Abstract: Neuronal activity results in release of K+ into the extracellular space of the central nervous system. If the excess K+ is allowed to accumulate, neuronal firing will be compromised by the ensuing neuronal membrane depolarization. The surrounding glial cells are involved in clearing K+ from the extracellular space by molecular mechanism(s), the identity of which have been a matter of controversy for over half a century. Kir4.1-mediated spatial buffering of K+ has been promoted as a major contributor to K+ removal although its quantitative and temporal contribution has remained undefined. We discuss the biophysical and experimental challenges regarding determination of the contribution of Kir4.1 to extracellular K+ management during neuronal activity. It is concluded that 1) the geometry of the experimental preparation is crucial for detection of Kir4.1-mediated spatial buffering and 2) Kir4.1 enacts spatial buffering of K+ during but not after neuronal activity.

Abstract: Death of murine T cells induced by extracellular ATP is mainly triggered by activation of purinergic P2X 7 receptors (P2X 7Rs). However, a link between P2X 7Rs and pannexin1 (Panx1) channels, which are non-selective, has been recently demonstrated in other cell types. In this work, we characterized the expression and cellular distribution of pannexin family members (Panxs 1, 2 and 3) in isolated T cells. Panx1 was the main pannexin family member clearly detected in both helper (CD4+) and cytotoxic (CD8+) T cells, whereas low levels of Panx2 were found in both T-cell subsets. Using pharmacological and genetic approaches, Panx1 channels were found to mediate most ATP-induced ethidium uptake since this was drastically reduced by Panx1 channel blockers (10Panx1, Probenecid and low carbenoxolone concentration) and absent in T cells derived from Panx1-/- mice. Moreover, electrophysiological measurements in wild-type CD4+ cells treated with ATP unitary current events and pharmacological sensitivity compatible with Panx1 channels were found. In addition, ATP release from T cells treated with 4Br-A23187, a calcium ionophore, was completely blocked with inhibitors of both connexin hemichannels and Panx1 channels. Panx1 channel blockers drastically reduced the ATP-induced T-cell mortality, indicating that Panx1 channels mediate the ATP-induced T-cell death. However, mortality was not reduced in T cells of Panx1-/- mice, in which levels of P2X 7Rs and ATP-induced intracellular free Ca2+ responses were enhanced suggesting that P2X 7Rs take over Panx1 channels lose-function in mediating the onset of cell death induced by extracellular ATP.

Abstract: The DEG/ENaC gene family of ion channels is characterized by a high degree of structural similarity and an equally high degree of diversity concerning the physiological function. In humans and rodents, the DEG/ENaC family comprises 2 main subgroups: the subunits of the epithelial Na+ channel (ENaC) and the subunits of the acid sensing ion channels (ASICs). The bile acid-sensitive channel (BASIC), previously known as BLINaC or INaC, represents a third subgroup within the DEG/ENaC family. Although BASIC was identified more than a decade ago, very little is known about its physiological function. Recent progress in the characterization of this neglected member of the DEG/ENaC family, which is summarized in this focused review, includes the discovery of surprising species differences, its pharmacological characterization, and the identification of bile acids as putative natural activators.

Abstract: Eukaryotic voltage-gated sodium channels (VGSCs) are essential for the initiation and propagation of action potentials in electrically excitable cells, and are important pharmaceutical targets for the treatment of neurological disorders such as epilepsy, cardiac arrhythmias, and chronic pain Evidence suggests that small, hydrophobic, VGSC-blocking drugs can gain access to binding residues within the central cavity of these channels by passing through lateral, lipid-filled “fenestrations” which run between the exterior of the protein and its central pore Here, we use molecular dynamics simulations to investigate how the size and shape of fenestrations change over time in several bacterial VGSC models and a homology model of Nav14 We show that over the course of the simulations, the size of the fenestrations is primarily influenced by rapid protein motions, such as amino acid side-chain rotation, and highlight that differences between fenestration bottleneck-contributing residues are the primary cause of variations in fenestration size between the 6 bacterial models In the eukaryotic channel model, 2 fenestrations are wide, but 2 are narrow due to differences in the amino acid sequence in the 4 domains Lipid molecules are found to influence the size of the fenestrations by protruding acyl chains into the fenestrations and displacing amino acid side-chains Together, the results suggest that fenestrations provide viable pathways for small, flexible, hydrophobic drugs

Abstract: Genetic studies have linked alterations in Kir7.1 channel to diverse pathologies. We summarize functional relevance of Kir7.1 channel in retinal pigment epithelium (RPE), regulation of channel function by various cytoplasmic metabolites, and mutations that cause channelopathies. At the apical membrane of RPE, K(+) channels contribute to subretinal K(+) homeostasis and support Na(+)/K(+) pump and Na(+)-K(+)-2Cl(-) cotransporter function by providing a pathway for K(+) secretion. Electrophysiological studies have established that barium- and cesium-sensitive inwardly rectifying K(+) (Kir) channels make up a major component of the RPE apical membrane K(+) conductance. Native human RPE expresses transcripts for Kir1.1, Kir2.1, Kir2.2, Kir3.1, Kir3.4, Kir4.2, and Kir6.1, albeit at levels at least 50-fold lower than Kir7.1. Kir7.1 is structurally similar to other Kir channels, consisting of 2 trans-membrane domains, a pore-forming loop that contains the selectivity filter, and 2 cytoplasmic polar tails. Within the cytoplasmic structure, clusters of amino acid sequences form regulatory domains that interact with cellular metabolites and control the opening and closing of the channel. Recent evidence indicated that intrinsic sequence motifs present in Kir7.1 control surface expression. Mutant Kir7.1 channels are associated with inherited eye pathologies such as Snowflake Vitreoretinal Degeneration (SVD) and Lebers Congenital Amaurosis (LCA16). Based on the current evidence, mutations implicated in channelopathies have the potential to be used for genetic testing to diagnose blindness due to Kir7.1.

Abstract: The acid-sensitive outwardly rectifying (ASOR) anion channel has been found in non-neuronal cell types and was shown to be involved in acidotoxic death of epithelial cells. We have recently shown t...

Abstract: As exceptionally calcium selective store-operated channels, Orai channels play a prominent role in cellular calcium signaling. While most studied in the immune system, we are beginning to recognize...

Abstract: The presence of Ca2+-activated Cl– currents (ICl(Ca)) in vascular smooth muscle cells (VSMCs) is well established. ICl(Ca) are supposedly important for arterial contraction by linking changes in [Ca2+]i and membrane depolarization. Bestrophins and some members of the TMEM16 protein family were recently associated with ICl(Ca). Two distinct ICl(Ca) are characterized in VSMCs; the cGMP-dependent ICl(Ca) dependent upon bestrophin expression and the ‘classical’ Ca2+-activated Cl– current, which is bestrophin-independent. Interestingly, TMEM16A is essential for both the cGMP-dependent and the classical ICl(Ca). Furthermore, TMEM16A has a role in arterial contraction while bestrophins do not. TMEM16A’s role in the contractile response cannot be explained however only by a simple suppression of the depolarization by Cl– channels. It is suggested that TMEM16A expression modulates voltage-gated Ca2+ influx in a voltage-independent manner and recent studies also demonstrate a complex role of TMEM16A in modulating other membrane proteins.

Abstract: Connexin40 (Cx40) and connexin43 (Cx43) are co-expressed in the cardiovascular system, yet their ability to form functional heterotypic Cx43/Cx40 gap junctions remains controversial. We paired Cx43 or Cx40 stably-transfected N2a cells to examine the formation and biophysical properties of heterotypic Cx43/Cx40 gap junction channels. Dual whole cell patch clamp recordings demonstrated that Cx43 and Cx40 form functional heterotypic gap junctions with asymmetric transjunctional voltage (Vj) dependent gating properties. The heterotypic Cx43/Cx40 gap junctions exhibited less Vj gating when the Cx40 cell was positive and pronounced gating when negative. Endogenous N2a cell connexin expression levels were 1,000-fold lower than exogenously expressed Cx40 and Cx43 levels, measured by real-time PCR and Western blotting methods, suggestive of heterotypic gap junction formation by exogenous Cx40 and Cx43. Imposing a [KCl] gradient across the heterotypic gap junction modestly diminished the asymmetry of the macroscopic normalized junctional conductance - voltage (Gj-Vj) curve when [KCl] was reduced by 50% on the Cx43 side and greatly exacerbated the Vj gating asymmetries when lowered on the Cx40 side. Pairing wild-type (wt) Cx43 with the Cx40 E9,13K mutant protein produced a nearly symmetrical heterotypic Gj-Vj curve. These studies conclusively demonstrate the ability of Cx40 and Cx43 to form rectifying heterotypic gap junctions, owing primarily to alternate amino-terminal (NT) domain acidic and basic amino acid differences that may play a significant role in the physiology and/or pathology of the cardiovascular tissues including cardiac conduction properties and myoendothelial intercellular communication.

Abstract: In organisms from all kingdoms of life, ammonia and its conjugated ion ammonium are transported across membranes by proteins of the AMT/Rh family. Efficient and successful growth often depends on sufficient ammonium nutrition. The proteins mediating this transport, the so called Ammonium Transporter (AMT) or Rhesus like (Rh) proteins, share a very similar trimeric overall structure and a high sequence similarity even throughout the kingdoms. Even though structural components of the transport mechanism, like an external substrate recruitment site, an essential twin histidine pore motif, a phenylalanine gate and the hydrophobic pore are strongly conserved and have been analyzed in detail by molecular dynamic simulations and mutational studies, the substrate(s), which pass the central pores of the AMT/Rh subunits, NH4+, NH3 + H+, NH4+ + H+ or NH3, are still a matter of debate for most proteins, including the best characterized AmtB protein from Escherichia coli. The lack of a robust expression system for fun...

Abstract: In pancreatic β-cells, KATP channels consisting of Kir6.2 and SUR1 couple cell metabolism to membrane excitability and regulate insulin secretion. Sulfonylureas, insulin secretagogues used to treat...

Abstract: Mechanosensitive (MS) ion channels are molecular sensors that detect and transduce signals across prokaryotic and eukaryotic cell membranes arising from external mechanical stimuli or osmotic gradients. They play an integral role in mechanosensory responses including touch, hearing, and proprioception by opening or closing in order to facilitate or prevent the flow of ions and organic osmolytes. In this study we use a linear force model of MS channel gating to determine the gating membrane tension (γ) and the gating area change (ΔA) associated with the energetics of MscS channel gating in giant spheroplasts and azolectin liposomes. Analysis of Boltzmann distribution functions describing the dependence of MscS channel gating on membrane tension indicated that the gating area change (ΔA) was the same for MscS channels recorded in both preparations. The comparison of the membrane tension (γ) gating the channel, however, showed a significant difference between the MscS channel activities in these two preparations.

Abstract: According to live-cell calcium-imaging experiments, the Sec61 complex is a passive calcium-leak channel in the human endoplasmic reticulum (ER) membrane that is regulated by ER luminal immunoglobulin heavy chain binding protein (BiP) and cytosolic Ca2+-calmodulin. In single channel measurements, the open Sec61 complex is Ca2+ permeable. It can be closed not only by interaction with BiP or Ca2+-calmodulin, but also with Pseudomonas aeruginosa Exotoxin A which can enter human cells by retrograde transport. Exotoxin A has been shown to interact with the Sec61 complex and, thereby, inhibit ER export of immunogenic peptides into the cytosol. Here, we show that Exotoxin A also inhibits passive Ca2+ leakage from the ER in human cells, and we characterized the N-terminus of the Sec61 α-subunit as the relevant binding site for Exotoxin A.

Abstract: Novel immune function for the TRPV1 channel in T lymphocytes Samuel Bertin, Petrus Rudolf de Jong, Wilfred A Jefferies & Eyal Raz a Department of Medicine, UCSD; La Jolla, CA USA b Sanford-Burnham Medical Research Institute (SBMRI); La Jolla, CA USA c Michael Smith Laboratories, Center for Blood Research; The Brain Research Center; Department of Medical Genetics: Department of Microbiology and Immunology and Department of Zoology; University of British Columbia; Vancouver, Canada Accepted author version posted online: 21 Dec 2014.Published online: 23 Jan 2015.

Abstract: Control of plasma membrane connexin hemichannel opening is indispensable, and is achieved by physiological extracellular divalent ion concentrations. Here, we explore the differences between regula...

Abstract: Transient outward K+ current (Ito) plays a crucial role in the early phase of cardiac action potential repolarization. Kv4.3 K+ channel is an important component of Ito. The function and expression...

Abstract: Acid-sensing ion channels (ASICs) are proton-gated cation channels that are widely expressed in both the peripheral and central nervous systems. ASICs contribute to a variety of pathophysiological conditions that involve tissue acidosis, such as ischemic stroke, epileptic seizures and multiple sclerosis. Although much progress has been made in researching the structure-function relationship and pharmacology of ASICs, little is known about the trafficking of ASICs and its contribution to ASIC function. The recent identification of the mechanism of membrane insertion and endocytosis of ASIC1a highlights the emerging role of ASIC trafficking in regulating its pathophysiological functions. In this review, we summarize the recent advances and discuss future directions on this topic.

Abstract: Nervous system (NS) activity participates in metabolic homeostasis by detecting peripheral signal molecules derived from food intake and energy balance. High quality diets are thought to include fiber-rich foods like whole grain rice, breads, cereals, and grains. Several studies have associated high consumption of fiber-enriched diets with a reduced risk of diabetes, obesity, and gastrointestinal disorders. In the lower intestine, anaerobic fermentation of soluble fibers by microbiota produces short chain fatty acids (SCFAs), key energy molecules that have a recent identified leading role in the intestinal gluconeogenesis, promoting beneficial effects on glucose tolerance and insulin resistance. SCFAs are also signaling molecules that bind to specific G-protein coupled receptors (GPCRs) named Free Fatty Acid Receptor 3 (FFA3, GPR41) and 2 (FFA2, GPR43). However, how SCFAs impact NS activity through their GPCRs is poorly understood. Recently, studies have demonstrated the presence of FFA2 and FFA3 in the sympathetic NS of rat, mouse and human. Two studies have showed that FFA3 activation by SCFAs increases firing and norepinephrine (NE) release from sympathetic neurons. However, the recent study from the Ikeda Laboratory revealed that activation of FFA3 by SCFAs impairs N-type calcium channel (NTCC) activity, which contradicts the idea of FFA3 activation leading to increased action potential evoked NE release. Here we will discuss the scope of the latter study and the putative physiological role of SCFAs and FFAs in the sympathetic NS.

Abstract: Many physiological processes such as cell division, endocytosis and exocytosis cause severe local curvature of the cell membrane. Local curvature has been shown experimentally to modulate numerous mechanosensitive (MS) ion channels. In order to quantify the effects of local curvature we introduced a coarse grain representative volume element for the bacterial mechanosensitive ion channel of large conductance (MscL) using continuum elasticity. Our model is designed to be consistent with the channel conformation in the closed and open states to capture its major continuum rheological behavior in response to the local membrane curvature. Herein we show that change in the local curvature of the lipid bilayer can modulate MscL activity considerably by changing both bilayer thickness and lateral pressure profile. Intriguingly, although bending in any direction results in almost the same free-energy cost, inward (cytoplasmic) bending favors channel opening, whereas outward (periplasmic) bending facilitates closing of the narrowest part of the MscL pore. This quantitative study using MscL as a model channel may have wide reaching consequences for the effect of local curvature on the physiological function of other types of prokaryotic and eukaryotic membrane proteins.

Abstract: An increasing number of studies have implicated that the activation of innate immune system and inflammatory mechanisms are of importance in the pathogenesis of numerous diseases. The innate immune system is present in almost all multicellular organisms in response to pathogens or tissue injury, which is performed via germ-line encoded pattern-recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) or dangers-associated molecular patterns (DAMPs). Intracellular pathways linking immune and inflammatory response to ion channel expression and function have been recently identified. Among ion channels, transient receptor potential (TRP) channels are a major family of non-selective cation-permeable channels that function as polymodal cellular sensors involved in many physiological and pathological processes. In this review, we summarize current knowledge about classifications, functions, and interactions of TRP channels and PRRs, which may provide new insights into their roles in the pathogenesis of inflammatory diseases.

Abstract: We recently reported gating currents recorded from hERG channels expressed in mammalian TSA cells and assessed the kinetics at different voltages. We detected 2 distinct components of charge movement with the bulk of the charge being carried by a slower component. Here we compare our findings in TSA cells with recordings made from oocytes using the Cut Open Vaseline Gap clamp (COVG) and go on to directly compare activation of gating charge and ionic currents at 0 and +60 mV. The data show that gating charge saturates and moves more rapidly than ionic current activates suggesting a transition downstream from the movement of the bulk of gating charge is rate limiting for channel opening.

Abstract: A diffusion barrier segregates the plasma membrane of the rod photoreceptor outer segment into 2 domains; one which is optimized for the conductance of ions in the phototransduction cascade and another for disk membrane synthesis. We propose the former to be named “phototransductive plasma membrane domain," and the latter to be named “disk morphogenic plasma membrane domain." Within the phototransductive plasma membrane, cGMP-gated channels are concentrated in striated membrane features, which are proximally located to the sites of active cGMP production within the disk membranes. For proper localization of cGMP-gated channel to the phototransductive plasma membrane, the glutamic acid-rich protein domain encoded in the β subunit plays a critical role. Quantitative study suggests that the disk morphogenic domain likely plays an important role in enriching rhodopsin prior to its sequestration into closed disk membranes. Thus, this and our previous studies provide new insight into the mechanism that spatiall...

Abstract: We demonstrated previously that BK (KCa1.1) channel activity (NPo) increases in response to bisphenol A (BPA). Moreover, BK channels containing regulatory β1 subunits were more sensitive to the stimulatory effect of BPA. How BPA increases BK channel NPo remains mostly unknown. Estradiol activates BK channels by binding to an extracellular site, but neither the existence nor location of a BPA binding site has been demonstrated. We tested the hypothesis that an extracellular binding site is responsible for activation of BK channels by BPA. We synthesized membrane-impermeant BPA-monosulfate (BPA-MS) and used patch clamp electrophysiology to study channels composed of α or α + β1 subunits in cell-attached (C-A), whole-cell (W-C), and inside-out (I-O) patches. In C-A patches, bath application of BPA-MS (100 μM) had no effect on the NPo of BK channels, regardless of their subunit composition. Importantly, however, subsequent addition of membrane-permeant BPA (100 μM) increased the NPo of both α and α + β1 channels in C-A patches. The C-A data indicate that in order to alter BK channel NPo, BPA must interact with the channel itself (or some closely associated partner) and diffusible messengers are not involved. In W-C patches, 100 μM BPA-MS activated current in cells expressing α subunits, whereas cells expressing α + β1 subunits responded similarly to a log-order lower concentration (10 μM). The W-C data suggest that an extracellular activation site exists, but do not eliminate the possibility that an intracellular site may also be present. In I-O patches, where the cytoplasmic face was exposed to the bath, BPA-MS had no effect on the NPo of BK α subunits, but BPA increased it. BPA-MS increased the NPo of α + β1 channels in I-O patches, but not as much as BPA. We conclude that BPA activates BK α via an extracellular site and that BPA-sensitivity is increased by the β1 subunit, which may also constitute part of an intracellular binding site.

Abstract: Acid-sensing ion channels (ASICs) are proton-sensitive, sodium-selective channels expressed in the nervous system that sense changes in extracellular pH. These ion channels are sensitive to an increasing number of nonproton ligands that include natural venom peptides and guanidine compounds. In the case of chicken ASIC1, the spider toxin Psalmotoxin-1 (PcTx1) activates the channel, resulting in an inward current. Furthermore, a growing class of ligands containing a guanidine group has been identified that stimulate peripheral ASICs (ASIC3), but exert subtle influence on other ASIC subtypes. The effects of the guanidine compounds on cASIC1 have not been the focus of previous study. Here, we investigated the interaction of the guanidine compound 2-guanidine-4-methylquinazoline (GMQ) on cASIC1 proton activation and PcTx1 stimulation. Exposure of expressed cASIC1 to PcTx1 resulted in biphasic currents consisting of a transient peak followed by an irreversible cASIC1 PcTx1 persistent current. This cASIC1 PcTx1 persistent current may be the result of locking the cASIC1 protein into a desensitized transition state. The guanidine compound GMQ increased the apparent affinity of protons on cASIC1 and decreased the half-maximal constant of the cASIC1 steady-state desensitization profile. Furthermore, GMQ stimulated the cASIC1 PcTx1 persistent current in a concentration-dependent manner, which resulted in a non-desensitizing inward current. Our data suggests that GMQ may have multiple sites within cASIC1 and may act as a "molecular wedge" that forces the PcTx1-desensitized ASIC into an open state. Our findings indicate that guanidine compounds, such as GMQ, may alter acid-sensing ion channel activity in combination with other stimuli, and that additional ASIC subtypes (along with ASIC3) may serve to sense and mediate signals from multiple stimuli.

Abstract: In order to shuttle substrates across the lipid bilayer, membrane proteins undergo a series of conformation changes that are influenced by protein structure, ligands, and the lipid environment. To test the effect of lipid on conformation change of the ABC transporter MolBC, EPR studies were conducted in lipids and detergents of variable composition. In both a detergent and lipid environment, MolBC underwent the same general conformation changes as detected by site-directed EPR spectroscopy. However, differences in activity and the details of the EPR analysis indicate conformational rigidity that is dependent on the lipid environment. From these observations, we conclude that native-like lipid mixtures provide the transporter with greater activity and conformational flexibility as well as technical advantages such as reconstitution efficiency and protein stability.

Abstract: We reported recently a new mechanism by which the neuronal N-type Ca2+ (CaV2.2) channel expression may be regulated by ubiquitination. This mechanism involves the interaction between the channel and the light chain (LC1) of the microtubule associated protein B (MAP1B). We also showed that MAP1B-LC1 could interact with the ubiquitin-conjugating E2 enzyme UBE2L3 and that the ubiquitination/degradation mechanism triggered by MAP1B-LC1 could be prevented by inhibiting the ubiquitin-proteasome proteolytic pathway. We now report that MAP1B-LC1 can interact with the 2 main variants of the CaV2.2 channels (CaV2.2e37a and CaV2.2e37b) and that the MAP1B-LC1-mediated regulation most likely involves an internalization of the channels via a dynamin and clathrin-dependent pathway. In addition, here we propose that this novel mechanism of CaV channel regulation might be conserved among N-type and P/Q-type channels.

Abstract: Small conductance Ca2+-sensitive potassium (SK2) channels are voltage-independent, Ca2+-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and syn...

Abstract: In the retina, the firing behaviors that ganglion cells exhibit when exposed to light stimuli are very important due to the significant roles they play in encoding the visual information. However, the detailed mechanisms, especially the intrinsic properties that generate and modulate these firing behaviors is not completely clear yet. In this study, 2 typical firing behaviors—i.e., tonic and phasic activities, which are widely observed in retinal ganglion cells (RGCs)—are investigated. A modified computational model was developed to explore the possible ionic mechanisms that underlie the generation of these 2 firing patterns. Computational results indicate that the generation of tonic and phasic activities may be attributed to the collective actions of 2 kinds of adaptation currents, i.e., an inactivating sodium current and a delayed-rectifier potassium current. The concentration of magnesium ions has crucial but differential effects in the modulation of tonic and phasic firings, when the model neuron is driven by N-methyl-D-aspartate (NMDA) -type synaptic input instead of constant current injections. The proposed model has robust features that account for the ionic mechanisms underlying the tonic and phasic firing behaviors, and it may also be used as a good candidate for modeling some other firing patterns in RGCs.

Abstract: Pannexins (Panxs) are a multifaceted family of ion and metabolite channels that play key roles in a number of physiological and pathophysiological settings. These single membrane large-pore channels exhibit a variety of tissue, cell type, and subcellular distributions. The lifecycles of Panxs are complex, yet must be understood to accurately target these proteins for future therapeutic use. Here we review the basics of Panx function and localization, and then analyze the recent advances in knowledge regarding Panx trafficking. We examine several intrinsic features of Panxs including specific post-translational modifications, the divergent C-termini, and oligomerization, all of which contribute to Panx anterograde transport pathways. Further, we examine the potential influence of extrinsic factors, such as protein-protein interactions, on Panx trafficking. Finally, we highlight what is currently known with respect to Panx internalization and retrograde transport, and present new data illustrating Panx1 internalization following an activating stimulus.