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The TRP superfamily of channels (nomenclature as agreed by NC-IUPHAR [176, 1072]), whose founder member is the Drosophila Trp channel, exists in mammals as six families; TRPC, TRPM, TRPV, TRPA, TRPP and TRPML based on amino acid homologies. TRP subunits contain six putative TM domains and assemble as homo- or hetero-tetramers to form cation selective channels with diverse modes of activation and varied permeation properties (reviewed by [730]). Established, or potential, physiological functions of the individual members of the TRP families are discussed in detail in the recommended reviews and in a number of books [401, 686, 1155, 256]. The established, or potential, involvement of TRP channels in disease [1126] is reviewed in [448, 685], [688] and [464], together with a special edition of Biochemica et Biophysica Acta on the subject [685]. Additional disease related reviews, for pain [633], stroke [1135], sensation and inflammation [988], itch [130], and airway disease [310, 1051], are available. The pharmacology of most TRP channels has been advanced in recent years. Broad spectrum agents are listed in the tables along with more selective, or recently recognised, ligands that are flagged by the inclusion of a primary reference. See Rubaiy (2019) for a review of pharmacological tools for TRPC1/C4/C5 channels [805]. Most TRP channels are regulated by phosphoinostides such as PtIns(4,5)P2 although the effects reported are often complex, occasionally contradictory, and likely to be dependent upon experimental conditions, such as intracellular ATP levels (reviewed by [1009, 689, 801]). Such regulation is generally not included in the tables.When thermosensitivity is mentioned, it refers specifically to a high Q10 of gating, often in the range of 10-30, but does not necessarily imply that the channel's function is to act as a 'hot' or 'cold' sensor. In general, the search for TRP activators has led to many claims for temperature sensing, mechanosensation, and lipid sensing. All proteins are of course sensitive to energies of binding, mechanical force, and temperature, but the issue is whether the proposed input is within a physiologically relevant range resulting in a response. TRPA (ankyrin) familyTRPA1 is the sole mammalian member of this group (reviewed by [293]). TRPA1 activation of sensory neurons contribute to nociception [414, 890, 602]. Pungent chemicals such as mustard oil (AITC), allicin, and cinnamaldehyde activate TRPA1 by modification of free thiol groups of cysteine side chains, especially those located in its amino terminus [575, 60, 365, 577]. Alkenals with α, β-unsaturated bonds, such as propenal (acrolein), butenal (crotylaldehyde), and 2-pentenal can react with free thiols via Michael addition and can activate TRPA1. However, potency appears to weaken as carbon chain length increases [26, 60]. Covalent modification leads to sustained activation of TRPA1. Chemicals including carvacrol, menthol, and local anesthetics reversibly activate TRPA1 by non-covalent binding [424, 511, 1081, 1080]. TRPA1 is not mechanosensitive under physiological conditions, but can be activated by cold temperatures [425, 212]. The electron cryo-EM structure of TRPA1 [740] indicates that it is a 6-TM homotetramer. Each subunit of the channel contains two short ‘pore helices’ pointing into the ion selectivity filter, which is big enough to allow permeation of partially hydrated Ca2+ ions. TRPC (canonical) familyMembers of the TRPC subfamily (reviewed by [284, 778, 18, 4, 94, 446, 739, 70]) fall into the subgroups outlined below. TRPC2 is a pseudogene in humans. It is generally accepted that all TRPC channels are activated downstream of Gq/11-coupled receptors, or receptor tyrosine kinases (reviewed by [765, 953, 1072]). A comprehensive listing of G-protein coupled receptors that activate TRPC channels is given in [4]. Hetero-oligomeric complexes of TRPC channels and their association with proteins to form signalling complexes are detailed in [18] and [447]. TRPC channels have frequently been proposed to act as store-operated channels (SOCs) (or compenents of mulimeric complexes that form SOCs), activated by depletion of intracellular calcium stores (reviewed by [741, 18, 770, 820, 1121, 157, 726, 64, 158]). However, the weight of the evidence is that they are not directly gated by conventional store-operated mechanisms, as established for Stim-gated Orai channels. TRPC channels are not mechanically gated in physiologically relevant ranges of force. All members of the TRPC family are blocked by 2-APB and SKF96365 [347, 346]. Activation of TRPC channels by lipids is discussed by [70]. Important progress has been recently made in TRPC pharmacology [805, 619, 436, 102, 851, 191, 291]. TRPC channels regulate a variety of physiological functions and are implicated in many human diseases [295, 71, 885, 1031, 1025, 154, 103, 561, 913, 409]. TRPC1/C4/C5 subgroup TRPC1 alone may not form a functional ion channel [229]. TRPC4/C5 may be distinguished from other TRP channels by their potentiation by micromolar concentrations of La3+. TRPC2 is a pseudogene in humans, but in other mammals appears to be an ion channel localized to microvilli of the vomeronasal organ. It is required for normal sexual behavior in response to pheromones in mice. It may also function in the main olfactory epithelia in mice [1114, 723, 724, 1115, 539, 1168, 1109].TRPC3/C6/C7 subgroup All members are activated by diacylglycerol independent of protein kinase C stimulation [347].TRPM (melastatin) familyMembers of the TRPM subfamily (reviewed by [275, 346, 741, 1151]) fall into the five subgroups outlined below. TRPM1/M3 subgroupIn darkness, glutamate released by the photoreceptors and ON-bipolar cells binds to the metabotropic glutamate receptor 6 , leading to activation of Go . This results in the closure of TRPM1. When the photoreceptors are stimulated by light, glutamate release is reduced, and TRPM1 channels are more active, resulting in cell membrane depolarization. Human TRPM1 mutations are associated with congenital stationary night blindness (CSNB), whose patients lack rod function. TRPM1 is also found melanocytes. Isoforms of TRPM1 may present in melanocytes, melanoma, brain, and retina. In melanoma cells, TRPM1 is prevalent in highly dynamic intracellular vesicular structures [398, 708]. TRPM3 (reviewed by [714]) exists as multiple splice variants which differ significantly in their biophysical properties. TRPM3 is expressed in somatosensory neurons and may be important in development of heat hyperalgesia during inflammation (see review [941]). TRPM3 is frequently coexpressed with TRPA1 and TRPV1 in these neurons. TRPM3 is expressed in pancreatic beta cells as well as brain, pituitary gland, eye, kidney, and adipose tissue [713, 940]. TRPM3 may contribute to the detection of noxious heat [1017]. TRPM2TRPM2 is activated under conditions of oxidative stress (respiratory burst of phagocytic cells). The direct activators are calcium, adenosine diphosphate ribose (ADPR) [970] and cyclic ADPR (cADPR) [1118]. As for many ion channels, PI(4,5)P2 must also be present [1109]. Numerous splice variants of TRPM2 exist which differ in their activation mechanisms [239]. Recent studies have reported structures of human (hs) TRPM2, which demonstrate two ADPR binding sites in hsTRPM2, one in the N-terminal MHR1/2 domain and the other in the C-terminal NUDT9-H domain. In addition, one Ca2+ binding site in the intracellular S2-S3 loop is revealed and proposed to mediate Ca2+ binding that induces conformational changes leading the ADPR-bound closed channel to open [387, 1027]. Meanwhile, a quadruple-residue motif (979FGQI982) was identified as the ion selectivity filter and a gate to control ion permeation in hsTRPM2 [1120]. TRPM2 is involved in warmth sensation [848], and contributes to several diseases [76]. TRPM2 interacts with extra synaptic NMDA receptors (NMDAR) and enhances NMDAR activity in ischemic stroke [1164]. Activation of TRPM2 in macrophages promotes atherosclerosis [1165, 1147]. Moreover, silica nanoparticles induce lung inflammation in mice via ROS/PARP/TRPM2 signaling-mediated lysosome impairment and autophagy dysfunction [1028]. Recent studies have designed various compounds for their potential to selectively inhibit the TRPM2 channel, including ACA derivatives A23, and 2,3-dihydroquinazolin-4(1H)-one derivatives [1137, 1139]. TRPM4/5 subgroupTRPM4 and TRPM5 have the distinction within all TRP channels of being impermeable to Ca2+ [1072]. A splice variant of TRPM4 (i.e.TRPM4b) and TRPM5 are molecular candidates for endogenous calcium-activated cation (CAN) channels [327]. TRPM4 is active in the late phase of repolarization of the cardiac ventricular action potential. TRPM4 deletion or knockout enhances beta adrenergic-mediated inotropy [593]. Mutations are associated with conduction defects [404, 593, 879]. TRPM4 has been shown to be an important regulator of Ca2+ entry in to mast cells [993] and dendritic cell migration [52]. TRPM5 in taste receptor cells of the tongue appears essential for the transduction of sweet, amino acid and bitter stimuli [537] TRPM5 contributes to the slow afterdepolarization of layer 5 neurons in mouse prefrontal cortex [513]. Both TRPM4 and TRPM5 are required transduction of taste stimuli [246]. TRPM6/7 subgroupTRPM6 and 7 combine channel and enzymatic activities (‘chanzymes’) [172]. These channels have the unusual property of permeation by divalent (Ca2+, Mg2+, Zn2+) and monovalent cations, high single channel conductances, but overall extremely small inward conductance when expressed to the plasma membrane. They are inhibited by internal Mg2+ at ~0.6 mM, around the free level of Mg2+ in cells. Whether they contribute to Mg2+ homeostasis is a contentious issue. PIP2 is required for TRPM6 and TRPM7 activation [810, 1077]. When either gene is deleted in mice, the result is embryonic lethality [413, 1065]. The C-terminal kinase region of TRPM6 and TRPM7 is cleaved under unknown stimuli, and the kinase phosphorylates nuclear histones [479, 480]. TRPM7 is responsible for oxidant- induced Zn2+ release from intracellular vesicles [3] and contributes to intestinal mineral absorption essential for postnatal survival [622]. The putative metal transporter proteins CNNM1-4 interact with TRPM7 and regulate TRPM7 channel activity [40, 467]. TRPM8Is a channel activated by cooling and pharmacological agents evoking a ‘cool’ sensation and participates in the thermosensation of cold temperatures [63, 178, 224] reviewed by [1011, 562, 457, 649]. Direct chemical agonists include menthol and icilin[1086]. Besides, linalool can promote ERK phosphorylation in human dermal microvascular endothelial cells, down-regulate intracellular ATP levels, and activate TRPM8 [68]. Recent studies have found that TRPM8 has typical S4-S5 connectomes with clear selective filters and exowell rings [512], and have identified cryo-electron microscopy structures of mouse TRPM8 in closed, intermediate, and open states along the ligand- and PIP2-dependent gated pathways [1111]. Moreover, the last 36 amino acids at the carboxyl terminal of TRPM8 are key protein sequences for TRPM8's temperature-sensitive function [194]. TRPM8 deficiency reduced the expression of S100A9 and increased the expression of HNF4α in the liver of mice, which reduced inflammation and fibrosis progression in mice with liver fibrosis, and helped to alleviate the symptoms of bile duct disease [556]. Channel deficiency also shortens the time of hypersensitivity reactions in migraine mouse models by promoting the recovery of normal sensitivity [12]. A cyclic peptide DeC‐1.2 was designed to inhibit ligand activation of TRPM8 but not cold activation, which can eliminate the side effects of cold dysalgesia in oxaliplatin-treated mice without changing body temperature [9]. Analysis of clinical data shows that TRPM8-specific blockers WS12 can reduce tumor growth in colorectal cancer xenografted mice by reducing transcription and activation of Wnt signaling regulators and β-catenin and its target oncogenes, such as C-Myc and Cyclin D1 [732]. TRPML (mucolipin) familyThe TRPML family [782, 1132, 775, 1084, 190] consists of three mammalian members (TRPML1-3). TRPML channels are probably restricted to intracellular vesicles and mutations in the gene (MCOLN1) encoding TRPML1 (mucolipin-1) cause the neurodegenerative disorder mucolipidosis type IV (MLIV) in man. TRPML1 is a cation selective ion channel that is important for sorting/transport of endosomes in the late endocytotic pathway and specifically, fission from late endosome-lysosome hybrid vesicles and lysosomal exocytosis [822]. TRPML2 and TRPML3 show increased channel activity in low luminal sodium and/or increased luminal pH, and are activated by similar small molecules [319, 147, 877]. A naturally occurring gain of function mutation in TRPML3 (i.e. A419P) results in the varitint waddler (Va) mouse phenotype (reviewed by [782, 690]). TRPP (polycystin) familyThe TRPP family (reviewed by [216, 214, 300, 1061, 374]) or PKD2 family is comprised of PKD2 (PC2), PKD2L1 (PC2L1), PKD2L2 (PC2L2), which have been renamed TRPP1, TRPP2 and TRPP3, respectively [1072]. It should also be noted that the nomenclature of PC2 was TRPP2 in old literature. However, PC2 has been uniformed to be called TRPP2 [345]. PKD2 family channels are clearly distinct from the PKD1 family, whose function is unknown. PKD1 and PKD2 form a hetero-oligomeric complex with a 1:3 ratio. [905]. Although still being sorted out, TRPP family members appear to be 6TM spanning nonselective cation channels. TRPV (vanilloid) familyMembers of the TRPV family (reviewed by [995]) can broadly be divided into the non-selective cation channels, TRPV1-4 and the more calcium selective channels TRPV5 and TRPV6. TRPV1-V4 subfamilyTRPV1 is involved in the development of thermal hyperalgesia following inflammation and may contribute to the detection of noxius heat (reviewed by [762, 882, 922]). Numerous splice variants of TRPV1 have been described, some of which modulate the activity of TRPV1, or act in a dominant negative manner when co-expressed with TRPV1 [844]. The pharmacology of TRPV1 channels is discussed in detail in [329] and [1015]. TRPV2 is probably not a thermosensor in man [736], but has recently been implicated in innate immunity [547]. Functional TRPV2 expression is described in placental trophoblast cells of mouse [204]. TRPV3 and TRPV4 are both thermosensitive. There are claims that TRPV4 is also mechanosensitive, but this has not been established to be within a physiological range in a native environment [127, 530]. TRPV5/V6 subfamily TRPV5 and TRPV6 are highly expressed in placenta, bone, and kidney. Under physiological conditions, TRPV5 and TRPV6 are calcium selective channels involved in the absorption and reabsorption of calcium across intestinal and kidney tubule epithelia (reviewed by [1057, 205, 651, 270]).TRPV6 is reported to play a key role in calcium transport in the mouse placenta [1056].

BackgroundIn vascular smooth muscle cells (VSMCs), LRRC8A volume regulated anion channels (VRACs) are activated by inflammatory and pro-contractile stimuli including tumor necrosis factor alpha (TNFα), angiotensin II and stretch. LRRC8A physically associates with NADPH oxidase 1 (Nox1) and supports its production of extracellular superoxide (O2-•).Methods and ResultsMice lacking LRRC8A exclusively in VSMCs (Sm22α-Cre, KO) were used to assess the role of VRACs in TNFα signaling and vasomotor function. KO mesenteric vessels contracted normally to KCl and phenylephrine, but relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) was enhanced compared to wild type (WT). 48 hours ofex vivoexposure to TNFα (10ng/ml) markedly impaired dilation to ACh and SNP in WT but not KO vessels. VRAC blockade (carbenoxolone, CBX, 100 μM, 20 min) enhanced dilation of control rings and restored impaired dilation following TNFα exposure. Myogenic tone was absent in KO rings. LRRC8A immunoprecipitation followed by mass spectroscopy identified 35 proteins that interacted with LRRC8A. Pathway analysis revealed actin cytoskeletal regulation as the most closely associated function of these proteins. Among these proteins, the Myosin Phosphatase Rho-Interacting protein (MPRIP) links RhoA, MYPT1 and actin. LRRC8A-MPRIP co-localization was confirmed by confocal imaging of tagged proteins, Proximity Ligation Assays, and IP/western blots which revealed LRRC8A binding at the second Pleckstrin Homology domain of MPRIP. siLRRC8A or CBX treatment decreased RhoA activity in cultured VSMCs, and MYPT1 phosphorylation at T853 was reduced in KO mesenteries suggesting that reduced ROCK activity contributes to enhanced relaxation. MPRIP was a target of redox modification, becoming oxidized (sulfenylated) after TNFα exposure.ConclusionsInteraction of Nox1/LRRC8A with MPRIP/RhoA/MYPT1/actin may allow redox regulation of the cytoskeleton and link Nox1 activation to both inflammation and vascular contractility.

There are three identified indigenous poultry breeds in Nepal; Sakini, Ghanti Khuile, Pwankh Ulte. They are reared for dual purpose i.e. meat & egg. To access various non-genetic factors affecting growth performance of indigenous chicken, different relevant research papers, review papers, annual progress reports, and statistical year book were reviewed & analyzed. Results revealed that local breeds are hardy & subject to be affected with environmental factors. They are reared on natural food than formulated feed which meant that they were hardy & rich in high quality protein. They had better weight as they found food continuously in free ranging system. It was found that chicken reared in cool climate had higher body weight than the ones grown in hot period due to less respiratory loss & low body maintenance required. Natural daylight supplemented light at night time was being deployed by farmers & had satisfactory performance. Due to well-developed thick feathers on their body, indigenous chicken were deployed for brooding than artificial methods. Body weight and feed intake was found higher when floor space of 0.279/m2 per bird and flock size of 30 chickens were maintained. Despite any vaccinations, the indigenous breeds still had minimum disease incidence which may be linked to their better adaptability.

The TRP superfamily of channels (nomenclature as agreed by NC-IUPHAR [159, 999]), whose founder member is the Drosophila Trp channel, exists in mammals as six families; TRPC, TRPM, TRPV, TRPA, TRPP and TRPML based on amino acid homologies. TRP subunits contain six putative TM domains and assemble as homo- or hetero-tetramers to form cation selective channels with diverse modes of activation and varied permeation properties (reviewed by [679]). Established, or potential, physiological functions of the individual members of the TRP families are discussed in detail in the recommended reviews and in a number of books [371, 635, 1066, 236]. The established, or potential, involvement of TRP channels in disease is reviewed in [412, 634] and [637], together with a special edition of Biochemica et Biophysica Acta on the subject [634]. Additional disease related reviews, for pain [585], stroke [1052], sensation and inflammation [921], itch [117], and airway disease [284, 979], are available. The pharmacology of most TRP channels has been advanced in recent years. Broad spectrum agents are listed in the tables along with more selective, or recently recognised, ligands that are flagged by the inclusion of a primary reference. See Rubaiy (2019) for a review of pharmacological tools for TRPC1/C4/C5 channels [751]. Most TRP channels are regulated by phosphoinostides such as PtIns(4,5)P2 although the effects reported are often complex, occasionally contradictory, and likely to be dependent upon experimental conditions, such as intracellular ATP levels (reviewed by [941, 638, 747]). Such regulation is generally not included in the tables.When thermosensitivity is mentioned, it refers specifically to a high Q10 of gating, often in the range of 10-30, but does not necessarily imply that the channel's function is to act as a 'hot' or 'cold' sensor. In general, the search for TRP activators has led to many claims for temperature sensing, mechanosensation, and lipid sensing. All proteins are of course sensitive to energies of binding, mechanical force, and temperature, but the issue is whether the proposed input is within a physiologically relevant range resulting in a response. TRPA (ankyrin) familyTRPA1 is the sole mammalian member of this group (reviewed by [268]). TRPA1 activation of sensory neurons contribute to nociception [382, 831, 555]. Pungent chemicals such as mustard oil (AITC), allicin, and cinnamaldehyde activate TRPA1 by modification of free thiol groups of cysteine side chains, especially those located in its amino terminus [529, 51, 336, 531]. Alkenals with α, β-unsaturated bonds, such as propenal (acrolein), butenal (crotylaldehyde), and 2-pentenal can react with free thiols via Michael addition and can activate TRPA1. However, potency appears to weaken as carbon chain length increases [23, 51]. Covalent modification leads to sustained activation of TRPA1. Chemicals including carvacrol, menthol, and local anesthetics reversibly activate TRPA1 by non-covalent binding [391, 470, 1007, 1006]. TRPA1 is not mechanosensitive under physiological conditions, but can be activated by cold temperatures [392, 193]. The electron cryo-EM structure of TRPA1 [688] indicates that it is a 6-TM homotetramer. Each subunit of the channel contains two short ‘pore helices’ pointing into the ion selectivity filter, which is big enough to allow permeation of partially hydrated Ca2+ ions. TRPC (canonical) familyMembers of the TRPC subfamily (reviewed by [261, 726, 15, 4, 84, 410, 687, 60]) fall into the subgroups outlined below. TRPC2 is a pseudogene in humans. It is generally accepted that all TRPC channels are activated downstream of Gq/11-coupled receptors, or receptor tyrosine kinases (reviewed by [713, 889, 999]). A comprehensive listing of G-protein coupled receptors that activate TRPC channels is given in [4]. Hetero-oligomeric complexes of TRPC channels and their association with proteins to form signalling complexes are detailed in [15] and [411]. TRPC channels have frequently been proposed to act as store-operated channels (SOCs) (or compenents of mulimeric complexes that form SOCs), activated by depletion of intracellular calcium stores (reviewed by [689, 15, 718, 765, 1039, 141, 675, 55, 142]). However, the weight of the evidence is that they are not directly gated by conventional store-operated mechanisms, as established for Stim-gated Orai channels. TRPC channels are not mechanically gated in physiologically relevant ranges of force. All members of the TRPC family are blocked by 2-APB and SKF96365 [319, 318]. Activation of TRPC channels by lipids is discussed by [60]. Important progress has been recently made in TRPC pharmacology [751, 571, 400, 92]. TRPC channels regulate a variety of physiological functions and are implicated in many human diseases [270, 61, 827, 960]. TRPC1/C4/C5 subgroup TRPC1 alone may not form a functional ion channel [210]. TRPC4/C5 may be distinguished from other TRP channels by their potentiation by micromolar concentrations of La3+. TRPC2 is a pseudogene in humans, but in other mammals appears to be an ion channel localized to microvilli of the vomeronasal organ. It is required for normal sexual behavior in response to pheromones in mice. It may also function in the main olfactory epithelia in mice [1036, 672, 673, 1037, 496, 1077, 1032].TRPC3/C6/C7 subgroup All members are activated by diacylglycerol independent of protein kinase C stimulation [319].TRPM (melastatin) familyMembers of the TRPM subfamily (reviewed by [252, 318, 689, 1064]) fall into the five subgroups outlined below. TRPM1/M3 subgroupIn darkness, glutamate released by the photoreceptors and ON-bipolar cells binds to the metabotropic glutamate receptor 6 , leading to activation of Go . This results in the closure of TRPM1. When the photoreceptors are stimulated by light, glutamate release is reduced, and TRPM1 channels are more active, resulting in cell membrane depolarization. Human TRPM1 mutations are associated with congenital stationary night blindness (CSNB), whose patients lack rod function. TRPM1 is also found melanocytes. Isoforms of TRPM1 may present in melanocytes, melanoma, brain, and retina. In melanoma cells, TRPM1 is prevalent in highly dynamic intracellular vesicular structures [368, 657]. TRPM3 (reviewed by [663]) exists as multiple splice variants which differ significantly in their biophysical properties. TRPM3 is expressed in somatosensory neurons and may be important in development of heat hyperalgesia during inflammation (see review [878]). TRPM3 is frequently coexpressed with TRPA1 and TRPV1 in these neurons. TRPM3 is expressed in pancreatic beta cells as well as brain, pituitary gland, eye, kidney, and adipose tissue [662, 877]. TRPM3 may contribute to the detection of noxious heat [949].TRPM2TRPM2 is activated under conditions of oxidative stress (respiratory burst of phagocytic cells) and ischemic conditions. However, the direct activators are ADPR(P) and calcium. As for many ion channels, PIP2 must also be present (reviewed by [1020]). Numerous splice variants of TRPM2 exist which differ in their activation mechanisms [219]. The C-terminal domain contains a TRP motif, a coiled-coil region, and an enzymatic NUDT9 homologous domain. TRPM2 appears not to be activated by NAD, NAAD, or NAADP, but is directly activated by ADPRP (adenosine-5'-O-disphosphoribose phosphate) [902]. TRPM2 is involved in warmth sensation [789], and contributes to neurological diseases [66]. Recent study shows that 2'-deoxy-ADPR is an endogenous TRPM2 superagonist [253]. TRPM4/5 subgroupTRPM4 and TRPM5 have the distinction within all TRP channels of being impermeable to Ca2+ [999]. A splice variant of TRPM4 (i.e.TRPM4b) and TRPM5 are molecular candidates for endogenous calcium-activated cation (CAN) channels [301]. TRPM4 is active in the late phase of repolarization of the cardiac ventricular action potential. TRPM4 deletion or knockout enhances beta adrenergic-mediated inotropy [546]. Mutations are associated with conduction defects [374, 546, 821]. TRPM4 has been shown to be an important regulator of Ca2+ entry in to mast cells [926] and dendritic cell migration [43]. TRPM5 in taste receptor cells of the tongue appears essential for the transduction of sweet, amino acid and bitter stimuli [494] TRPM5 contributes to the slow afterdepolarization of layer 5 neurons in mouse prefrontal cortex [471]. Both TRPM4 and TRPM5 are required transduction of taste stimuli [226].TRPM6/7 subgroupTRPM6 and 7 combine channel and enzymatic activities (‘chanzymes’). These channels have the unusual property of permeation by divalent (Ca2+, Mg2+, Zn2+) and monovalent cations, high single channel conductances, but overall extremely small inward conductance when expressed to the plasma membrane. They are inhibited by internal Mg2+ at ~0.6 mM, around the free level of Mg2+ in cells. Whether they contribute to Mg2+ homeostasis is a contentious issue. When either gene is deleted in mice, the result is embryonic lethality. The C-terminal kinase region is cleaved under unknown stimuli, and the kinase phosphorylates nuclear histones. TRPM7 is responsible for oxidant- induced Zn2+ release from intracellular vesicles [3] and contributes to intestinal mineral absorption essential for postnatal survival [574]. TRPM8Is a channel activated by cooling and pharmacological agents evoking a ‘cool’ sensation and participates in the thermosensation of cold temperatures [54, 161, 205] reviewed by [943, 516, 420, 599]. TRPML (mucolipin) familyThe TRPML family [729, 1049, 723, 1010, 173] consists of three mammalian members (TRPML1-3). TRPML channels are probably restricted to intracellular vesicles and mutations in the gene (MCOLN1) encoding TRPML1 (mucolipin-1) cause the neurodegenerative disorder mucolipidosis type IV (MLIV) in man. TRPML1 is a cation selective ion channel that is important for sorting/transport of endosomes in the late endocytotic pathway and specifically, fission from late endosome-lysosome hybrid vesicles and lysosomal exocytosis [766]. TRPML2 and TRPML3 show increased channel activity in low extracellular sodium and are activated by similar small molecules [293]. A naturally occurring gain of function mutation in TRPML3 (i.e. A419P) results in the varitint waddler (Va) mouse phenotype (reviewed by [729, 639]). TRPP (polycystin) familyThe TRPP family (reviewed by [197, 195, 275, 988, 345]) or PKD2 family is comprised of PKD2 (PC2), PKD2L1 (PC2L1), PKD2L2 (PC2L2), which have been renamed TRPP1, TRPP2 and TRPP3, respectively [999]. It should also be noted that the nomenclature of PC2 was TRPP2 in old literature. However, PC2 has been uniformed to be called TRPP2 [317]. PKD2 family channels are clearly distinct from the PKD1 family, whose function is unknown. PKD1 and PKD2 form a hetero-oligomeric complex with a 1:3 ratio. [845]. Although still being sorted out, TRPP family members appear to be 6TM spanning nonselective cation channels. TRPV (vanilloid) familyMembers of the TRPV family (reviewed by [928]) can broadly be divided into the non-selective cation channels, TRPV1-4 and the more calcium selective channels TRPV5 and TRPV6.TRPV1-V4 subfamilyTRPV1 is involved in the development of thermal hyperalgesia following inflammation and may contribute to the detection of noxius heat (reviewed by [710, 824, 860]). Numerous splice variants of TRPV1 have been described, some of which modulate the activity of TRPV1, or act in a dominant negative manner when co-expressed with TRPV1 [787]. The pharmacology of TRPV1 channels is discussed in detail in [303] and [947]. TRPV2 is probably not a thermosensor in man [684], but has recently been implicated in innate immunity [503]. TRPV3 and TRPV4 are both thermosensitive. There are claims that TRPV4 is also mechanosensitive, but this has not been established to be within a physiological range in a native environment [114, 488].TRPV5/V6 subfamily TRPV5 and TRPV6 are highly expressed in placenta, bone, and kidney. Under physiological conditions, TRPV5 and TRPV6 are calcium selective channels involved in the absorption and reabsorption of calcium across intestinal and kidney tubule epithelia (reviewed by [984, 185, 601, 248]).

LRRC8 family proteins on the plasma membrane play a critical role in cellular osmoregulation by forming volume-regulated anion channels (VRACs) necessary to prevent necrotic cell death. We demonstrate that intracellular LRRC8 proteins acting within lysosomes also play an essential role in cellular osmoregulation. LRRC8 proteins on lysosome membranes generate large lysosomal volume-regulated anion channel (Lyso-VRAC) currents in response to low cytoplasmic ionic strength conditions. When a double-leucine L(706)L(707) motif at the C terminus of LRRC8A was mutated to alanines, normal plasma membrane VRAC currents were still observed, but Lyso-VRAC currents were absent. We used this targeting mutant, as well as pharmacological tools, to demonstrate that Lyso-VRAC currents are necessary for the formation of large lysosome-derived vacuoles, which store and then expel excess water to maintain cytosolic water homeostasis. Thus, Lyso-VRACs allow lysosomes of mammalian cells to act as the cell`s “bladder.” When Lyso-VRAC current was selectively eliminated, the extent of necrotic cell death to sustained stress was greatly increased, not only in response to hypoosmotic stress, but also to hypoxic and hypothermic stresses. Thus Lyso-VRACs play an essential role in enabling cells to mount successful homeostatic responses to multiple stressors.

Total ceramide levels in cardiac tissue relate to cardiac dysfunction in animal models. However, emerging evidence suggests that the fatty acyl chain length of ceramides also impacts their relationship to cardiac function. This review explores evidence regarding the relationship between ceramides and left ventricular dysfunction and heart failure. It further explores possible mechanisms underlying these relationships.In large, community-based cohorts, a higher ratio of specific plasma ceramides, C16 : 0/C24 : 0, related to worse left ventricular dysfunction. Increased left ventricular mass correlated with plasma C16 : 0/C24 : 0, but this relationship became nonsignificant after adjustment for multiple comparisons. Decreased left atrial function and increased left atrial size also related to C16 : 0/C24 : 0. Furthermore, increased incident heart failure, overall cardiovascular disease (CVD) mortality and all-cause mortality were associated with higher C16 : 0/C24 : 0 (or lower C24 : 0/C16 : 0). Finally, a number of possible biological mechanisms are outlined supporting the link between C16 : 0/C24 : 0 ceramides, ceramide signalling and CVD.High cardiac levels of total ceramides are noted in heart failure. In the plasma, C16 : 0/C24 : 0 ceramides may be a valuable biomarker of preclinical left ventricular dysfunction, remodelling, heart failure and mortality. Continued exploration of the mechanisms underlying these profound relationships may help develop specific lipid modulators to combat cardiac dysfunction and heart failure.

The TRP superfamily of channels (nomenclature as agreed by NC-IUPHAR [159, 997]), whose founder member is the Drosophila Trp channel, exists in mammals as six families; TRPC, TRPM, TRPV, TRPA, TRPP and TRPML based on amino acid homologies. TRP subunits contain six putative TM domains and assemble as homo- or hetero-tetramers to form cation selective channels with diverse modes of activation and varied permeation properties (reviewed by [679]). Established, or potential, physiological functions of the individual members of the TRP families are discussed in detail in the recommended reviews and in a number of books [371, 635, 1064, 236]. The established, or potential, involvement of TRP channels in disease is reviewed in [412, 634] and [637], together with a special edition of Biochemica et Biophysica Acta on the subject [634]. Additional disease related reviews, for pain [585], stroke [1050], sensation and inflammation [919], itch [117], and airway disease [284, 977], are available. The pharmacology of most TRP channels has been advanced in recent years. Broad spectrum agents are listed in the tables along with more selective, or recently recognised, ligands that are flagged by the inclusion of a primary reference. See Rubaiy (2019) for a review of pharmacological tools for TRPC1/C4/C5 channels [751]. Most TRP channels are regulated by phosphoinostides such as PtIns(4,5)P2 although the effects reported are often complex, occasionally contradictory, and likely to be dependent upon experimental conditions, such as intracellular ATP levels (reviewed by [939, 638, 747]). Such regulation is generally not included in the tables.When thermosensitivity is mentioned, it refers specifically to a high Q10 of gating, often in the range of 10-30, but does not necessarily imply that the channel's function is to act as a 'hot' or 'cold' sensor. In general, the search for TRP activators has led to many claims for temperature sensing, mechanosensation, and lipid sensing. All proteins are of course sensitive to energies of binding, mechanical force, and temperature, but the issue is whether the proposed input is within a physiologically relevant range resulting in a response. TRPA (ankyrin) familyTRPA1 is the sole mammalian member of this group (reviewed by [268]). TRPA1 activation of sensory neurons contribute to nociception [382, 829, 555]. Pungent chemicals such as mustard oil (AITC), allicin, and cinnamaldehyde activate TRPA1 by modification of free thiol groups of cysteine side chains, especially those located in its amino terminus [529, 51, 336, 531]. Alkenals with α, β-unsaturated bonds, such as propenal (acrolein), butenal (crotylaldehyde), and 2-pentenal can react with free thiols via Michael addition and can activate TRPA1. However, potency appears to weaken as carbon chain length increases [23, 51]. Covalent modification leads to sustained activation of TRPA1. Chemicals including carvacrol, menthol, and local anesthetics reversibly activate TRPA1 by non-covalent binding [391, 470, 1005, 1004]. TRPA1 is not mechanosensitive under physiological conditions, but can be activated by cold temperatures [392, 193]. The electron cryo-EM structure of TRPA1 [688] indicates that it is a 6-TM homotetramer. Each subunit of the channel contains two short ‘pore helices’ pointing into the ion selectivity filter, which is big enough to allow permeation of partially hydrated Ca2+ ions. TRPC (canonical) familyMembers of the TRPC subfamily (reviewed by [261, 726, 15, 4, 84, 410, 687, 60]) fall into the subgroups outlined below. TRPC2 is a pseudogene in humans. It is generally accepted that all TRPC channels are activated downstream of Gq/11-coupled receptors, or receptor tyrosine kinases (reviewed by [713, 887, 997]). A comprehensive listing of G-protein coupled receptors that activate TRPC channels is given in [4]. Hetero-oligomeric complexes of TRPC channels and their association with proteins to form signalling complexes are detailed in [15] and [411]. TRPC channels have frequently been proposed to act as store-operated channels (SOCs) (or compenents of mulimeric complexes that form SOCs), activated by depletion of intracellular calcium stores (reviewed by [689, 15, 718, 764, 1037, 141, 675, 55, 142]). However, the weight of the evidence is that they are not directly gated by conventional store-operated mechanisms, as established for Stim-gated Orai channels. TRPC channels are not mechanically gated in physiologically relevant ranges of force. All members of the TRPC family are blocked by 2-APB and SKF96365 [319, 318]. Activation of TRPC channels by lipids is discussed by [60]. Important progress has been recently made in TRPC pharmacology [751, 571, 400, 92]. TRPC channels regulate a variety of physiological functions and are implicated in many human diseases [270, 61, 825, 958]. TRPC1/C4/C5 subgroup TRPC1 alone may not form a functional ion channel [210]. TRPC4/C5 may be distinguished from other TRP channels by their potentiation by micromolar concentrations of La3+. TRPC2 is a pseudogene in humans, but in other mammals appears to be an ion channel localized to microvilli of the vomeronasal organ. It is required for normal sexual behavior in response to pheromones in mice. It may also function in the main olfactory epithelia in mice [1034, 672, 673, 1035, 496, 1075, 1030].TRPC3/C6/C7 subgroup All members are activated by diacylglycerol independent of protein kinase C stimulation [319].TRPM (melastatin) familyMembers of the TRPM subfamily (reviewed by [252, 318, 689, 1062]) fall into the five subgroups outlined below. TRPM1/M3 subgroupIn darkness, glutamate released by the photoreceptors and ON-bipolar cells binds to the metabotropic glutamate receptor 6 , leading to activation of Go . This results in the closure of TRPM1. When the photoreceptors are stimulated by light, glutamate release is reduced, and TRPM1 channels are more active, resulting in cell membrane depolarization. Human TRPM1 mutations are associated with congenital stationary night blindness (CSNB), whose patients lack rod function. TRPM1 is also found melanocytes. Isoforms of TRPM1 may present in melanocytes, melanoma, brain, and retina. In melanoma cells, TRPM1 is prevalent in highly dynamic intracellular vesicular structures [368, 657]. TRPM3 (reviewed by [663]) exists as multiple splice variants which differ significantly in their biophysical properties. TRPM3 is expressed in somatosensory neurons and may be important in development of heat hyperalgesia during inflammation (see review [876]). TRPM3 is frequently coexpressed with TRPA1 and TRPV1 in these neurons. TRPM3 is expressed in pancreatic beta cells as well as brain, pituitary gland, eye, kidney, and adipose tissue [662, 875]. TRPM3 may contribute to the detection of noxious heat [947].TRPM2TRPM2 is activated under conditions of oxidative stress (respiratory burst of phagocytic cells) and ischemic conditions. However, the direct activators are ADPR(P) and calcium. As for many ion channels, PIP2 must also be present (reviewed by [1018]). Numerous splice variants of TRPM2 exist which differ in their activation mechanisms [219]. The C-terminal domain contains a TRP motif, a coiled-coil region, and an enzymatic NUDT9 homologous domain. TRPM2 appears not to be activated by NAD, NAAD, or NAADP, but is directly activated by ADPRP (adenosine-5'-O-disphosphoribose phosphate) [900]. TRPM2 is involved in warmth sensation [788], and contributes to neurological diseases [66]. Recent study shows that 2'-deoxy-ADPR is an endogenous TRPM2 superagonist [253]. TRPM4/5 subgroupTRPM4 and TRPM5 have the distinction within all TRP channels of being impermeable to Ca2+ [997]. A splice variant of TRPM4 (i.e.TRPM4b) and TRPM5 are molecular candidates for endogenous calcium-activated cation (CAN) channels [301]. TRPM4 is active in the late phase of repolarization of the cardiac ventricular action potential. TRPM4 deletion or knockout enhances beta adrenergic-mediated inotropy [546]. Mutations are associated with conduction defects [374, 546, 819]. TRPM4 has been shown to be an important regulator of Ca2+ entry in to mast cells [924] and dendritic cell migration [43]. TRPM5 in taste receptor cells of the tongue appears essential for the transduction of sweet, amino acid and bitter stimuli [494] TRPM5 contributes to the slow afterdepolarization of layer 5 neurons in mouse prefrontal cortex [471]. Both TRPM4 and TRPM5 are required transduction of taste stimuli [226].TRPM6/7 subgroupTRPM6 and 7 combine channel and enzymatic activities (‘chanzymes’). These channels have the unusual property of permeation by divalent (Ca2+, Mg2+, Zn2+) and monovalent cations, high single channel conductances, but overall extremely small inward conductance when expressed to the plasma membrane. They are inhibited by internal Mg2+ at ~0.6 mM, around the free level of Mg2+ in cells. Whether they contribute to Mg2+ homeostasis is a contentious issue. When either gene is deleted in mice, the result is embryonic lethality. The C-terminal kinase region is cleaved under unknown stimuli, and the kinase phosphorylates nuclear histones. TRPM7 is responsible for oxidant- induced Zn2+ release from intracellular vesicles [3] and contributes to intestinal mineral absorption essential for postnatal survival [574]. TRPM8Is a channel activated by cooling and pharmacological agents evoking a ‘cool’ sensation and participates in the thermosensation of cold temperatures [54, 161, 205] reviewed by [941, 516, 420, 599]. TRPML (mucolipin) familyThe TRPML family [729, 1047, 723, 1008, 173] consists of three mammalian members (TRPML1-3). TRPML channels are probably restricted to intracellular vesicles and mutations in the gene (MCOLN1) encoding TRPML1 (mucolipin-1) cause the neurodegenerative disorder mucolipidosis type IV (MLIV) in man. TRPML1 is a cation selective ion channel that is important for sorting/transport of endosomes in the late endocytotic pathway and specifically, fission from late endosome-lysosome hybrid vesicles and lysosomal exocytosis [765]. TRPML2 and TRPML3 show increased channel activity in low extracellular sodium and are activated by similar small molecules [293]. A naturally occurring gain of function mutation in TRPML3 (i.e. A419P) results in the varitint waddler (Va) mouse phenotype (reviewed by [729, 639]). TRPP (polycystin) familyThe TRPP family (reviewed by [197, 195, 275, 986, 345]) or PKD2 family is comprised of PKD2 (PC2), PKD2L1 (PC2L1), PKD2L2 (PC2L2), which have been renamed TRPP1, TRPP2 and TRPP3, respectively [997]. It should also be noted that the nomenclature of PC2 was TRPP2 in old literature. However, PC2 has been uniformed to be called TRPP2 [317]. PKD2 family channels are clearly distinct from the PKD1 family, whose function is unknown. PKD1 and PKD2 form a hetero-oligomeric complex with a 1:3 ratio. [843]. Although still being sorted out, TRPP family members appear to be 6TM spanning nonselective cation channels. TRPV (vanilloid) familyMembers of the TRPV family (reviewed by [926]) can broadly be divided into the non-selective cation channels, TRPV1-4 and the more calcium selective channels TRPV5 and TRPV6.TRPV1-V4 subfamilyTRPV1 is involved in the development of thermal hyperalgesia following inflammation and may contribute to the detection of noxius heat (reviewed by [710, 822, 858]). Numerous splice variants of TRPV1 have been described, some of which modulate the activity of TRPV1, or act in a dominant negative manner when co-expressed with TRPV1 [786]. The pharmacology of TRPV1 channels is discussed in detail in [303] and [945]. TRPV2 is probably not a thermosensor in man [684], but has recently been implicated in innate immunity [503]. TRPV3 and TRPV4 are both thermosensitive. There are claims that TRPV4 is also mechanosensitive, but this has not been established to be within a physiological range in a native environment [114, 488].TRPV5/V6 subfamily TRPV5 and TRPV6 are highly expressed in placenta, bone, and kidney. Under physiological conditions, TRPV5 and TRPV6 are calcium selective channels involved in the absorption and reabsorption of calcium across intestinal and kidney tubule epithelia (reviewed by [982, 185, 601, 248]).

International audience; The understanding of the electrophysiological mechanisms that underlie mechanosensitivity of the sinoatrial node (SAN), the primary pacemaker of the heart, has been evolving over the past century. The heart is constantly exposed to a dynamic mechanical environment; as such, the SAN has numerous canonical and emerging mechanosensitive ion channels and signaling pathways that govern its ability to respond to both fast (within second or on beat-to-beat manner) and slow (minutes) timescales. This review summarizes the effects of mechanical loading on the SAN activity and reviews putative candidates, including fast mechanoactivated channels (Piezo, TREK, and BK) and slow mechanoresponsive ion channels [including volume-regulated chloride channels and transient receptor potential (TRP)], as well as the components of mechanochemical signal transduction, which may contribute to SAN mechanosensitivity. Furthermore, we examine the structural foundation for both mechano-electrical and mechanochemical signal transduction and discuss the role of specialized membrane nanodomains, namely, caveolae, in mechanical regulation of both membrane and calcium clock components of the so-called coupled-clock pacemaker system responsible for SAN automaticity. Finally, we emphasize how these mechanically activated changes contribute to the pathophysiology of SAN dysfunction and discuss controversial areas necessitating future investigations. Though the exact mechanisms of SAN mechanosensitivity are currently unknown, identification of such components, their impact into SAN pacemaking, and pathological remodeling may provide new therapeutic targets for the treatment of SAN dysfunction and associated rhythm abnormalities.

The leucine-rich repeat-containing family 8 member A (LRRC8A) is an essential subunit of the volume-regulated anion channel (VRAC). VRAC is critical for cell volume control, but its broader physiological functions remain under investigation. Recent studies in the field indicate that Lrrc8a disruption in the brain astrocytes reduces neuronal excitability, impairs synaptic plasticity and memory, and protects against cerebral ischemia. In the present work, we generated brain-wide conditional LRRC8A knockout mice (LRRC8A bKO) using NestinCre -driven Lrrc8aflox/flox excision in neurons, astrocytes, and oligodendroglia. LRRC8A bKO animals were born close to the expected Mendelian ratio and developed without overt histological abnormalities, but, surprisingly, all died between 5 and 9 weeks of age with a seizure phenotype, which was confirmed by video and EEG recordings. Brain slice electrophysiology detected changes in the excitability of pyramidal cells and modified GABAergic inputs in the hippocampal CA1 region of LRRC8A bKO. LRRC8A-null hippocampi showed increased immunoreactivity of the astrocytic marker GFAP, indicating reactive astrogliosis. We also found decreased whole-brain protein levels of the GABA transporter GAT-1, the glutamate transporter GLT-1, and the astrocytic enzyme glutamine synthetase. Complementary HPLC assays identified reduction in the tissue levels of the glutamate and GABA precursor glutamine. Together, these findings suggest that VRAC provides vital control of brain excitability in mouse adolescence. VRAC deletion leads to a lethal phenotype involving progressive astrogliosis and dysregulation of astrocytic uptake and supply of amino acid neurotransmitters and their precursors.

Obesity is becoming a global epidemic, predisposing to Type 2 diabetes, cardiovascular disease, fatty liver disease, pulmonary disease, osteoarthritis and cancer. Therefore, understanding the biology of adipocyte expansion in response to overnutrition is critical to devising strategies to treat obesity, and the associated burden of morbidity and mortality. Through exploratory patch-clamp experiments in freshly isolated primary murine and human adipocytes, we recently determined that SWELL1/LRRC8a, a leucine-rich repeat containing transmembrane protein, functionally encoded an ion channel signalling complex (the volume-regulated anion channel, or VRAC) on the adipocyte plasma membrane. The SWELL1-/LRRC8 channel complex activates in response to increases in adipocyte volume and in the context of obesity. SWELL1 is also required for insulin-PI3K-AKT2 signalling to regulate adipocyte growth and systemic glycaemia. This commentary delves further into our working models for the molecular mechanisms of adipocyte SWELL1-mediated VRAC activation, proposed signal transduction mechanisms, and putative impact on adipocyte hypertrophy during caloric excess.

Perilipin 2 (PLIN2) is a lipid droplet (LD) protein in β cells that increases under nutritional stress. Downregulation of PLIN2 is often sufficient to reduce LD accumulation. To determine whether PLIN2 positively or negatively affects β cell function under nutritional stress, PLIN2 was downregulated in mouse β cells, INS1 cells, and human islet cells. β Cell–specific deletion of PLIN2 in mice on a high-fat diet reduced glucose-stimulated insulin secretion (GSIS) in vivo and in vitro. Downregulation of PLIN2 in INS1 cells blunted GSIS after 24-hour incubation with 0.2 mM palmitic acid. Downregulation of PLIN2 in human pseudoislets cultured at 5.6 mM glucose impaired both phases of GSIS, indicating that PLIN2 is critical for GSIS. Downregulation of PLIN2 decreased specific OXPHOS proteins in all 3 models and reduced oxygen consumption rates in INS1 cells and mouse islets. Moreover, we found that PLIN2-deficient INS1 cells increased the distribution of a fluorescent oleic acid analog to mitochondria and showed signs of mitochondrial stress, as indicated by susceptibility to fragmentation and alterations of acyl-carnitines and glucose metabolites. Collectively, PLIN2 in β cells has an important role in preserving insulin secretion, β cell metabolism, and mitochondrial function under nutritional stress.

Crystal structures activate innate immune cells, especially macrophages and initiate inflammatory responses. We aimed to understand the role of the mechanosensitive TRPV4 channel in crystal-induced inflammation. Real-time RT-PCR, RNAscope in situ hybridisation, and Trpv4eGFP mice were used to examine TRPV4 expression and whole-cell patch-clamp recording and live-cell Ca2+ imaging were used to study TRPV4 function in mouse synovial macrophages and human peripheral blood mononuclear cells (PBMCs). Both genetic deletion and pharmacological inhibition approaches were used to investigate the role of TRPV4 in NLRP3 inflammasome activation induced by diverse crystals in vitro and in mouse models of crystal-induced pain and inflammation in vivo. TRPV4 was functionally expressed by synovial macrophages and human PBMCs and TRPV4 expression was upregulated by stimulation with monosodium urate (MSU) crystals and in human PBMCs from patients with acute gout flares. MSU crystal-induced gouty arthritis were significantly reduced by either genetic ablation or pharmacological inhibition of TRPV4 function. Mechanistically, TRPV4 mediated the activation of NLRP3 inflammasome by diverse crystalline materials but not non-crystalline NLRP3 inflammasome activators, driving the production of inflammatory cytokine interleukin-1β which elicited TRPV4-dependent inflammatory responses in vivo. Moreover, chemical ablation of the TRPV1-expressing nociceptors significantly attenuated the MSU crystal-induced gouty arthritis. In conclusion, TRPV4 is a common mediator of inflammatory responses induced by diverse crystals through NLRP3 inflammasome activation in macrophages. TRPV4-expressing resident macrophages are critically involved in MSU crystal-induced gouty arthritis. A neuroimmune interaction between the TRPV1-expressing nociceptors and the TRPV4-expressing synovial macrophages contributes to the generation of acute gout flares.

Type 2 diabetes (T2D) is associated with insulin resistance, impaired insulin secretion from the pancreatic β-cell, and nonalcoholic fatty liver disease (NAFLD). SWELL1 (LRRC8a) ablation impairs adipose and skeletal muscle insulin-pAKT2 signaling, β-cell insulin secretion and glycemic control - suggesting that SWELL1-LRRC8 complex dysfunction contributes to T2D pathogenesis. Here, we show that ICl,SWELLand SWELL1 protein are reduced in adipose and β-cells in murine and human T2D. Combining cryo-electron microscopy, molecular docking, medicinal chemistry, and functional studies, we define a structure activity relationship to rationally-designed active derivatives (SN-40X) of a SWELL1 channel inhibitor (DCPIB/SN-401), that bind the SWELL1-LRRC8 hexameric complex, restore SWELL1-LRRC8 protein, plasma membrane trafficking, signaling and islet insulin secretion via SWELL1-dependent mechanisms.In vivo, SN-401 and active SN-40X compounds restore glycemic control and prevents NAFLD by improving insulin-sensitivity and insulin secretion in murine T2D. These findings demonstrate that small molecule SWELL1 modulators restore SWELL1-dependent insulin-sensitivity and insulin secretion in T2D and may represent a first-in-class therapeutic approach for T2D and NAFLD.

Perilipin 2 (PLIN2) is the lipid droplet (LD) protein in β cells that increases under nutritional stress. Down-regulation of PLIN2 is often sufficient to reduce LD accumulation. To determine whether PLIN2 positively or negatively affects β cell function under nutritional stress, PLIN2 was down-regulated in mouse β cells, INS1 cells, and human islet cells. β cell specific deletion of PLIN2 in mice on a high fat diet reduced glucose-stimulated insulin secretion (GSIS) in vivo and in vitro. Down-regulation of PLIN2 in INS1 cells blunted GSIS after 24 h incubation with 0.2 mM palmitic acids. Down-regulation of PLIN2 in human pseudoislets cultured at 5.6 mM glucose impaired both phases of GSIS, indicating that PLIN2 is critical for GSIS. Down-regulation of PLIN2 decreased specific OXPHOS proteins in all three models and reduced oxygen consumption rates in INS1 cells and mouse islets. Moreover, we found that PLIN2 deficient INS1 cells increased the distribution of a fluorescent oleic acid analog to mitochondria and showed signs of mitochondrial stress as indicated by susceptibility to fragmentation and alterations of acyl-carnitines and glucose metabolites. Collectively, PLIN2 in β cells have an important role in preserving insulin secretion, β cell metabolism and mitochondrial function under nutritional stress.

Calcium signals are initiated in immune cells by the process of store-operated calcium entry (SOCE), where receptor activation triggers transient calcium release from the endoplasmic reticulum, followed by opening of plasma-membrane calcium-release activated calcium (CRAC) channels. ORAI1, ORAI2, and ORAI3 are known to comprise the CRAC channel; however, the contributions of individual isoforms to neutrophil function are not well understood. Here, we show that loss of ORAI1 partially decreases calcium influx, while loss of both ORAI1 and ORAI2 completely abolishes SOCE. In other immune-cell types, loss of ORAI2 enhances SOCE. In contrast, we find that ORAI2-deficient neutrophils display decreased calcium influx, which is correlated with measurable differences in the regulation of neutrophil membrane potential via KCa3.1. Decreased SOCE in ORAI1-, ORAI2-, and ORAI1/2-deficient neutrophils impairs multiple neutrophil functions, including phagocytosis, degranulation, leukotriene, and reactive oxygen species (ROS) production, rendering ORAI1/2-deficient mice highly susceptible to staphylococcal infection. This study demonstrates that ORAI1 and ORAI2 are the primary components of the neutrophil CRAC channel and identifies subpopulations of neutrophils where cell-membrane potential functions as a rheostat to modulate the SOCE response. These findings have implications for mechanisms that modulate neutrophil function during infection, acute and chronic inflammatory conditions, and cancer.

Maintenance of skeletal muscle is beneficial in obesity and Type 2 diabetes. Mechanical stimulation can regulate skeletal muscle differentiation, growth and metabolism; however, the molecular mechanosensor remains unknown. Here, we show that SWELL1 (Lrrc8a) functionally encodes a swell-activated anion channel that regulates PI3K-AKT, ERK1/2, mTOR signaling, muscle differentiation, myoblast fusion, cellular oxygen consumption, and glycolysis in skeletal muscle cells. LRRC8A over-expression in Lrrc8a KO myotubes boosts PI3K-AKT-mTOR signaling to supra-normal levels and fully rescues myotube formation. Skeletal muscle-targeted Lrrc8a KO mice have smaller myofibers, generate less force ex vivo, and exhibit reduced exercise endurance, associated with increased adiposity under basal conditions, and glucose intolerance and insulin resistance when raised on a high-fat diet, compared to wild-type (WT) mice. These results reveal that the LRRC8 complex regulates insulin-PI3K-AKT-mTOR signaling in skeletal muscle to influence skeletal muscle differentiation in vitro and skeletal myofiber size, muscle function, adiposity and systemic metabolism in vivo., eLife digest Skeletal muscles – the force-generating tissue attached to bones – must maintain their mass and health for the body to work properly. It is therefore necessary to understand how an organism can regulate the way skeletal muscles form, grow and heal. A multitude of factors can control how muscles form, including mechanical signals. The molecules that can sense these mechanical stimuli, however, remain unknown. Mechanoresponsive ion channels provide possible candidates for these molecular sensors. These proteins are studded through the cell membranes, where they can respond to mechanical changes by opening and allowing the flow of ions in and out of a cell, or by changing interactions with other proteins. The SWELL1 protein is a component of an ion channel known as VRAC, which potentially responds to mechanical stimuli. This channel is associated with many biological processes such as cells multiplying, migrating, growing and dying, but it is still unclear how. Here, Kumar et al. first tested whether SWELL1 controls how skeletal muscle precursors mature into their differentiated and functional form. These experiments showed that SWELL1 regulates this differentiation process under the influence of the hormone insulin, as well as mechanical signals such as cell stretching. In addition, this work revealed that SWELL1 relies on an adaptor molecule called GRB2 to relay these signals in the cell. Next, Kumar et al. genetically engineered mice lacking SWELL1 only in skeletal muscle. These animals had smaller muscle cells, as well as muscles that were weaker and less enduring. When raised on a high-calorie diet, the mutant mice also got more obese and developed resistance to insulin, which is an important step driving obesity-induced diabetes. Together, these findings show that SWELL1 helps to regulate the formation and function of muscle cells, and highlights how an ion channel participates in these processes. Healthy muscles are key for overall wellbeing, as they also protect against obesity and obesity-related conditions such as type 2 diabetes or nonalcoholic fatty liver disease. This suggests that targeting SWELL1 could prove advantageous in a clinical setting.

Maintenance of skeletal muscle is beneficial in obesity and Type 2 diabetes. Mechanical stimulation can regulate skeletal muscle differentiation, growth and metabolism, however the molecular mechanosensor remains unknown. Here, we show that SWELL1 (LRRC8a) functionally encodes a swell-activated anion channel that regulates PI3K-AKT, ERK1/2, mTOR signaling, muscle differentiation, myoblast fusion, cellular oxygen consumption, and glycolysis in skeletal muscle cells. SWELL1 over-expression in SWELL1 KO myotubes boosts PI3K-AKT-mTOR signaling to supra-normal levels and fully rescues myotube formation. Skeletal muscle targeted SWELL1 KO mice have smaller myofibers, generate less forceex vivo, and exhibit reduced exercise endurance, associated with increased adiposity under basal conditions, and glucose intolerance and insulin resistance when raised on a high-fat diet, compared to WT mice. These results reveal that the SWELL1-LRRC8 complex regulates insulin-PI3K-AKT-mTOR signalling in skeletal muscle to influence skeletal muscle differentiationin vitroand skeletal myofiber size, muscle function, adiposity and systemic metabolismin vivo.

Volume-regulated anion channels (VRACs) are critically involved in regulating cell volume, and leucine-rich repeat-containing protein 8A (LRRC8A, SWELL1) is an obligatory subunit of VRACs. Cell swelling occurs early after brain ischemia, but it is unclear whether neuronal LRRC8a contributes to ischemia-induced glutamate release and brain injury. We found that Lrrc8a conditional knockout (Lrrc8a-cKO) mice produced by crossing NestinCre+/− with Lrrc8aflox+/+ mice died 7–8 weeks of age, indicating an essential role of neuronal LRRC8A for survival. Middle cerebral artery occlusion (MCAO) caused an early increase in LRRC8A protein levels in the hippocampus in wild-type (WT) mice. Whole-cell patch-clamp recording in brain slices revealed that oxygen-glucose deprivation significantly increased the amplitude of VRAC currents in hippocampal CA1 neurons in WT but not in Lrrc8a-cKO mice. Hypotonicity increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in hippocampal CA1 neurons in WT mice, and this was abolished by DCPIB, a VRAC blocker. But in Lrrc8a-cKO mice, hypotonic solution had no effect on the frequency of sEPSCs in these neurons. Furthermore, the brain infarct volume and neurological severity score induced by MCAO were significantly lower in Lrrc8a-cKO mice than in WT mice. In addition, MCAO-induced increases in cleaved caspase-3 and calpain activity, two biochemical markers of neuronal apoptosis and death, in brain tissues were significantly attenuated in Lrrc8a-cKO mice compared with WT mice. These new findings indicate that cerebral ischemia increases neuronal LRRC8A-dependent VRAC activity and that VRACs contribute to increased glutamatergic input to hippocampal neurons and brain injury caused by ischemic stroke.

Insulin secretion is initiated by activation of voltage-gated Ca2+ channels (VGCC) to trigger Ca2+-mediated insulin vesicle fusion with the β-cell plasma membrane. The firing of VGCC requires β-cell membrane depolarization, which is regulated by a balance of depolarizing and hyperpolarizing ionic currents. Here, we show that SWELL1 mediates a swell-activated, depolarizing chloride current (ICl,SWELL) in both murine and human β-cells. Hypotonic and glucose-stimulated β-cell swelling activates SWELL1-mediated ICl,SWELL and this contributes to membrane depolarization and activation of VGCC-dependent intracellular calcium signaling. SWELL1 depletion in MIN6 cells and islets significantly impairs glucose-stimulated insulin secretion. Tamoxifen-inducible β-cell-targeted Swell1 KO mice have normal fasting serum glucose and insulin levels but impaired glucose-stimulated insulin secretion and glucose tolerance; and this is further exacerbated in mild obesity. Our results reveal that β-cell SWELL1 modulates insulin secretion and systemic glycaemia by linking glucose-mediated β-cell swelling to membrane depolarization and activation of VGCC-triggered calcium signaling., Insulin secretion by β-cells is stimulated by glucose and is dependent on the induction of β-cell membrane depolarization, mainly driven by the closure of KATP channels, which in turn promotes voltage-gated Ca2+ channel opening. Here Kang et al. show that the volume-regulated anion channel, SWELL1, is involved in glucose-stimulated calcium increase and insulin secretion.

Cardiac macrophages represent a heterogeneous cell population with distinct origins, dynamics, and functions. Recent studies have revealed that C-C Chemokine Receptor 2 positive (CCR2(+)) macrophages derived from infiltrating monocytes regulate myocardial inflammation and heart failure pathogenesis. Comparatively little is known about the functions of tissue resident (CCR2(−)) macrophages. Herein, we identified an essential role for CCR2(−) macrophages in the chronically failing heart. Depletion of CCR2(−) macrophages in mice with dilated cardiomyopathy accelerated mortality and impaired ventricular remodeling and coronary angiogenesis, adaptive changes necessary to maintain cardiac output in the setting of reduced cardiac contractility. Mechanistically, CCR2(−) macrophages interacted with neighboring cardiomyocytes via focal adhesion complexes and were activated in response to mechanical stretch through a transient receptor potential vanilloid 4 (TRPV4) dependent pathway that controlled growth factor expression. These findings establish a role for tissue resident macrophages in adaptive cardiac remodeling and implicate mechanical sensing in cardiac macrophage activation.

Pancreatic beta cells synthesize and secrete the neurotransmitter GABA (gamma-aminobutyric acid) as a paracrine and autocrine signal to help regulate hormone secretion and islet homeostasis. Islet GABA release has classically been described as a secretory-vesicle-mediated event. Yet, a limitation of the hypothesized vesicular GABA release from islets is the lack of expression of a vesicular GABA transporter in beta cells. Consequentially, GABA accumulates in the cytosol. Here, we provide evidence that the human beta cell effluxes GABA from a cytosolic pool in a pulsatile manner, imposing a synchronizing rhythm on pulsatile insulin secretion. The volume regulatory anion channel, functionally encoded by LRRC8A or Swell1, is critical for pulsatile GABA secretion. GABA content in beta cells is depleted and secretion is disrupted in islets from patients with type 1 and type 2 diabetes, suggesting that loss of GABA as a synchronizing signal for hormone output may correlate with diabetes pathogenesis.

The TRP superfamily of channels (nomenclature as agreed by NC-IUPHAR [145, 915]), whose founder member is the Drosophila Trp channel, exists in mammals as six families; TRPC, TRPM, TRPV, TRPA, TRPP and TRPML based on amino acid homologies. TRP subunits contain six putative transmembrane domains and assemble as homo- or hetero-tetramers to form cation selective channels with diverse modes of activation and varied permeation properties (reviewed by [630]). Established, or potential, physiological functions of the individual members of the TRP families are discussed in detail in the recommended reviews and in a number of books [344, 589, 979, 216]. The established, or potential, involvement of TRP channels in disease is reviewed in [384, 588] and [591], together with a special edition of Biochemica et Biophysica Acta on the subject [588]. Additional disease related reviews, for pain [542], stroke [967], sensation and inflammation [843], itch [109], and airway disease [261, 896], are available. The pharmacology of most TRP channels has been advanced in recent years. Broad spectrum agents are listed in the tables along with more selective, or recently recognised, ligands that are flagged by the inclusion of a primary reference. See Rubaiy (2019) for a review of pharmacological tools for TRPC1/C4/C5 channels [692]. Most TRP channels are regulated by phosphoinostides such as PtIns(4,5)P2 although the effects reported are often complex, occasionally contradictory, and likely to be dependent upon experimental conditions, such as intracellular ATP levels (reviewed by [862, 592, 689]). Such regulation is generally not included in the tables.When thermosensitivity is mentioned, it refers specifically to a high Q10 of gating, often in the range of 10-30, but does not necessarily imply that the channel's function is to act as a 'hot' or 'cold' sensor. In general, the search for TRP activators has led to many claims for temperature sensing, mechanosensation, and lipid sensing. All proteins are of course sensitive to energies of binding, mechanical force, and temperature, but the issue is whether the proposed input is within a physiologically relevant range resulting in a response. TRPA (ankyrin) familyTRPA1 is the sole mammalian member of this group (reviewed by [246]). TRPA1 activation of sensory neurons contribute to nociception [356, 763, 516]. Pungent chemicals such as mustard oil (AITC), allicin, and cinnamaldehyde activate TRPA1 by modification of free thiol groups of cysteine side chains, especially those located in its amino terminus [491, 47, 311, 493]. Alkenals with α, β-unsaturated bonds, such as propenal (acrolein), butenal (crotylaldehyde), and 2-pentenal can react with free thiols via Michael addition and can activate TRPA1. However, potency appears to weaken as carbon chain length increases [21, 47]. Covalent modification leads to sustained activation of TRPA1. Chemicals including carvacrol, menthol, and local anesthetics reversibly activate TRPA1 by non-covalent binding [364, 438, 923, 922]. TRPA1 is not mechanosensitive under physiological conditions, but can be activated by cold temperatures [365, 175]. The electron cryo-EM structure of TRPA1 [639] indicates that it is a 6-TM homotetramer. Each subunit of the channel contains two short ‘pore helices’ pointing into the ion selectivity filter, which is big enough to allow permeation of partially hydrated Ca2+ ions. TRPC (canonical) familyMembers of the TRPC subfamily (reviewed by [239, 673, 14, 4, 79, 382, 638, 55]) fall into the subgroups outlined below. TRPC2 is a pseudogene in humans. It is generally accepted that all TRPC channels are activated downstream of Gq/11-coupled receptors, or receptor tyrosine kinases (reviewed by [661, 814, 915]). A comprehensive listing of G-protein coupled receptors that activate TRPC channels is given in [4]. Hetero-oligomeric complexes of TRPC channels and their association with proteins to form signalling complexes are detailed in [14] and [383]. TRPC channels have frequently been proposed to act as store-operated channels (SOCs) (or compenents of mulimeric complexes that form SOCs), activated by depletion of intracellular calcium stores (reviewed by [640, 14, 665, 703, 954, 132, 626, 51, 133]). However, the weight of the evidence is that they are not directly gated by conventional store-operated mechanisms, as established for Stim-gated Orai channels. TRPC channels are not mechanically gated in physiologically relevant ranges of force. All members of the TRPC family are blocked by 2-APB and SKF96365 [295, 294]. Activation of TRPC channels by lipids is discussed by [55]. Important progress has been recently made in TRPC pharmacology [692, 529, 372, 87]. TRPC channels regulate a variety of physiological functions and are implicated in many human diseases [248, 56, 759, 879]. TRPC1/C4/C5 subgroup TRPC1 alone may not form a functional ion channel [191]. TRPC4/C5 may be distinguished from other TRP channels by their potentiation by micromolar concentrations of La3+. TRPC2 is a pseudogene in humans, but in other mammals appears to be an ion channel localized to microvilli of the vomeronasal organ. It is required for normal sexual behavior in response to pheromones in mice. It may also function in the main olfactory epithelia in mice [951, 625, 624, 952, 462, 988, 947].TRPC3/C6/C7 subgroup All members are activated by diacylglycerol independent of protein kinase C stimulation [295].TRPM (melastatin) familyMembers of the TRPM subfamily (reviewed by [230, 294, 640, 978]) fall into the five subgroups outlined below. TRPM1/M3 subgroupIn darkness, glutamate released by the photoreceptors and ON-bipolar cells binds to the metabotropic glutamate receptor 6 , leading to activation of Go . This results in the closure of TRPM1. When the photoreceptors are stimulated by light, glutamate release is reduced, and TRPM1 channels are more active, resulting in cell membrane depolarization. Human TRPM1 mutations are associated with congenital stationary night blindness (CSNB), whose patients lack rod function. TRPM1 is also found melanocytes. Isoforms of TRPM1 may present in melanocytes, melanoma, brain, and retina. In melanoma cells, TRPM1 is prevalent in highly dynamic intracellular vesicular structures [341, 609]. TRPM3 (reviewed by [615]) exists as multiple splice variants which differ significantly in their biophysical properties. TRPM3 is expressed in somatosensory neurons and may be important in development of heat hyperalgesia during inflammation (see review [803]). TRPM3 is frequently coexpressed with TRPA1 and TRPV1 in these neurons. TRPM3 is expressed in pancreatic beta cells as well as brain, pituitary gland, eye, kidney, and adipose tissue [614, 802]. TRPM3 may contribute to the detection of noxious heat [870].TRPM2TRPM2 is activated under conditions of oxidative stress (respiratory burst of phagocytic cells) and ischemic conditions. However, the direct activators are ADPR(P) and calcium. As for many ion channels, PIP2 must also be present (reviewed by [935]). Numerous splice variants of TRPM2 exist which differ in their activation mechanisms [200]. The C-terminal domain contains a TRP motif, a coiled-coil region, and an enzymatic NUDT9 homologous domain. TRPM2 appears not to be activated by NAD, NAAD, or NAADP, but is directly activated by ADPRP (adenosine-5'-O-disphosphoribose phosphate) [827]. TRPM2 is involved in warmth sensation [724], and contributes to neurological diseases [61]. Recent study shows that 2'-deoxy-ADPR is an endogenous TRPM2 superagonist [231]. TRPM4/5 subgroupTRPM4 and TRPM5 have the distinction within all TRP channels of being impermeable to Ca2+ [915]. A splice variant of TRPM4 (i.e.TRPM4b) and TRPM5 are molecular candidates for endogenous calcium-activated cation (CAN) channels [278]. TRPM4 is active in the late phase of repolarization of the cardiac ventricular action potential. TRPM4 deletion or knockout enhances beta adrenergic-mediated inotropy [507]. Mutations are associated with conduction defects [347, 507, 753]. TRPM4 has been shown to be an important regulator of Ca2+ entry in to mast cells [847] and dendritic cell migration [39]. TRPM5 in taste receptor cells of the tongue appears essential for the transduction of sweet, amino acid and bitter stimuli [460] TRPM5 contributes to the slow afterdepolarization of layer 5 neurons in mouse prefrontal cortex [439]. Both TRPM4 and TRPM5 are required transduction of taste stimuli [206].TRPM6/7 subgroupTRPM6 and 7 combine channel and enzymatic activities (‘chanzymes’). These channels have the unusual property of permeation by divalent (Ca2+, Mg2+, Zn2+) and monovalent cations, high single channel conductances, but overall extremely small inward conductance when expressed to the plasma membrane. They are inhibited by internal Mg2+ at ~0.6 mM, around the free level of Mg2+ in cells. Whether they contribute to Mg2+ homeostasis is a contentious issue. When either gene is deleted in mice, the result is embryonic lethality. The C-terminal kinase region is cleaved under unknown stimuli, and the kinase phosphorylates nuclear histones. TRPM7 is responsible for oxidant- induced Zn2+ release from intracellular vesicles [3] and contributes to intestinal mineral absorption essential for postnatal survival [532]. TRPM8Is a channel activated by cooling and pharmacological agents evoking a ‘cool’ sensation and participates in the thermosensation of cold temperatures [50, 147, 186] reviewed by [864, 481, 391, 556]. TRPML (mucolipin) familyThe TRPML family [676, 964, 670, 926, 156] consists of three mammalian members (TRPML1-3). TRPML channels are probably restricted to intracellular vesicles and mutations in the gene (MCOLN1) encoding TRPML1 (mucolipin-1) cause the neurodegenerative disorder mucolipidosis type IV (MLIV) in man. TRPML1 is a cation selective ion channel that is important for sorting/transport of endosomes in the late endocytotic pathway and specifically, fission from late endosome-lysosome hybrid vesicles and lysosomal exocytosis [704]. TRPML2 and TRPML3 show increased channel activity in low extracellular sodium and are activated by similar small molecules [270]. A naturally occurring gain of function mutation in TRPML3 (i.e. A419P) results in the varitint waddler (Va) mouse phenotype (reviewed by [676, 593]). TRPP (polycystin) familyThe TRPP family (reviewed by [179, 177, 252, 905, 320]) or PKD2 family is comprised of PKD2 (PC2), PKD2L1 (PC2L1), PKD2L2 (PC2L2), which have been renamed TRPP1, TRPP2 and TRPP3, respectively [915]. It should also be noted that the nomenclature of PC2 was TRPP2 in old literature. However, PC2 has been uniformed to be called TRPP2 [293]. PKD2 family channels are clearly distinct from the PKD1 family, whose function is unknown. PKD1 and PKD2 form a hetero-oligomeric complex with a 1:3 ratio. [775]. Although still being sorted out, TRPP family members appear to be 6TM spanning nonselective cation channels. TRPV (vanilloid) familyMembers of the TRPV family (reviewed by [849]) can broadly be divided into the non-selective cation channels, TRPV1-4 and the more calcium selective channels TRPV5 and TRPV6.TRPV1-V4 subfamilyTRPV1 is involved in the development of thermal hyperalgesia following inflammation and may contribute to the detection of noxius heat (reviewed by [660, 756, 786]). Numerous splice variants of TRPV1 have been described, some of which modulate the activity of TRPV1, or act in a dominant negative manner when co-expressed with TRPV1 [722]. The pharmacology of TRPV1 channels is discussed in detail in [280] and [868]. TRPV2 is probably not a thermosensor in man [635], but has recently been implicated in innate immunity [469]. TRPV3 and TRPV4 are both thermosensitive. There are claims that TRPV4 is also mechanosensitive, but this has not been established to be within a physiological range in a native environment [106, 454].TRPV5/V6 subfamily TRPV5 and TRPV6 are highly expressed in placenta, bone, and kidney. Under physiological conditions, TRPV5 and TRPV6 are calcium selective channels involved in the absorption and reabsorption of calcium across intestinal and kidney tubule epithelia (reviewed by [901, 168, 558, 227]).

BACKGROUND: Atrial fibrillation (AF) is associated with elevated risk for ischemic stroke and myocardial infarction (MI). The aim of the study is to assess the role of insulin use on the risk of stroke and MI in AF patients with diabetes. METHODS: We identified Medicare beneficiaries with new AF in 2011-2013. Primary outcomes were ischemic stroke and MI. Multivariate Cox regression models were used to assess the association between AF and time to stroke and MI. We adjusted for anticoagulant as a time-dependent covariate. RESULTS: Out of 798,592 AF patients, 53212 (6.7%) were insulin-requiring diabetics (IRD), 250214 (31.3%) were non-insulin requiring diabetics (NIRD) and 495166 (62%) were non-diabetics (ND). IRD had a higher risk of stroke when compared to NIRD (adjusted HR: 1.15, 95% CI 1.10-1.21) and ND (aHR 1.24, 95% CI 1.18-1.31) (P

Obesity is associated with a loss of insulin-sensitivity and systemic dysglycemia, resulting in Type 2 diabetes, however the molecular mechanisms underlying this association are unclear. Through adipocyte patch-clamp studies, we recently showed that SWELL1 is required for the Volume-Regulated Anion Current (VRAC) in adipocytes and that SWELL1-mediated VRAC is activated by both mechanical and pathophysiological adipocyte expansion. We also demonstrated that adipocyte SWELL1 is required for maintaining insulin signaling and glucose homeostasis, particularly in the setting of obesity. Here we show that SWELL1 protein expression is induced in subcutaneous fat, visceral fat and liver in the setting of obesity. Long- term AAV/rec2-shRNA mediated SWELL1 knock-down in both fat and liver are associated with increased weight gain, increased adiposity and exacerbated insulin resistance in mice raised on a high-fat diet. These data further support the notion that SWELL1 induction occurs in insulin- sensitive tissues (liver and adipose) in the setting of over-nutrition and contributes to improved systemic glycemia by supporting enhanced insulin-sensitivity.

TRPM7 (transient receptor potential cation channel subfamily M member 7) is required for normal organ development but also mediates anoxic neuronal death. TRPM7 contains a channel that conducts cations into the cytosol and C-terminal kinase that can phosphorylate multiple substrates. The kinase is cleaved to regulate gene expression and apoptosis. The link between TRPM7’s channel and its organismal function remains the least understood aspect of TRPM7. Here, we identify intracellular Zn2+ storage vesicles that contain the majority of TRPM7 protein. TRPM7 senses reactive oxygen species (ROS) to release Zn2+ from these vesicles. Just as the endoplasmic reticulum sequesters and releases Ca2+, we propose that these vesicles fulfill a similar function for Zn2+ and that TRPM7 coordinates fluctuations in cellular [Zn2+] and ROS during development and injury.

Significance TRPM7 (transient receptor potential cation channel subfamily M member 7) is required for normal organ development but also mediates anoxic neuronal death. TRPM7 contains a channel that conducts cations into the cytosol and C-terminal kinase that can phosphorylate multiple substrates. The kinase is cleaved to regulate gene expression and apoptosis. The link between TRPM7’s channel and its organismal function remains the least understood aspect of TRPM7. Here, we identify intracellular Zn 2+ storage vesicles that contain the majority of TRPM7 protein. TRPM7 senses reactive oxygen species (ROS) to release Zn 2+ from these vesicles. Just as the endoplasmic reticulum sequesters and releases Ca 2+ , we propose that these vesicles fulfill a similar function for Zn 2+ and that TRPM7 coordinates fluctuations in cellular [Zn 2+ ] and ROS during development and injury.

Insulin secretion from the pancreatic β-cell initiated by activation of voltage-gated Ca2+ channels (VGCC) to trigger Ca2+-mediated insulin vesicle fusion with the β-cell plasma membrane. The firing of VGCC depends on the β-cell membrane potential, which is in turn mediated by the balance of depolarizing (excitatory) and hyperpolarizing (inhibitory) ionic currents1-3. While much attention has focused on inhibitory potassium currents4-10 there is little knowledge about the excitatory currents required to depolarize the β-cell, including the molecular identity of these excitatory currents3. Here we show that SWELL1 (LRRC8a) mediates a swell-activated, depolarizing chloride current (ICl,SWELL) in β-cells. Hypotonic and glucose-stimulated β-cell swelling activates SWELL1-mediated ICl,SWELL and this is required for both glucose-stimulated and hypotonic swell-mediated activation of VGCC-dependent intracellular calcium signaling in β-cells. SWELL1 KO MIN6 cells and β-cell targeted SWELL1 KO murine islets exhibit significantly impaired glucose-stimulated insulin secretion, with preserved insulin content in vitro. Tamoxifen-inducible β-cell targeted SWELL1 KO mice have normal fasting insulin levels but display markedly impaired glucose-stimulated insulin secretion. Our results reveal a physiological role for SWELL1 as a glucose sensor - linking glucose-mediated β-cell swelling to SWELL1-dependent activation of VGCC-triggered calcium signaling, and highlights SWELL1-mediated “swell-secretion” coupling as required for glucose-stimulated insulin secretion.

The leucine rich repeat containing protein 8A (LRRC8A), or SWELL1, is an essential component of the volume-regulated anion channel (VRAC) that is activated by cell swelling and ionic strength. We report here for the first time to our knowledge its expression in a primary cell culture of nodose ganglia neurons and its localization in the soma, neurites, and neuronal membrane. We show that this neuronal VRAC/SWELL1 senses low external pH (pHo) in addition to hypoosmolarity. A robust sustained chloride current is seen in 77% of isolated nodose neurons following brief exposures to extracellular acid pH. Its activation involves proton efflux, intracellular alkalinity, and an increase in NOX-derived H2O2. The molecular identity of both the hypoosmolarity-induced and acid pHo-conditioned VRAC as LRRC8A (SWELL1) was confirmed by Cre-flox-mediated KO, shRNA-mediated knockdown, and CRISPR/Cas9-mediated LRRC8A deletion in HEK cells and in primary nodose neuronal cultures. Activation of VRAC by low pHo reduces neuronal injury during simulated ischemia and N-methyl-D-aspartate-induced (NMDA-induced) apoptosis. These results identify the VRAC (LRRC8A) as a dual sensor of hypoosmolarity and low pHo in vagal afferent neurons and define the mechanisms of its activation and its neuroprotective potential.

The patch-clamp technique allows for the study of ion channel activity in the native adipocyte environment to better understand the contributions of ion channels to adipocyte signaling. Here, we describe methods for isolating primary mature adipocytes from both mouse and human white adipose tissues (subcutaneous and visceral). From the same preparation, we describe how to culture and differentiate preadipocytes isolated from the stromal vascular fraction. We then describe in detail patch-clamp methods, including both whole-cell and perforated-patch configurations.

Background— Transient receptor potential ( TRP ) channels are a superfamily of broadly expressed ion channels with diverse physiological roles. TRPC1, TRPC3 , and TRPC6 are believed to contribute to cardiac hypertrophy in mouse models. Human mutations in TRPM4 have been linked to progressive familial heart block. TRPM7 is a divalent-permeant channel and kinase of unknown function, recently implicated in the pathogenesis of atrial fibrillation; however, its function in ventricular myocardium remains unexplored. Methods and Results— We generated multiple cardiac-targeted knockout mice to test the hypothesis that TRPM7 is required for normal ventricular function. Early cardiac Trpm7 deletion (before embryonic day 9; TnT / Isl1-C re) results in congestive heart failure and death by embryonic day 11.5 as a result of hypoproliferation of the compact myocardium. Remarkably, Trpm7 deletion late in cardiogenesis (about embryonic day 13; αMHC-Cre ) produces viable mice with normal adult ventricular size, function, and myocardial transcriptional profile. Trpm7 deletion at an intermediate time point results in 50% of mice developing cardiomyopathy associated with heart block, impaired repolarization, and ventricular arrhythmias. Microarray analysis reveals elevations in transcripts of hypertrophy/remodeling genes and reductions in genes important for suppressing hypertrophy ( Hdac9) and for ventricular repolarization ( Kcnd2 ) and conduction ( Hcn4 ). These transcriptional changes are accompanied by action potential prolongation and reductions in transient outward current ( I to ; Kcnd2 ). Similarly, the pacemaker current ( I f ; Hcn4 ) is suppressed in atrioventricular nodal cells, accounting for the observed heart block. Conclusions— Trpm7 is dispensable in adult ventricular myocardium under basal conditions but is critical for myocardial proliferation during early cardiogenesis. Loss of Trpm7 at an intermediate developmental time point alters the myocardial transcriptional profile in adulthood, impairing ventricular function, conduction, and repolarization.

A 60-year-old man without significant past medical history presented with 1 month of gradually increasing dyspnea on exertion, associated with generalized fatigue and pedal edema. He denied orthopnea, paroxysmal nocturnal dyspnea, chest pain, fever, cough, or weight loss. One month before this presentation, he could walk for miles and ride a road bike without difficulty. His social history was notable for 45 pack-year smoking and intravenous drug abuse. He worked as a refuse collector and roof mechanic for 2 to 3 years. Vital signs on presentation revealed a blood pressure 97/72 mm Hg, pulse 112 beats per minute, temperature 36.9°C, and respiratory rate of 24 breaths per minute. The oxygen saturation was 98% on room air. Cardiovascular examination revealed mild jugular venous distention to 10 cm, distant heart sounds with no murmurs, an impalpable apical impulse, and 2+ bilateral lower extremity edema. Pulmonary examination was unremarkable with clear breath sounds on auscultation bilaterally. Abdominal and neurological examination was unremarkable. Laboratory testing revealed negative troponins and mildly positive D-Dimer at 0.93 μg/mL. Although N-terminal pro b-type natriuretic peptide was elevated at 1382 pg/mL, a 2-view chest radiograph showed normal cardio-mediastinal silhouette and pulmonary vasculature. An ECG on admission showed sinus tachycardia with a rate of 110 beats per minute, right axis deviation, low-voltage complexes, and T wave inversions in leads V4, V5, and V6. Based on examination findings and initial testing, a computed tomographic …

Adipocytes undergo considerable volumetric expansion in the setting of obesity. It has been proposed that such marked increases in adipocyte size may be sensed via adipocyte-autonomous mechanisms to mediate size-dependent intracellular signalling. Here, we show that SWELL1 (LRRC8a), a member of the Leucine-Rich Repeat Containing protein family, is an essential component of a volume-sensitive ion channel (VRAC) in adipocytes. We find that SWELL1-mediated VRAC is augmented in hypertrophic murine and human adipocytes in the setting of obesity. SWELL1 regulates adipocyte insulin-PI3K-AKT2-GLUT4 signalling, glucose uptake and lipid content via SWELL1 C-terminal leucine-rich repeat domain interactions with GRB2/Cav1. Silencing GRB2 in SWELL1 KO adipocytes rescues insulin-pAKT2 signalling. In vivo, shRNA-mediated SWELL1 knockdown and adipose-targeted SWELL1 knockout reduce adiposity and adipocyte size in obese mice while impairing systemic glycaemia and insulin sensitivity. These studies identify SWELL1 as a cell-autonomous sensor of adipocyte size that regulates adipocyte growth, insulin sensitivity and glucose tolerance.