on isolated cardiomyocytes, ELISA on tissue homogenates, and immunostaining of cardiac tissue, whereas their mRNAs were detected by RT-qPCR. Both TRPM6 and TRPM7 were present in all chamber walls, with TRPM7 being more abundant. TRPM6 was co-expressed with TRPM7. The expression levels were dependent on cell incubation conditions (presence or absence of divalent cations, pH of the extracellular milieu, presence of TRP channel inhibitors 2-aminoethoxydiphenyl-borate and carvacrol). These drugs reduced TRPM7 immunofluorescence but increased that of TRPM6. TRPM6 and TRPM7 expression was increased in tissues from IHD patients. This is the first demonstration of the presence and co-expression of TRPM6 and TRPM7 in cardiomyocytes from all chamber walls of the human heart. The increased TRPM6 and TRPM7 expression in IHD suggests that the chanzymes are involved in the pathophysiology of the disease. ispartof: SCIENTIFIC REPORTS vol:11 issue:1 ispartof: location:England status: published

In the current numerical study, various wall shape effects are investigated on the thermal-hydraulic characteristics of different channels filled with water-based graphite-SiO2 hybrid nanofluid. In this work, the performance evaluation criteria (PEC) index is employed as the target parameter to attain optimum geometry. Six different cases are studied in this research, and each case has different geometrical dimensions. The inlet temperature for the fluids in the channel is 300 K, over a range of different flow velocities. According to the obtained results, an increase in the volume fraction of nanoparticles results in higher PEC values. In addition, an increase in Reynolds number to Re = leads to an increase in the PEC index. The results clearly show that increasing the Reynolds number has two consequences: on the one hand, it increases the pressure drop penalty, on the other hand, it improves heat transfer. Therefore, the maximum value of the PEC index occurs at Re = 15,000.

Kv1.3 is a voltage gated potassium channel located in the plasma membrane, as well as at intracellular levels, such as mitochondria (mitoKv1.3), nucleus and Golgi apparatus. The plasma membrane channel has been shown to be important for cell proliferation, while the mitochondrial counterpart has been related to modulation of cell death. Moreover, altered expression of Kv1.3 was observed in various tumors and Kv1.3 seems to be involved in development and progression of various cancerous forms. Recent experimental evidences have proved that pharmacological inhibition of the mitoKv1.3 succeeded in reducing up to 90% of tumor volume in vivo in orthotopic mouse model. Furthermore, mitoKv1.3 modulation could impact on cell proliferation as well as on regulation of intracellular signaling pathways. Indeed, the treatment with sub-lethal doses of mitoKv1.3 inhibitors can downregulate Wnt-β catenin signaling by reducing mitochondrial ATP production and triggering ER-stress. In this review, we describe the role of the mitoKv1.3 in cell death, cancer and intracellular signaling. We will discuss how pharmacological modulation of mitochondrial potassium fluxes impact on mitochondrial membrane potential, reactive oxygen species production and ATP synthesis. All these changes in mitochondrial fitness are related to cell proliferation as well as to cell death and finally on cancer development and progression, so Kv1.3 (and mitoKv1.3) could be now considered a new oncological target.

Whether the mitochondrial permeability transition pore (PTP), also called mitochondrial megachannel (MMC), originates from the F-ATP synthase is a matter of controversy. This hypothesis is supported both by site-directed mutagenesis of specific residues of F-ATP synthase affecting regulation of the PTP/MMC and by deletion of specific subunits causing dramatic changes in channel conductance. In contrast, human cells lacking an assembled F-ATP synthase apparently display persistence of the PTP. We discuss recent data that shed new light on this controversy, supporting the conclusion that the PTP/MMC originates from a Ca2+ -dependent conformational change in F-ATP synthase allowing its reversible transformation into a high-conductance channel.

Background: Glycogen synthase kinase-3β (GSK3β) is a key regulator of cellular homeostasis. In neurons, GSK3β contributes to the control of neuronal transmission and plasticity, but its role in epilepsy remains to be defined. Methods: Biochemical and electrophysiological methods were used to assess the role of GSK3β in regulating neuronal transmission and epileptogenesis. GSK3β activity was increased genetically in GSK3β[S9A] mice. Its effects on neuronal transmission and epileptogenesis induced by kainic acid were assessed by field potential recordings in mice brain slices and video electroencephalography in vivo. The ion channel expression was measured in brain samples from mice and followed by analysis in samples from patients with temporal lobe epilepsy or focal cortical dysplasia in correlation to GSK3β phosphorylation. Findings: Higher GSK3β activity decreased the susceptibility to kainic acid-induced epileptiform discharges and the progression of epileptogenesis. At the biochemical level, higher GSK3β activity increased the expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel 4 and decreased phosphorylation of the glutamate α±-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA1. The phosphorylation of GluA1 at Serine 831 and Serine 845 was lower in GSK3β[S9A] mice than in wildtype controls, under basal conditions and in the epileptic mouse brain, respectively. Moreover, we found a significant correlation between higher inhibitory GSK3β phosphorylation at Serine 9 and higher activating GluA1 phosphorylation at Serine 845 in brain samples from epileptic patients. Interpretation: Our data imply GSK3β activity in the protection of neuronal networks from hyper-activation and indicate that the anti-epileptogenic function of GSK3I² involves decreasing the synaptic AMPA receptors pool. Funding: Research was supported by the Polish National Science Centre (no. 2011/01/N/NZ3/05409 to MU; no. 2017/26/D/NZ4/00159 to PK-G) and FP7 European Union grants (no. 223276, “NeuroGSK3” to FvL and JJ; no. 229676, “HealthProt” to JJ; no. 602102, “Epitarget” to EV and EA.). This study was also co-financed by European Union funds from the European Social Fund (MU). The work of MU was partially financed by IUVENTUS IP2012 037872. The work of MU, KKotulska, SJ, EA and JJ was partly supported by 7FP grant “EPISTOP” (grant agreement no. 602391), and the Polish Ministerial funds for science (years 2014-2019) for the implementation of international co-financed projects. The work of AG was financed by the Polish National Science Centre (grant no. 2011/01/B/NZ3/05397). KKotulska, SJ and JJ were partly financed by the Polish National Center for Research and Development (grant EPIMARKER, no. STRATEGMED3/306306/4/2017). MU was a recipient of a L’OrealUNESCO for Women and Science Fellowship in Poland and EMBO Short Term Fellowship. JJ and MU were also recipients of a Foundation for Polish Science "Mistrz" Professorial Subsidy and Fellowship, respectively. Declaration of Interest: SJ received Advisory Board honoraria from Novartis Oncology. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethical Approval: The study was approved by the local Ethics Board in Warsaw (approval no. 223/KBE/2015).

Additive manufacturing (3D printing) is a disruptive technology that is changing production systems globally. In addition, microfluidic devices are increasingly being used for chemical analysis and continuous production of chemicals. Printing of materials such as polymers and metals is already a reality, but additive manufacturing of glass for microfluidic systems has received minor attention. We characterize microfluidic devices (channel cross-section dimensions down to a scale of 100 mm) that have been produced by additive manufacturing of molten soda-lime glass in tens of minutes and report their mass spectrometric and Raman spectroscopic analysis examples. The functionality of a microfluidic glass microreactor is shown with online mass spectrometric analysis of linezolid synthesis. Additionally, the performance of a direct infusion device is demonstrated by mass spectrometric analysis of drugs. Finally, the excellent optical quality of the glass structures is demonstrated with in-line Raman spectroscopic measurements. Our results promise a bright future for additively manufactured glass microdevices in diverse fields of science.

Recently, we found that the deletion of TRPC5 leads to increased inflammation and pain-related behaviour in two animal models of arthritis. (-)-Englerin A (EA), an extract from the East African plant Phyllanthus engleri has been identified as a TRPC4/5 agonist. Here, we studied whether or not EA has any anti-inflammatory and analgesic properties via TRPC4/5 in the carrageenan model of inflammation. We found that EA treatment in CD1 mice inhibited thermal hyperalgesia and mechanical allodynia in a dose-dependent manner. Furthermore, EA significantly reduced the volume of carrageenan-induced paw oedema and the mass of the treated paws. Additionally, in dorsal root ganglion (DRG) neurons cultured from WT 129S1/SvIm mice, EA induced a dose-dependent cobalt uptake that was surprisingly preserved in cultured DRG neurons from 129S1/SvIm TRPC5 KO mice. Likewise, EA-induced anti-inflammatory and analgesic effects were preserved in the carrageenan model in animals lacking TRPC5 expression or in mice treated with TRPC4/5 antagonist ML204.This study demonstrates that while EA activates a sub-population of DRG neurons, it induces a novel TRPC4/5-independent analgesic and anti-inflammatory effect in vivo. Future studies are needed to elucidate the molecular and cellular mechanisms underlying EA’s anti-inflammatory and analgesic effects.

Significance The unique aspects of brain mitochondria composition and function are incompletely understood, and new approaches are required to understand their relationship. We addressed this challenge through a dual-process isolation procedure that yields both semipure brain mitochondria suitable for functional studies but not protein localization and ultrapure brain mitochondria that are suitable for determining protein localization but functionally compromised. We observed that contrary to the published literature, N-methyl-D-aspartate receptor, ryanodine receptor 1, glyceraldehyde 3-phosphate dehydrogenase, monocarboxylate transporter 1, excitatory amino acid transporter 1, and the L-type calcium channel Cav1.2α1 subunit are absent from brain mitochondria. Utilization of this dual-process brain mitochondria isolation technique will foster clarity on mechanisms of brain health and disease., The brain requires continuously high energy production to maintain ion gradients and normal function. Mitochondria critically undergird brain energetics, and mitochondrial abnormalities feature prominently in neuropsychiatric disease. However, many unique aspects of brain mitochondria composition and function are poorly understood. Developing improved neuroprotective therapeutics thus requires more comprehensively understanding brain mitochondria, including accurately delineating protein composition and channel–transporter functional networks. However, obtaining pure mitochondria from the brain is especially challenging due to its distinctive lipid and cell structure properties. As a result, conflicting reports on protein localization to brain mitochondria abound. Here we illustrate this problem with the neuropsychiatric disease-associated L-type calcium channel Cav1.2α1 subunit previously observed in crude mitochondria. We applied a dual-process approach to obtain functionally intact versus compositionally pure brain mitochondria. One branch utilizes discontinuous density gradient centrifugation to isolate semipure mitochondria suitable for functional assays but unsuitable for protein localization because of endoplasmic reticulum (ER) contamination. The other branch utilizes self-forming density gradient ultracentrifugation to remove ER and yield ultrapure mitochondria that are suitable for investigating protein localization but functionally compromised. Through this process, we evaluated brain mitochondria protein content and observed the absence of Cav1.2α1 and other previously reported mitochondrial proteins, including the NMDA receptor, ryanodine receptor 1, monocarboxylate transporter 1, excitatory amino acid transporter 1, and glyceraldehyde 3-phosphate dehydrogenase. Conversely, we confirmed mitochondrial localization of several plasma membrane proteins previously reported to also localize to mitochondria. We expect this dual-process isolation procedure will enhance understanding of brain mitochondria in both health and disease.

Inspired by the natural phospholipid structures for cell membrane, a synthetic phospholipid LC with an ion recognition group benzo-18-crown-6 (B18C6) moiety was prepared which has been demonstrated to be able to transport ions across the lipid bilayers. Fluorescent vesicle assay shows that LC has an excellent transport activity, and the EC50 value for K+ is 11.2 μM. The voltage clamp measurement exhibits regular square-like current signals with considerably long opening times, which indicates that LC achieves efficient ion transport through a channel mechanism and its single channel conductivity is 17 pS. Both of the vesicle assay and patch clamp tests indicate that LC has selectivity for Rb+, whose ionic radius is larger than the cavity of crown ether. It suggests that the sandwich interaction may play a key role in the ion transport across lipid bilayers. All these results help us to speculate that LC transports ions via a channel mechanism with a tetrameric aggregate as the active structure. In addition, LC had obvious toxicity to HeLa cells, and the IC50 was 100.0 μM after coculture for 36 h. We hope that this simple synthetic phospholipid will offer novel perspectives in the development of more efficient and selective ion transporters.

Summary: Loss-of-function mutations in proline-rich transmembrane protein-2 (PRRT2) cause paroxysmal disorders associated with defective Ca2+ dependence of glutamatergic transmission. We find that either acute or constitutive PRRT2 deletion induces a significant decrease in the amplitude of evoked excitatory postsynaptic currents (eEPSCs) that is insensitive to extracellular Ca2+ and associated with a reduced contribution of P/Q-type Ca2+ channels to the EPSC amplitude. This synaptic phenotype parallels a decrease in somatic P/Q-type Ca2+ currents due to a decreased membrane targeting of the channel with unchanged total expression levels. Co-immunoprecipitation, pull-down assays, and proteomics reveal a specific and direct interaction of PRRT2 with P/Q-type Ca2+ channels. At presynaptic terminals lacking PRRT2, P/Q-type Ca2+ channels reduce their clustering at the active zone, with a corresponding decrease in the P/Q-dependent presynaptic Ca2+ signal. The data highlight the central role of PRRT2 in ensuring the physiological Ca2+ sensitivity of the release machinery at glutamatergic synapses.

ispartof: MOLECULAR CATALYSIS vol:522 status: published

Marked age- and development- related differences have been observed in morphology and characteristics of action potentials (AP) of neonatal and adult sinoatrial node (SAN) cells. These may be attributable to a different set of ion channel interactions between the different ages. However, the underlying mechanism(s) have yet to be elucidated. The objective of this study was to determine the mechanisms underlying different spontaneous APs and heart rate between neonatal and adult SAN cells of the rabbit heart by biophysical modeling approaches. A mathematical model of neonatal rabbit SAN cells was developed by modifying the current densities and/or kinetics of ion channels and transporters in an adult cell model based on available experimental data obtained from neonatal SAN cells. The single cell models were then incorporated into a multi-cellular, two-dimensional model of the intact SAN-atrium to investigate the functional impact of altered ion channels during maturation on pacemaking electrical activities and their conduction at the tissue level. Effects of the neurotransmitter acetylcholine on the pacemaking activities in neonatal cells were also investigated and compared to those in the adult. Our results showed: (1) the differences in ion channel properties between neonatal and adult SAN cells are able to account for differences in their APs and the heart rate, providing mechanistic insight into understanding the reduced pacemaking rate of the rabbit sinoatrial node during postnatal development; (2) in the 2D model of the intact SAN-atria, it was shown that cellular changes during postnatal development impaired pacemaking activity through increasing the activation time and reducing the conduction velocity across the SAN; (3) the neonatal SAN model, with its faster beating rates, showed a greater sensitivity to parasympathetic modulation in response to acetylcholine than did the adult model. These results provide novel insights into the understanding of the cellular mechanisms underlying the differences in the cardiac pacemaking activities of the neonatal and adult SAN.

Gap junction channels mediate the direct intercellular passage of small ions as well as larger solutes such as second messengers. A family of proteins called connexins make up the subunits of gap junction channels in chordate animals. Each individual connexin forms channels that exhibit distinct permeability to molecules that influence cellular signaling, such as calcium ions, cyclic nucleotides, or inositol phosphates. In this review, we examine the permeability of connexin channels containing Cx43, Cx46, and Cx50 to signaling molecules and attempt to relate the observed differences in permeability to possible in vivo consequences that were revealed by studies of transgenic animals where these connexin genes have been manipulated. Taken together, these data suggest that differences in the permeability of individual connexin channels to larger solutes like 3′,5′-cyclic adenosine monophosphate (cAMP) and inositol 1,4,5-trisphosphate (IP3) could play a role in regulating epithelial cell division, differentiation, and homeostasis in organs like the ocular lens.

The cone snails (family Conidae) are the best known and most intensively studied venomous marine gastropods. However, of the total biodiversity of venomous marine mollusks (superfamily Conoidea, &gt, 20,000 species), cone snails comprise a minor fraction. The venoms of the family Drilliidae, a highly diversified family in Conoidea, have not previously been investigated. In this report, we provide the first biochemical characterization of a component in a Drilliidae venom and define a gene superfamily of venom peptides. A bioactive peptide, cdg14a, was purified from the venom of Clavus davidgilmouri Fedosov and Puillandre, 2020. The peptide is small (23 amino acids), disulfide-rich (4 cysteine residues) and belongs to the J-like drillipeptide gene superfamily. Other members of this superfamily share a conserved signal sequence and the same arrangement of cysteine residues in their predicted mature peptide sequences. The cdg14a peptide was chemically synthesized in its bioactive form. It elicited scratching and hyperactivity, followed by a paw-thumping phenotype in mice. Using the Constellation Pharmacology platform, the cdg14a drillipeptide was shown to cause increased excitability in a majority of non-peptidergic nociceptors, but did not affect other subclasses of dorsal root ganglion (DRG) neurons. This suggests that the cdg14a drillipeptide may be blocking a specific molecular isoform of potassium channels. The potency and selectivity of this biochemically characterized drillipeptide suggest that the venoms of the Drilliidae are a rich source of novel and selective ligands for ion channels and other important signaling molecules in the nervous system.

Summary Fast purinergic signaling is mediated by ATP and ATP-gated ionotropic P2X receptors (P2XRs), and it is implicated in pain-related behaviors. The properties exhibited by P2XRs vary between those expressed in heterologous cells and in vivo. Several modulators of ligand-gated ion channels have recently been identified, suggesting that there are P2XR functional modulators in vivo. Here, we establish a genome-wide open reading frame (ORF) collection and perform functional screening to identify modulators of P2XR activity. We identify TMEM163, which specifically modulates the channel properties and pharmacology of P2XRs. We also find that TMEM163 is required for full function of the neuronal P2XR and a pain-related ATP-evoked behavior. These results establish TMEM163 as a critical modulator of P2XRs in vivo and a potential target for the discovery of drugs for treating pain.

Cancerous tumors comprise cells showing metabolic heterogeneity. Among numerous efforts to understand this property, little attention has been paid to the possibility that cancer cells take up and utilize otherwise unusable substrates as fuel. Here we discuss this issue by focusing on l-glucose, the mirror image isomer of naturally occurring d-glucose; l-glucose is an unmetabolizable sugar except in some bacteria. By combining relatively small fluorophores with l-glucose, we generated fluorescence-emitting l-glucose tracers (fLGs). To our surprise, 2-NBDLG, one of these fLGs, which we thought to be merely a control substrate for the fluorescent d-glucose tracer 2-NBDG, was specifically taken up into tumor cell aggregates (spheroids) that exhibited nuclear heterogeneity, a major cytological feature of malignancy in cancer diagnosis. Changes in mitochondrial activity were also associated with the spheroids taking up fLG. To better understand these phenomena, we review here the Warburg effect as well as key studies regarding glucose uptake. We also discuss tumor heterogeneity involving aberrant uptake of glucose and mitochondrial changes based on the data obtained by fLG. We then consider the use of fLGs as novel markers for visualization and characterization of malignant tumor cells.

Conformational changes in amyloidogenic proteins, such as β-amyloid protein, prion proteins, and α-synuclein, play a critical role in the pathogenesis of numerous neurodegenerative diseases, including Alzheimer’s disease, prion disease, and Lewy body disease. The disease-associated proteins possess several common characteristics, including the ability to form amyloid oligomers with β-pleated sheet structure, as well as cytotoxicity, although they differ in amino acid sequence. Interestingly, these amyloidogenic proteins all possess the ability to bind trace metals, can regulate metal homeostasis, and are co-localized at the synapse, where metals are abundantly present. In this review, we discuss the physiological roles of these amyloidogenic proteins in metal homeostasis, and we propose hypothetical models of their pathogenetic role in the neurodegenerative process as the loss of normal metal regulatory functions of amyloidogenic proteins. Notably, these amyloidogenic proteins have the capacity to form Ca2+-permeable pores in membranes, suggestive of a toxic gain of function. Therefore, we focus on their potential role in the disruption of Ca2+ homeostasis in amyloid-associated neurodegenerative diseases.

Cystic fibrosis (CF) is a genetic disease characterized by the lack of cystic fibrosis transmembrane conductance regulator (CFTR) protein expressed in epithelial cells. The resulting defective chloride and bicarbonate secretion and imbalance of the transepithelial homeostasis lead to abnormal airway surface liquid (ASL) composition and properties. The reduced ASL volume impairs ciliary beating with the consequent accumulation of sticky mucus. This situation prevents the normal mucociliary clearance, favouring the survival and proliferation of bacteria and contributing to the genesis of CF lung disease. Here, we have explored the potential of small molecules capable of facilitating the transmembrane transport of chloride and bicarbonate in order to replace the defective transport activity elicited by CFTR in CF airway epithelia. Primary human bronchial epithelial cells obtained from CF and non-CF patients were differentiated into a mucociliated epithelia in order to assess the effects of our compounds on some key properties of ASL. The treatment of these functional models with non-toxic doses of the synthetic anionophores improved the periciliary fluid composition, reducing the fluid re-absorption, correcting the ASL pH and reducing the viscosity of the mucus, thus representing promising drug candidates for CF therapy.

Several studies support, both in vitro and in vivo, the anti-cancer effects of cannabidiol (CBD), a transient receptor potential vanilloid 2 (TRPV2) ligand. TRPV2, often dysregulated in tumors, is associated with altered cell proliferation and aggressiveness. Endometrial cancer (EC) is historically divided in type I endometrioid EC and type II non-endometrioid EC, associated with poor prognosis. Treatment options with chemotherapy and combinations with radiation showed only limited efficacy. Since no data are reported concerning TRPV2 expression as well as CBD potential effects in EC, the aim of this study was to evaluate the expression of TRPV2 in biopsies and cell lines as well as the effects of CBD in in vitro models. Overall survival (OS), progression-free survival (PFS), cell viability, migration, and chemo-resistance have been evaluated. Results show that TRPV2 expression increased with the malignancy of the cancer tissue and correlated with shorter PFS (p = 0.0224). Moreover, in vitro TRPV2 over-expression in Ishikawa cell line increased migratory ability and response to cisplatin. CBD reduced cell viability, activating predominantly apoptosis in type I cells and autophagy in mixed type EC cells. The CBD improved chemotherapeutic drugs cytotoxic effects, enhanced by TRPV2 over-expression. Hence, TRPV2 could be considered as a marker for optimizing the therapy and CBD might be a useful therapeutic option as adjuvant therapy. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:21 issue:15 ispartof: location:Switzerland status: published

Chloroplasts are endosymbiotic organelles and play crucial roles in energy supply and metabolism of eukaryotic photosynthetic organisms (algae and land plants). They harbor channels and transporters in the envelope and thylakoid membranes, mediating the exchange of ions and metabolites with the cytosol and the chloroplast stroma and between the different chloroplast subcompartments. In secondarily evolved algae, three or four envelope membranes surround the chloroplast, making more complex the exchange of ions and metabolites. Despite the importance of transport proteins for the optimal functioning of the chloroplast in algae, and that many land plant homologues have been predicted, experimental evidence and molecular characterization are missing in most cases. Here, we provide an overview of the current knowledge about ion and metabolite transport in the chloroplast from algae. The main aspects reviewed are localization and activity of the transport proteins from algae and/or of homologues from other organisms including land plants. Most chloroplast transporters were identified in the green alga Chlamydomonas reinhardtii, reside in the envelope and participate in carbon acquisition and metabolism. Only a few identified algal transporters are located in the thylakoid membrane and play role in ion transport. The presence of genes for putative transporters in green algae, red algae, diatoms, glaucophytes and cryptophytes is discussed, and roles in the chloroplast are suggested. A deep knowledge in this field is required because algae represent a potential source of biomass and valuable metabolites for industry, medicine and agriculture. Electronic supplementary material The online version of this article (10.1007/s00018-018-2793-0) contains supplementary material, which is available to authorized users.

Neuroblastoma (NB) is the most common extra-cranial pediatric solid tumor. High-risk NB is difficult to treat due to the lack of response to current therapies and aggressive disease progression. Despite novel drugs, alternative treatments and multi-modal treatments, finding an effective treatment strategy for these patients continues to be a major challenge. The current study focuses on examining the effects of FTY-720 or fingolimod, a drug that is FDA-approved for refractory multiple sclerosis, in NB. The results showed that FTY-720 regulates multiple pathways that result in various effects on calcium signaling, ion channel activation and cell survival/death pathways. FTY-720 rapidly inhibits TRPM7 channel activity, and inhibited TRPM7 kinase activity, modulates calcium signaling, induces a loss of mitochondrial membrane potential and opening of the mitochondrial permeability transition pore, and ultimately leads to cell death. Interestingly, the data also showed that low concentrations of FTY-720 sensitized drug-resistant NB cells to antineoplastic drugs. These results suggest that FTY-720 may be an attractive alternative for the treatment of NB.

Pyroelectrification (PE) has been proposed as a low-cost electrode-free tool for orienting and aligning dipole molecules in polymer layers, by means of the electric field produced by a pyroelectric crystal under appropriate thermal stimulations. Probing and assessing the coherent polarization arrangement is of fundamental importance for the process control and optimization but is also a challenging task. In fact, the probing operation must be noninvasive and avoid any disarrangement the dipoles and thus without interfering with the resulting surface charges. Here we show that the PE-induced polar ordering can be probed in situ by an electrode-free analysis based on the measurement of the intensity of the second-harmonic optical wave generated by the polymer film. In fact, the results show a substantial enhancement of the second-order susceptibility of the polymer layer, caused by the PE-induced dipoles alignment. Moreover, thanks to this approach, it is demonstrated the ability of PE process to polarize polymer layers even in the "noncontact configuration", i.e., when a dielectric spacer (glass substrate) is placed between the pyroelectric crystal and the polymer film.

Voltage-dependent inactivation of ion channels contributes to the regulation of the membrane potential of excitable cells. Mouse polycystic kidney disease 2-like 1 (PKD2L1) forms voltage-dependent nonselective cation channels, which are activated but subsequently inactivated in response to membrane depolarization. Here, we found that the mutation of an asparagine 533 residue (N533Q) in the outer pore loop region of PKD2L1 caused a marked increase in outward currents induced by depolarization. In addition, the tail current analysis demonstrated that the N533Q mutants are activated during depolarization but the subsequent inactivation does not occur. Interestingly, the N533Q mutants lacked the channel activation triggered by the removal of stimuli such as extracellular alkalization and heating. Our findings suggest that the N533 residue in the outer pore loop region of PKD2L1 has a key role in the voltage-dependent channel inactivation. ispartof: FEBS Open Bio vol:7 issue:9 pages:1392-1401 ispartof: location:England status: published

The present study tested the hypotheses that nephrotic syndrome (NS) leads to renal K+ loss because of augmented epithelial Na+ channel (ENaC) activity followed by downregulation of renal K+ secretory pathways by suppressed aldosterone. The hypotheses were addressed by determining K+ balance and kidney abundance of K+ and Na+ transporter proteins in puromycin aminonucleoside (PAN)-induced rat nephrosis. The effects of amiloride and angiotensin II type 1 receptor and mineralocorticoid receptor (MR) antagonists were tested. Glucocorticoid-dependent MR activation was tested by suppression of endogenous glucocorticoid with dexamethasone. Urine and plasma samples were obtained from pediatric patients with NS in acute and remission phases. PAN-induced nephrotic rats had ENaC-dependent Na+ retention and displayed lower renal K+ excretion but elevated intestinal K+ secretion that resulted in less cumulated K+ in NS. Aldosterone was suppressed at day 8. The NS-associated changes in intestinal, but not renal, K+ handling responded to suppression of corticosterone, whereas angiotensin II type 1 receptor and MR blockers and amiloride had no effect on urine K+ excretion during NS. In PAN-induced nephrosis, kidney protein abundance of the renal outer medullary K+ channel and γ-ENaC were unchanged, whereas the Na+-Cl− cotransporter was suppressed and Na+-K+-ATPase increased. Pediatric patients with acute NS displayed suppressed urine Na+-to-K+ ratios compared with remission and elevated plasma K+ concentration, whereas fractional K+ excretion did not differ. Acute NS is associated with less cumulated K+ in a rat model, whereas patients with acute NS have elevated plasma K+ and normal renal fractional K+ excretion. In NS rats, K+ balance is not coupled to ENaC activity but results from opposite changes in renal and fecal K+ excretion with a contribution from corticosteroid MR-driven colonic secretion The present study tested the hypotheses that nephrotic syndrome (NS) leads to renal K+ loss because of augmented epithelial Na+ channel (ENaC) activity followed by downregulation of renal K+ secretory pathways by suppressed aldosterone. The hypotheses were addressed by determining K+ balance and kidney abundance of K+ and Na+ transporter proteins in puromycin aminonucleoside (PAN)-induced rat nephrosis. The effects of amiloride and angiotensin II type 1 receptor and mineralocorticoid receptor (MR) antagonists were tested. Glucocorticoid-dependent MR activation was tested by suppression of endogenous glucocorticoid with dexamethasone. Urine and plasma samples were obtained from pediatric patients with NS in acute and remission phases. PAN-induced nephrotic rats had ENaC-dependent Na+ retention and displayed lower renal K+ excretion but elevated intestinal K+ secretion that resulted in less cumulated K+ in NS. Aldosterone was suppressed at day 8. The NS-associated changes in intestinal, but not renal, K+ handling responded to suppression of corticosterone, whereas angiotensin II type 1 receptor and MR blockers and amiloride had no effect on urine K+ excretion during NS. In PAN-induced nephrosis, kidney protein abundance of the renal outer medullary K+ channel and γ-ENaC were unchanged, whereas the Na+-Cl- cotransporter was suppressed and Na+-K+-ATPase increased. Pediatric patients with acute NS displayed suppressed urine Na+-to-K+ ratios compared with remission and elevated plasma K+ concentration, whereas fractional K+ excretion did not differ. Acute NS is associated with less cumulated K+ in a rat model, whereas patients with acute NS have elevated plasma K+ and normal renal fractional K+ excretion. In NS rats, K+ balance is not coupled to ENaC activity but results from opposite changes in renal and fecal K+ excretion with a contribution from corticosteroid MR-driven colonic secretion.

Bacterial type II secretion systems (T2SSs) translocate virulence factors, toxins and enzymes across the cell outer membrane. Here we use negative stain and cryo-electron microscopy to reveal the core architecture of an assembled T2SS from the pathogen Klebsiella pneumoniae. We show that 7 proteins form a ~2.4 MDa complex that spans the cell envelope. The outer membrane complex includes the secretin PulD, with all domains modelled, and the pilotin PulS. The inner membrane assembly platform components PulC, PulE, PulL, PulM and PulN have a relative stoichiometric ratio of 2:1:1:1:1. The PulE ATPase, PulL and PulM combine to form a flexible hexameric hub. Symmetry mismatch between the outer membrane complex and assembly platform is overcome by PulC linkers spanning the periplasm, with PulC HR domains binding independently at the secretin base. Our results show that the T2SS has a highly dynamic modular architecture, with implication for pseudo-pilus assembly and substrate loading., Bacterial type II secretion systems (T2SSs) translocate virulence factors, toxins and enzymes across the cell outer membrane. Here, Chernyatina and Low use negative stain and cryo-electron microscopy to reveal the core architecture of an assembled T2SS from the pathogen Klebsiella pneumoniae.

Plant development and fitness largely depend on the adequate availability of mineral elements in the soil. Most essential nutrients are available and can be membrane transported either as mono or divalent cations or as mono- or divalent anions. Trivalent cations are highly toxic to membranes, and plants have evolved different mechanisms to handle +3 elements in a safe way. The essential functional role of a few metal ions, with the possibility to gain a trivalent state, mainly resides in the ion’s redox activity; examples are iron (Fe) and manganese. Among the required nutrients, the only element with +3 as a unique oxidation state is the non-metal, boron. However, plants also can take up non-essential trivalent elements that occur in biologically relevant concentrations in soils. Examples are, among others, aluminum (Al), chromium (Cr), arsenic (As), and antimony (Sb). Plants have evolved different mechanisms to take up and tolerate these potentially toxic elements. This review considers recent studies describing the transporters, and specific and unspecific channels in different cell compartments and tissues, thereby providing a global vision of trivalent element homeostasis in plants.

N‐methyl‐D‐aspartate (NMDA) receptors are glutamate receptors with key roles in synaptic plasticity, due in part to their Mg2+ mediated voltage‐dependence. A large number of genetic variants affecting NMDA receptor subunits have been found in people with a range of neurodevelopmental disorders, including GluN2AN615K (GRIN2A C1845A) in two unrelated individuals with severe epileptic encephalopathy. This missense variant substitutes a lysine in place of an asparagine known to be important for blockade by Mg2+ and other small molecule channel blockers. We therefore measured the impact of GluN2AN615K on a range of NMDA receptor channel blockers using two‐electrode voltage clamp recordings made in Xenopus oocytes. We found that GluN2AN615K resulted in block by Mg2+ 1 mmol/L being greatly reduced (89% vs 8%), block by memantine 10 μmol/L (76% vs 27%) and amantadine 100 μmol/L (45% vs 17%) being substantially reduced, block by ketamine 10 μmol/L being modestly reduced (79% vs 73%) and block by dextromethorphan 10 μmol/L being enhanced (45% vs 55%). Coapplying Mg2+ with memantine or amantadine did not reduce the GluN2AN615K block seen with either small molecule. In addition, we measured single–channel conductance of GluN2AN615K–containing NMDA receptors in outside‐out patches pulled from Xenopus oocytes, finding a 4‐fold reduction in conductance (58 vs 15 pS). In conclusion, the GluN2AN615K variant is associated with substantial changes to important physiological and pharmacological properties of the NMDA receptor. Our findings are consistent with GluN2AN615K having a disease–causing role, and inform potential therapeutic strategies.

The concentration of sodium ions (Na(+)) is raised in solid tumours and can be measured at the cellular, tissue and patient levels. At the cellular level, the Na(+) gradient across the membrane powers the transport of H(+) ions and essential nutrients for normal activity. The maintenance of the Na(+) gradient requires a large proportion of the cell's ATP. Na(+) is a major contributor to the osmolarity of the tumour microenvironment, which affects cell volume and metabolism as well as immune function. Here, we review evidence indicating that Na(+) handling is altered in tumours, explore our current understanding of the mechanisms that may underlie these alterations and consider the potential consequences for cancer progression. Dysregulated Na(+) balance in tumours may open opportunities for new imaging biomarkers and re-purposing of drugs for treatment.

K IR2.1 potassium channels, producing inward rectifier potassium current (I K1), are important for final action potential repolarization and a stable resting membrane potential in excitable cells like cardiomyocytes. Abnormal K IR2.1 function, either decreased or increased, associates with diseases such as Andersen-Tawil syndrome, long and short QT syndromes. K IR2.1 ion channel protein trafficking and subcellular anchoring depends on intrinsic specific short amino acid sequences. We hypothesized that combining an evolutionary based sequence comparison and bioinformatics will identify new functional domains within the C-terminus of the KIR2.1 protein, which function could be determined by mutation analysis. We determined PEST domain signatures, rich in proline (P), glutamic acid (E), serine (S), and threonine (T), within K IR2.1 sequences using the "epestfind" webtool. WT and ΔPEST K IR2.1 channels were expressed in HEK293T and COS-7 cells. Patch-clamp electrophysiology measurements were performed in the inside-out mode on excised membrane patches and the whole cell mode using AxonPatch 200B amplifiers. K IR2.1 protein expression levels were determined by western blot analysis. Immunofluorescence microscopy was used to determine K IR2.1 subcellular localization. An evolutionary conserved PEST domain was identified in the C-terminus of the K IR2.1 channel protein displaying positive PEST scores in vertebrates ranging from fish to human. No similar PEST domain was detected in K IR2.2, K IR2.3, and K IR2.6 proteins. Deletion of the PEST domain in California kingsnake and human K IR2.1 proteins (ΔPEST), did not affect plasma membrane localization. Co-expression of WT and ΔPEST KIR2.1 proteins resulted in heterotetrameric channel formation. Deletion of the PEST domain did not increase protein stability in cycloheximide assays [T1/2 from 2.64 h (WT) to 1.67 h (ΔPEST), n.s.]. WT and ΔPEST channels, either from human or snake, produced typical I K1, however, human ΔPEST channels displayed stronger intrinsic rectification. The current observations suggest that the PEST sequence of K IR2.1 is not associated with rapid protein degradation, and has a role in the rectification behavior of I K1 channels.

Cysteine modifications emerge as important players in cellular signaling and homeostasis. Here, we present a chemical proteomics strategy for quantitative analysis of reversibly modified Cysteines using bioorthogonal cleavable-linker and switch technique (Cys-BOOST). Compared to iodoTMT for total Cysteine analysis, Cys-BOOST shows a threefold higher sensitivity and considerably higher specificity and precision. Analyzing S-nitrosylation (SNO) in S-nitrosoglutathione (GSNO)-treated and non-treated HeLa extracts Cys-BOOST identifies 8,304 SNO sites on 3,632 proteins covering a wide dynamic range of the proteome. Consensus motifs of SNO sites with differential GSNO reactivity confirm the relevance of both acid-base catalysis and local hydrophobicity for NO targeting to particular Cysteines. Applying Cys-BOOST to SH-SY5Y cells, we identify 2,151 SNO sites under basal conditions and reveal significantly changed SNO levels as response to early nitrosative stress, involving neuro(axono)genesis, glutamatergic synaptic transmission, protein folding/translation, and DNA replication. Our work suggests SNO as a global regulator of protein function akin to phosphorylation and ubiquitination., Reversible cysteine modifications play important roles in cellular redox signaling. Here, the authors develop a chemical proteomics strategy that enables the quantitative analysis of endogenous cysteine nitrosylation sites and their dynamic regulation under nitrosative stress conditions.

The flow rate of a pressure-driven liquid through a microchannel may be enhanced by texturing its no-slip boundaries with grooves aligned with the flow. In such cases, the grooves may contain vapour and/or an inert gas and the liquid is trapped in the Cassie state, resulting in (apparent) slip. The flow-rate enhancement is of benefit to different applications including the increase of throughput of a liquid in a lab-on-a-chip, and the reduction of thermal resistance associated with liquid metal cooling of microelectronics. At any given cross-section, the meniscus takes the approximate shape of a circular arc whose curvature is determined by the pressure difference across it. Hence, it typically protrudes into the grooves near the inlet of a microchannel and is gradually drawn into the microchannel as it is traversed and the liquid pressure decreases. For sufficiently large Reynolds numbers, the variation of the meniscus shape and hence the flow geometry necessitates the inclusion of inertial (non-parallel) flow effects. We capture them for a slender microchannel, where our small parameter is the ratio of ridge pitch-to-microchannel height, and order-one Reynolds numbers. This is done by using a hybrid analytical–numerical method to resolve the nonlinear three-dimensional (3-D) problem as a sequence of two-dimensional (2-D) linear ones in the microchannel cross-section, allied with non-local conditions that determine the slowly varying pressure distribution at leading and first orders. When the pressure difference across the microchannel is constrained by the advancing contact angle of the liquid on the ridges and its surface tension (which is high for liquid metals), inertial effects can significantly reduce the flow rate for realistic parameter values. For example, when the solid fraction of the ridges is 0.1, the microchannel height-to-(half) ridge pitch ratio is 6, the Reynolds number of the flow is 1 and the small parameter is 0.1, they reduce the flow rate of a liquid metal (Galinstan) by approximately 50 %. Conversely, for sufficiently large microchannel heights, they enhance it. Physical explanations of both of these phenomena are given.

Despite responses to initial treatment of photodynamic therapy (PDT) being promising, a recurrence rate exists. Thus, finding novel therapeutic targets to enhance PDT efficacy is an urgent need. Reports indicate that connexin (Cx) 40 plays an important role in tumor angiogenesis and growth. However, it is unknown whether Cx40-composed channels have effects on PDT efficacy. The study uniquely demonstrated that Cx40-formed channels could enhance the phototoxicity of PDT to malignant cells in vitro and in vivo. Specifically, Cx40-formed channels at high cell density could increase PDT photocytotoxicity. This action was substantially restricted when Cx40 expression was not induced or Cx40 channels were restrained. Additionally, the presence of Cx40-composed channels enhanced the phototoxicity of PDT in the tumor xenografts. The above results indicate that enhancing the function of Cx40-formed channels increases PDT efficacy. The enhancement of PDT efficacy mediated by Cx40 channels was related with intracellular pathways mediated by ROS and calcium pathways, but not the lipid peroxide-mediated pathway. This work demonstrates the capacity of Cx40-mediated channels to increase PDT efficacy and suggests that therapeutic strategies designed to maintain or enhance Cx40 expression and/or channels composed by Cx40 may increase the therapeutic efficacy of PDT.

International audience; Identifying the location of the active sites in a zeolite is a current challenge, impeding the design of optimal catalysts. In this work, we identify the location of the most active sites of 1-ethylcyclohexene isomerization in the EUO framework (10 MR channels, 12 MR side pockets) thanks to DFT calculations corroborated by experiments. Skeletal isomerization of cycloalkenes is a crucial industrial reaction for the bifunctional isomerization of ethylbenzene. Ethylcyclohexene is protonated by framework protons into cyclic carbenium ions, which undergo ring contraction–expansion reactions through protonated cyclopropane (PCP) like transition states. Ab initio calculations clearly show that the acid sites located at the intersection between the channel and the pocket stabilize much less the cyclic carbenium ions involved in the reaction than 12 MR pockets and 10 MR channel sites due to stronger dispersion stabilizing interactions. This computational finding is fully confirmed experimentally by the comparison of the catalytic performances of the H-EU-1 and H-ZSM-50 zeolites in ethylcyclohexane hydroisomerization. Both zeolites possess the EUO structure but with different location of the acid sites. The ratio in turnover frequencies is quantitatively rendered by the DFT-calculated free energy profiles. Diffusion measurements reveal similar ethylcyclohexane diffusion times for the two zeolites, supporting that the difference in activity is primarily driven by the location of the active sites.

The epithelial Na+ channel (ENaC) provides for Na+ absorption in various types of epithelia including the kidney, lung, and colon where ENaC is localized to the apical membrane to enable Na+ entry into the cell. The degree of Na+ entry via ENaC largely depends on the number of active channels localized to the cell membrane, and is tightly controlled by interactions with ubiquitin ligases, kinases, and G-proteins. While regulation of ENaC endocytosis has been well-studied, relatively little is understood of the proteins that govern ENaC exocytosis. We hypothesized that the annexin II light chain, p11, could participate in the transport of ENaC along the exocytic pathway. Our results demonstrate that all three ENaC channel subunits interacted with p11 in an in vitro binding assay. Furthermore, p11 was able to immunoprecipitate ENaC in epithelial cells. Quantitative mass spectrometry of affinity-purified ENaC-p11 complexes recovered several other trafficking proteins including HSP-90 and annexin A6. We also report that p11 exhibits a robust protein expression in cortical collecting duct epithelial cells. However, the expression of p11 in these cells was not influenced by either short-term or long-term exposure to aldosterone. To determine whether the p11 interaction affected ENaC function, we measured amiloride sensitive Na+ currents in Xenopus oocytes or mammalian epithelia co-expressing ENaC and p11 or a siRNA to p11. Results from these experiments showed that p11 significantly augmented ENaC current, whereas knockdown of p11 decreased current. Further, knockdown of p11 reduced ENaC cell surface population suggesting p11 promotes membrane insertion of ENaC. Overall, our findings reveal a novel protein interaction that controls the number of ENaC channels inserted at the membrane via the exocytic pathway.

Adenosine 5'-triphosphate (ATP) is an important extracellular signaling agent, operating in growth regulation, stomatal conductance, and wound response. With the first receptor for extracellular ATP now identified in plants (P2K1/DORN1) and a plasma membrane NADPH oxidase revealed as its target, the search continues for the components of the signaling cascades they command. The Arabidopsis root elongation zone epidermal plasma membrane has recently been shown to contain cation transport pathways (channel conductances) that operate downstream of P2K1 and could contribute to extracellular ATP (eATP) signaling. Here, patch clamp electrophysiology has been used to delineate two further conductances from the root elongation zone epidermal plasma membrane that respond to eATP, including one that would permit chloride transport. This perspective addresses how these conductances compare to those previously characterized in roots and how they might operate together to enable early events in eATP signaling, including elevation of cytosolic-free calcium as a second messenger. The role of the reactive oxygen species (ROS) that could arise from eATP's activation of NADPH oxidases is considered in a qualitative model that also considers the regulation of plasma membrane potential by the concerted action of the various cation and anion conductances. The molecular identities of the channel conductances in eATP signaling remain enigmatic but may yet be found in the multigene families of glutamate receptor-like channels, cyclic nucleotide-gated channels, annexins, and aluminum-activated malate transporters.

Sphingosine-1-phosphate (S1P) is a lipid mediator that is relatively abundant in plasma and plays an important role in the vascular and immune systems. To date, the only known mechanism for removing S1P from plasma has been dephosphorylation by phospholipid phosphatases (PLPPs) on the surface of cells in contact with the plasma. However, there remains a possibility that PLPP-independent dephosphorylation or direct S1P uptake into cells could occur. To examine these possibilities, here we generated triple KO (TKO) HAP1 cells that lacked all PLPPs (PLPP1-3) present in mammals. In the TKO cells, the intracellular metabolism of externally added deuterium-labeled S1P to ceramide was reduced to 17% compared with the WT cells, indicating that most extracellular S1P is dephosphorylated by PLPPs and then taken up into cells. However, this result also reveals the existence of a PLPP-independent S1P uptake pathway. Tracer experiments using [P-32]S1P showed the existence of a direct S1P uptake pathway that functions without prior dephosphorylation. Overexpression of sphingolipid transporter 2 (SPNS2) or of major facilitator superfamily domain containing 2B (MFSD2B), both known S1P efflux transporters, in TKO cells increased the direct uptake of S1P, whereas KO of MFSD2B in TKO cells reduced this uptake. These results suggest that these are channel-type transporters and capable of not only exporting but also importing S1P. Furthermore, we observed that erythroid cells expressing MFSD2B, exhibited high S1P uptake activity. Our findings describing direct S1P uptake may contribute to the elucidation of the molecular mechanisms that regulate plasma S1P concentration.