Inside-out Patches Research Articles

Cloning and characterization of the rat Slo3 (KCa 5.1) channel: From biophysics to pharmacology.

The Slo3 potassium (KCa 5.1) channel, which is specifically expressed in the testis and sperm, is essential for mammalian male fertilization. The sequence divergence of the bovine, mouse and human Slo3 α-subunit revealed a rapid evolution rate across different species. The rat Slo3 (rSlo3) channel has not been cloned and characterized previously. We used molecular cloning, electrophysiology (inside-out patches and outside-out patches) and mutagenesis to investigate the biophysical properties and pharmacological characteristics of the rSlo3 channel. The rat Slo3 channel (rSlo3) is gated by voltage and cytosolic pH rather than intracellular calcium. The characteristics of voltage-dependent, pH-sensitivity and activation kinetics of the rSlo3 channel differ from the characteristics of other Slo3 orthologues. In terms of pharmacology, the 4-AP blockade of the rSlo3 channel also shows properties distinct from its blockade of the mSlo3 channel. Iberiotoxin and progesterone weakly inhibit the rSlo3 channel. Finally, we found that propofol, one of the widely used general anaesthetics, blocks the rSlo3 channel from both intracellular and extracellular sides, whereas ketamine only blocks the rSlo3 channel at the extracellular side. Our findings suggest that the rSlo3 channel possesses unique biophysical and pharmacological properties. Our results provide new insights into the diversities of the Slo3 family of channels, which are valuable for estimating the effects of the use of these drugs to improve sperm quality.

EAG Channel PAS Domain Binder Inhibits Currents from EAG Channels and Decreases Tumor Growth in Zebrafish Xenograft Model

Ether-a-go-go (EAG) potassium selective channels regulate neuronal excitability and tumorigenesis. Inhibition of EAG channel currents has been shown to decrease cancer progression. EAG channels contain a Per-Arn-Sim (PAS) domain in their N-terminal region. The PAS domain is structurally similar to the PAS domains in other non-ion channel proteins, where these domains frequently function as ligand-binding domains. Despite the structural similarity, small molecule binders of the EAG PAS domain have not been identified. Using surface-plasmon resonance (SPR) based high-throughput screen we identified an EAG PAS domain small molecule binder which binds to the PAS domain in a concentration dependent manner. Application of the identified small molecule binder to the excised inside-out membrane patches expressing EAG channels inhibited currents recorded from EAG channels with an IC50 of 4.09 + 0.61 μM. Deletion of the PAS domain dramatically decreased the inhibition of EAG channel currents by the identified small molecule ligand, indicating that the effect on EAG currents is directly caused by binding of the ligand to the PAS domain. To investigate the therapeutic potential of the identified ligand we used zebrafish xenograft model. Treatment with the identified ligand decreased tumor growth in zebrafish implanted with cell lines known to overexpress EAG channels, while had statistically insignificant effect on tumor growth in zebrafish implanted with a cell line that does not overexpress EAG channels. Taken together, our study indicates that the PAS domain can function as a ligand-binding domain in EAG channels. Importantly, the identified ligand is an FDA approved drug. Thus, our study presents a case for repurposing the identified ligand for cancer treatment as an EAG channel inhibitor.

Toluene-Induced Inhibition of Smooth Muscle BK Channels and Middle Cerebral Artery Constriction

Signs and symptoms of toluene acute intoxication overlap with those of cerebral ischemia. Moreover, computed tomography shows that toluene acute intoxication causes brain hypoperfusion (Ryu et al., 1998). The targets underlying toluene-induced cerebrovascular ischemia are unknown. Using a cranial window in male adult rats, we demonstrate that acute toluene exposure (<10 mM) constricts middle cerebral arteries (MCA). This action is replicated in in vitro-pressurized MCA segments, underscoring that circulating factors are unnecessary. A similar outcome in de-endothelialized MCA indicates that toluene action is endothelium-independent but likely involves targets present in the MCA smooth muscle (SM). In this tissue, inhibition of Ca2+i/voltage-gated K+ channels of large conductance (BK) leads to vasoconstriction (Brayden & Nelson, 1992), raising the hypothesis that SM BK mediates toluene-induced MCA constriction. Indeed, pharmacological BK block with 1 μM paxilline ablates toluene-induced constriction of isolated MCAs. MCA myocyte BK complexes include channel-forming α (cbv1) and regulatory β1 subunits (Dopico et al., 2018). Toluene applied to inside-out patches from MCA SM cells reduces BK activity (NPo), this reduction being more robust in KCNMB1-/- than C57BL/6 mouse myocytes. Likewise, toluene-induced channel inhibition is more robust in cbv1 vs. cbv1+β1 expressed in CHO cells. Toluene inhibition of cbv1 channels reconstituted into 3:1 (w/w) POPE/POPS bilayers does not require Ca2+i but is modulated by this ion, reaching a maximum at ∼10 μM Ca2+i. Therefore, toluene at concentrations reached in blood during acute intoxication inhibits cerebrovascular SM BKs in absence of Ca2+i and β1 subunits, yet these two regulators of channel function fine-tune toluene action. Finally, truncation of cbv1 after S6 blunts toluene inhibition of cbv1 reconstituted into 3:1 (w/w) POPE/POPS bilayers. Data suggest that cbv1 is the primary target that mediates toluene-induced MCA constriction. Support: UTHSC Office of Research NGS.

Voltage-Gated Proton Channels Exist in the Plasma Membrane of Human Oocytes

Voltage gated proton channels (HV1) are highly expressed in human (but not mouse) sperm where they contribute to capacitation and motility (Lishko et al. 2010. Cell. 140:327; Musset et al. 2012. J. Biol. Chem. 287:9376). We investigated whether HV1 are also present in human oocytes. Frozen, thawed human metaphase II oocytes were obtained from material donated to the gamete repository at the Rush Center for Advanced Reproductive Care. Oocytes were retrieved by transvaginal ovarian follicle aspiration following treatment with recombinant follicle stimulating hormone and purified urinary gonadotropins with GnRH antagonist for controlled ovarian hyperstimulation. After sufficient 18-20 mm follicles were visualized by transvaginal ultrasound, human chorionic gonadotropin was administered to initiate the ovulatory cascade. Transvaginal aspiration of the follicles under ultrasound guidance was performed 36 h later. Human oocytes are large ∼110 μm diameter spheres surrounded by a thick extracellular glycoprotein matrix (the zona pellucida). Their size and zona pellucida make oocytes difficult to study by conventional patch-clamp techniques. To obtain an electrically tight seal we exposed bare oolemma by removing sections of the zona pellucida using a laser, followed by repeated pipetting to further separate the oocyte from the zona pellucida. Patch-clamp studies were performed using whole-cell configuration on oocytes, vesicles derived from oocytes, or inside-out patches of membrane, under conditions optimized for voltage-gated proton currents. Proton currents were present at significant levels in human oocytes where they exhibit properties similar to those reported in other human cells, as well as those in heterologous expression systems transfected with the HVCN1 gene. The gating kinetics, voltage dependence, and selectivity were indistinguishable from those of hHV1 in heterologous expression systems. Possible functions for voltage gated proton channels in human oocytes, most likely involving pH regulation, may now be contemplated.

Both Lobes of Calmodulin Bound to KCa2.2 Respond to Ca2+

An increase in intracellular Ca2+ opens KCa2.2 Ca2+-activated K+-channels (SK) to conduct K+ down its electrochemical gradient. SK activation requires bound calmodulin proteins to decode dynamic Ca2+ fluctuations in a cell. Bilobal calmodulin has four Ca2+ binding sites. A common model proposes that just the N-terminal domain (N-lobe) of calmodulin binds Ca2+ to open SK, while an immobile C-terminal domain (C-lobe) has constitutive, Ca2+-independent binding. Such a mechanism is compatible with x-ray and EM structures of intact SK channels and fragments. While molecular structures provide snapshots of the channels in different conformations with and without calcium, gating is a dynamic process involving several different conformations, calcium binding to calmodulin, interactions between calmodulin and the channel and channel opening. We measured SK activity using inside-out patch recordings from “calmodulin-disrupted” SK channels where calcium-dependent activation can be restored by exogenously applied full-length calmodulin. Isolated N and C lobes have little ability to activate SK. We measured calmodulin binding to a C-terminal SK2 peptide (SKp) using both composition-gradient multi-angle light scattering and tryptophan emission spectra. While they do not rescue activation, isolated lobes do bind to a SK cytoplasmic domain with high affinity. Consistent with earlier models, N-lobe binding to SKp is stronger in Ca2+, and C-lobe binding affinity is strong regardless of Ca2+. However, a native tryptophan in SKp is sensitive to Ca2+ binding to either the N- or C-lobe of calmodulin at Ca2+ concentrations that activate SK. Our results show that the calmodulin C-lobe bound to SK is Ca2+ sensitive and that both lobes of calmodulin bind SK in Ca2+-dependent states governed by the different properties of each lobe.

Dynamic Regulation of Bicarbonate Permeability through CFTR Channel by WNK1

Increase in relative HCO3− permeability (PHCO3/PCl) of cystic fibrosis transmembrane conductance regulator (CFTR) channel by[Cl−]i-sensitive kinases is essential for pancreatic HCO3− secretion. Defects in pancreatic HCO3− secretion caused by CFTR mutations are associated with cystic fibrosis and pancreatitis. However, the molecular mechanisms by which [Cl−]i-sensitive kinases regulate CFTR anion selectivity are unknown. We examined the mechanisms of CFTR PHCO3/PCl regulation by [Cl−]i-sensitive kinases using the electrophysiological and molecular approach, including whole-cell, outside-out, and inside-out patch clamp recordings and molecular dissection of WNK1 and CFTR proteins. Moreover, we analyzed the effects of pancreatitis-causing CFTR mutations on the WNK1-mediated regulation of CFTR. Although SPAK and/or OSR, which constitute a [Cl−]i-sensitive kinase cascade, wasn't expressed, the expression of WNK1 alone was sufficient to increase the CFTR PHCO3/PCl in patch clamp recordings. In molecular dissection of the WNK1 protein, the WNK1 kinase domain contributes CFTR PHCO3/PCl regulation by direct association with CFTR, while the surrounding N-terminal regions regulate the [Cl−]i-sensitivity of WNK1. Therefore, the inclusion of WNK1 N-terminal recombinant proteins in excised patch clamp recordings increased the CFTR PHCO3/PCl in a dose-dependent manner. The pancreatitis-causing R74Q and R75Q mutations in elbow helix 1 of CFTR hampered WNK1-CFTR physical associations and reduced WNK1-mediated CFTR PHCO3/PCl regulation. The CFTR PHCO3/PCl is regulated by [Cl−]i and a WNK1-dependent mechanism. In low [Cl−]i, physical associations between the N-terminal region of WNK1 and CFTR near p.R75 increase PHCO3/PCl. Defects in this process underlie the pathogenesis of some CFTR-related disorders.

Regulation of TRPC5 currents by intracellular ATP: Single channel studies.

TRPC5 channels are nonselective cation channels activated by G-protein-coupled receptors. It was previously found that recombinant TRPC5 currents are inhibited by intracellular ATP, when studied by whole-cell patch-clamp recording. In the present study, we investigated the mechanism of ATP inhibition at the single-channel levelusing patches from HEK-293 cells transiently transfected with TRPC5 and the M1 muscarinic receptor. In inside-out patches, application of ATP to the intracellular face of the membrane reduced TRPC5 channel activity at both positive and negative potentials without affecting the unitary current amplitude or open dwell time of the channel. The effect of ATP was rapidly reversible. These results suggest that ATP may bind to the channel protein and affect the ability of the channel to open or to remain in an open, nondesensitized state. The activity of TRPC5 channels may be influenced by cellular metabolism via changes in ATP levels.

Oxidation of methionine residues activates the high-threshold heat-sensitive ion channel TRPV2

Thermosensitive transient receptor potential (TRP) ion channels detect changes in ambient temperature to regulate body temperature and temperature-dependent cellular activity. Rodent orthologs of TRP vanilloid 2 (TRPV2) are activated by nonphysiological heat exceeding 50 °C, and human TRPV2 is heat-insensitive. TRPV2 is required for phagocytic activity of macrophages which are rarely exposed to excessive heat, but what activates TRPV2 in vivo remains elusive. Here we describe the molecular mechanism of an oxidation-induced temperature-dependent gating of TRPV2. While high concentrations of H2O2 induce a modest sensitization of heat-induced inward currents, the oxidant chloramine-T (ChT), ultraviolet A light, and photosensitizing agents producing reactive oxygen species (ROS) activate and sensitize TRPV2. This oxidation-induced activation also occurs in excised inside-out membrane patches, indicating a direct effect on TRPV2. The reducing agent dithiothreitol (DTT) in combination with methionine sulfoxide reductase partially reverses ChT-induced sensitization, and the substitution of the methionine (M) residues M528 and M607 to isoleucine almost abolishes oxidation-induced gating of rat TRPV2. Mass spectrometry on purified rat TRPV2 protein confirms oxidation of these residues. Finally, macrophages generate TRPV2-like heat-induced inward currents upon oxidation and exhibit reduced phagocytosis when exposed to the TRP channel inhibitor ruthenium red (RR) or to DTT. In summary, our data reveal a methionine-dependent redox sensitivity of TRPV2 which may be an important endogenous mechanism for regulation of TRPV2 activity and account for its pivotal role for phagocytosis in macrophages.

Voltage-gated proton channels exist in the plasma membrane of human oocytes.

Do human oocytes express voltage-gated proton channels? Human oocytes exhibit voltage-gated proton currents. Voltage-gated proton currents have been reported in human sperm, where they contribute to capacitation and motility. No such studies of human oocytes exist. Voltage-clamp studies were undertaken using entire oocytes and vesicles derived from oocytes and in excised patches of membrane from oocytes. Frozen, thawed human metaphase II oocytes were obtained from material donated to the gamete repository at the Rush Center for Advanced Reproductive Care. Prior to patch clamping, oocytes were warmed and equilibrated. Formation of an electrically tight seal requires exposing bare oolemma. Sections of the zona pellucida (ZP) were removed using a laser, followed by repeated pipetting, to further separate the oocyte from the ZP. Patch-clamp studies were performed using the whole-cell configuration on oocytes or vesicles derived from oocytes, and using inside-out patches of membrane, under conditions optimized to detect voltage-gated proton currents. Proton currents are present at significant levels in human oocytes where they exhibit properties similar to those reported in other human cells, as well as those in heterologous expression systems transfected with the HVCN1 gene that codes for the voltage-gated proton channel. N/A. Human oocytes are large cells, which limits our ability to control the intracellular solution. Subtle effects of cryopreservation by vitrification and subsequent warming on properties of HVCN1, the HVCN1 gene product, cannot be ruled out. Possible functions for voltage-gated proton channels in human oocytes may now be contemplated. NIH R35GM126902 (TED), Bears Care (DM). No competing interests. N/A.

P699Mechanisms of action of the small conductance Ca2+-activated K+-channel modulator AP30663, a novel compound being developed for treatment of atrial fibrillation in man

Abstract Background Small conductance Ca2+-activated K+-channels (SK-channels) are a promising new atrial selective target for treatment of atrial fibrillation (AF). AP30663 is a small molecule inhibitor of SK-channels that is effective in converting vernakalant-resistant AF in tachy-paced pigs. Detailed understanding of the molecular mechanism of AP30663 is important for the development of SK channel inhibition for use in man. Purpose To establish the electrophysiological profile, mechanism of action and efficacy in prolonging atrial refractoriness ex vivo of AP30663. Methods AP30663 potency and mechanism of action were established by whole cell and inside-out patch clamp recordings of expressed SK channels. The ion channel selectivity profile of AP30663 was investigated on heterologous expressed channels. Effects of AP30663 or vehicle (DMSO) on atrial refractoriness (AERP) and ventricular repolarization (QTcB) were investigated on isolated perfused guinea pig hearts. Results AP30663 was found to be a selective negative allosteric modulator of SK channels (IC50=0.77±x0.13 μM) with no or minor effects on a panel of other cardiac ion channels, including hERG/KV11.1, (IKr), KV7.1/KCNE1 (IKs), KV4.3/KChiP2 (Ito), Kir2.1 (IK1), Kir3.1/Kir3.4 (IKACh), KV1.5 (IKur), NaV1.5 (INa) and CaV1.2 (ICa). AP30663 inhibited the SK-channel by right-shifting the calcium activation curve of the SK-channel (the EC50 of Ca2+ increased from 0.43±0.02 μM (control, n=6) to 1.37±0.05 μM (in the presence of 7μM AP30663, n=6). In isolated guinea pig hearts, administration of vehicle had no effect on AERP or QTcB. AP30663 significantly prolonged the AERP in 3 μM (to 131±6% of baseline) and 10 μM (to 165±3% of baseline) without any effects on the QTcB. Conclusion AP30663 is a selective negative allosteric modulator of SK channels, acting by means of shifting the calcium dependence of SK-channel activation. AP30663 prolonged atrial refractoriness without affecting the QT-interval in isolated perfused heart preparations. These properties support continued development of AP30663 for treatment of AF in man. Acknowledgement/Funding Innovation Fund Denmark, Wellcome Trust

Regulation of CFTR Bicarbonate Channel Activity by WNK1: Implications for Pancreatitis and CFTR-Related Disorders.

Backgraoud & AimsAberrant epithelial bicarbonate (HCO3−) secretion caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is associated with several diseases including cystic fibrosis and pancreatitis. Dynamically regulated ion channel activity and anion selectivity of CFTR by kinases sensitive to intracellular chloride concentration ([Cl−]i) play an important role in epithelial HCO3− secretion. However, the molecular mechanisms of how [Cl−]i-dependent mechanisms regulate CFTR are unknown.MethodsWe examined the mechanisms of the CFTR HCO3− channel regulation by [Cl−]i-sensitive kinases using an integrated electrophysiological, molecular, and computational approach including whole-cell, outside-out, and inside-out patch clamp recordings and molecular dissection of WNK1 and CFTR proteins. In addition, we analyzed the effects of pancreatitis-causing CFTR mutations on the WNK1-mediated regulation of CFTR.ResultsAmong the WNK1, SPAK, and OSR1 kinases that constitute a [Cl−]i-sensitive kinase cascade, the expression of WNK1 alone was sufficient to increase the CFTR bicarbonate permeability (PHCO3/PCl) and conductance (GHCO3) in patch clamp recordings. Molecular dissection of the WNK1 domains revealed that the WNK1 kinase domain is responsible for CFTR PHCO3/PCl regulation by direct association with CFTR, while the surrounding N-terminal regions mediate the [Cl−]i-sensitivity of WNK1. Furthermore, the pancreatitis-causing R74Q and R75Q mutations in the elbow helix 1 of CFTR hampered WNK1-CFTR physical associations and reduced WNK1-mediated CFTR PHCO3/PCl regulation.ConclusionThe CFTR HCO3− channel activity is regulated by [Cl−]i and a WNK1-dependent mechanism. Our results provide new insights into the regulation of the ion selectivity of CFTR and the pathogenesis of CFTR-related disorders.

A network of phosphatidylinositol 4,5-bisphosphate binding sites regulates gating of the Ca2+-activated Cl− channel ANO1 (TMEM16A)

ANO1 (TMEM16A) is a Ca2+-activated Cl- channel that regulates diverse cellular functions including fluid secretion, neuronal excitability, and smooth muscle contraction. ANO1 is activated by elevation of cytosolic Ca2+ and modulated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Here, we describe a closely concerted experimental and computational study, including electrophysiology, mutagenesis, functional assays, and extended sampling of lipid-protein interactions with molecular dynamics (MD) to characterize PI(4,5)P2 binding modes and sites on ANO1. ANO1 currents in excised inside-out patches activated by 270 nM Ca2+ at +100 mV are increased by exogenous PI(4,5)P2 with an EC50 = 1.24 µM. The effect of PI(4,5)P2 is dependent on membrane voltage and Ca2+ and is explained by a stabilization of the ANO1 Ca2+-bound open state. Unbiased atomistic MD simulations with 1.4 mol% PI(4,5)P2 in a phosphatidylcholine bilayer identified 8 binding sites with significant probability of binding PI(4,5)P2 Three of these sites captured 85% of all ANO1-PI(4,5)P2 interactions. Mutagenesis of basic amino acids near the membrane-cytosol interface found 3 regions of ANO1 critical for PI(4,5)P2 regulation that correspond to the same 3 sites identified by MD. PI(4,5)P2 is stabilized by hydrogen bonding between amino acid side chains and phosphate/hydroxyl groups on PI(4,5)P2 Binding of PI(4,5)P2 alters the position of the cytoplasmic extension of TM6, which plays a crucial role in ANO1 channel gating, and increases the accessibility of the inner vestibule to Cl- ions. We propose a model consisting of a network of 3 PI(4,5)P2 binding sites at the cytoplasmic face of the membrane allosterically regulating ANO1 channel gating.

Entering the spotlight: Chitosan oligosaccharides as novel activators of CaCCs/TMEM16A

Calcium-activated chloride channels (CaCCs)/TMEM16A control diverse fundamental physiological functions, and abnormal function of TMEM16A will lead to various diseases including asthma, hypertension, gastrointestinal hypomotility and cancers. Therefore, TMEM16A as drug targets for related diseases has been increasingly concerned by researchers. In this work, COS were reported as novel natural activators of TMEM16A. It was demonstrated that COS can activate TMEM16A in a concentration dependent manner, with an EC50 of 74.5 μg/mL. Then, fluorescence experiments and inside-out patch clamp experiments were combined to confirm that COS can directly activate TMEM16A. Further, we compared the activation effects of COS monomers DP2 to DP6, with DP3 the best activator. Molecular simulation was performed to find that the binding sites between DP3 and TMEM16A are E143 and E146 in TMEM16A, and it was speculated that COS and TMEM16A may be combined by electrostatic interaction. Finally, we verified that guinea pig ileum contraction was promoted by COS and the monomers through activating TMEM16A. Collectively, COS are novel efficient natural activators of TMEM16A, with potential to be developed to treatment diseases caused by down-regulation of TMEM16A including gastrointestinal hypomotility.

24S-hydroxycholesterol alters activity of large-conductance Ca2+-dependent K+ (slo1 BK) channel through intercalation into plasma membrane

Oxysterols, oxidization products of cholesterol, are regarded as bioactive lipids affecting various physiological functions. However, little is known of their effects on ion channels. Using inside-out patch clamp recording, we found that naturally occurring side-chain oxidized oxysterols, 20S‑hydroxycholesterol, 22R‑hydroxycholesterol, 24S‑hydroxycholestero, 25‑hydroxycholesterol, and 27‑hydroxycholesterol, induced current reduction of large-conductance Ca2+- and voltage-activated K+ (slo1 BK) channels heterologously expressed in HEK293T cells. In contrast with side-chain oxidized oxysterols, naturally occurring ring oxidized ones, 7α‑hydroxycholesterol and 7‑ketocholesterol were without effect. By using 24S‑hydroxycholesterol (24S‑HC), the major brain oxysterol, we explored the inhibition mechanism. 24S‑HC inhibited Slo1 BK channels with an IC50 of ~2 μM, and decreased macroscopic current by ~60%. This marked current decrease was accompanied by a rightward shift in the conductance-voltage relationship and a slowed activation kinetics, with the deactivation kinetics unaltered. Furthermore, the membrane sterol scavenger γ‑cyclodextrin was found to rescue slo1 BK channels from the inhibition, implicating that 24S-HC may be intercalated into the plasma membrane to affect the channel. These findings unveil a novel physiological importance of oxysterols from a new angle that involves ion channel regulation.

Temperature-dependent increase in the calcium sensitivity and acceleration of activation of ANO6 chloride channel variants

Anoctamin-6 (ANO6) belongs to a family of calcium (Ca2+)-activated chloride channels (CaCCs), with three splicing variants (V1, V2, and V5) showing plasma membrane expression. Unlike other CaCCs, ANO6 requires a non-physiological intracellular free calcium concentration ([Ca2+]i > 1 μM) and several minutes for full activation under a whole-cell patch clamp. Therefore, its physiological role as an ion channel is uncertain and it is more commonly considered a Ca2+-dependent phospholipid scramblase. Here, we demonstrate that physiological temperature (37 °C) increases ANO6 Ca2+ sensitivity under a whole-cell patch clamp; V1 was activated by 1 μM [Ca2+]i, whereas V2 and V5 were activated by 300 nM [Ca2+]i. Increasing the temperature to 42 °C led to activation of all ANO6 variants by 100 nM [Ca2+]i. The delay time for activation of the three variants was significantly shortened at 37 °C. Notably, the temperature-dependent Ca2+-sensitisation of ANO6 became insignificant under inside-out patch clamp, suggesting critical roles of unknown cytosolic factors. Unlike channel activity, 27 °C but not 37 °C (physiological temperature) induced the scramblase activity of ANO6 at submicromolar [Ca2+]i (300 nM), irrespective of variant type. Our results reveal a physiological ion conducting property of ANO6 at 37 °C and suggest that ANO6 channel function acts separately from its scramblase activity.

Drosophila Subdued is a moonlighting transmembrane protein 16 (TMEM16) that transports ions and phospholipids

Transmembrane protein 16 (TMEM16) family members play numerous important physiological roles, ranging from controlling membrane excitability and secretion to mediating blood coagulation and viral infection. These diverse functions are largely due to their distinct biophysical properties. Mammalian TMEM16A and TMEM16B are Ca2+-activated Cl- channels (CaCCs), whereas mammalian TMEM16F, fungal afTMEM16, and nhTMEM16 are moonlighting (multifunctional) proteins with both Ca2+-activated phospholipid scramblase (CaPLSase) and Ca2+-activated, nonselective ion channel (CAN) activities. To further understand the biological functions of the enigmatic TMEM16 proteins in different organisms, here, by combining an improved annexin V-based CaPLSase-imaging assay with inside-out patch clamp technique, we thoroughly characterized Subdued, a Drosophila TMEM16 ortholog. We show that Subdued is also a moonlighting transport protein with both CAN and CaPLSase activities. Using a TMEM16F-deficient HEK293T cell line to avoid strong interference from endogenous CaPLSases, our functional characterization and mutagenesis studies revealed that Subdued is a bona fide CaPLSase. Our finding that Subdued is a moonlighting TMEM16 expands our understanding of the molecular mechanisms of TMEM16 proteins and their evolution and physiology in both Drosophila and humans.

An Ancient TRPM2 Ortholog is a True Channel-Enzyme, But its Catalytic Activity is Uncoupled from Pore Gating

Human TRPM2 (hTRPM2) is a non-selective Ca2+ permeable cation channel, expressed abundantly in brain, immune cells, and pancreatic β-cells. Under physiological conditions it plays a role in glucose-induced insulin secretion, and in the immune response. Besides, TRPM2 enhances the sensitivity of cells toward oxidative stress induced damage, e.g., facilitating neuronal cell death during brain ischemia/reperfusion. Nematostella vectensis TRPM2 (nvTRPM2) is an ancestor of hTRPM2. Both are activated by intracellular Ca2+ and ADP-ribose (ADPR), and contain a C-terminal NUDT9-H domain, which shows high sequence homology with the mitochondrial ADPR-hydrolase enzyme NUDT9. We have recently shown that the NUDT9-H domain of hTRPM2 has lost its ADPRase activity due to mutations of the conserved “Nudix box” motif.Interestingly, in nvTRPM2 the Nudix-box motif is still canonical. Correspondingly, we found that purified nvTRPM2 protein, or its isolated NUDT9-H domain, shows robust ADPRase activity. We next addressed whether nvTRPM2 enzymatic activity plays any role in channel gating. Enzymatic activity of isolated nvNUDT9-H could be reduced or abolished by point mutations, as well as by Mg2+ removal. On the other hand, in inside-out patch clamp experiments the kinetics of channel gating of full-length nvTRPM2 remained unaffected by these manipulations: none of them changed the apparent affinity of the currents towards ADPR, or the current relaxation time constant after sudden ADPR removal. We also investigated the effect of the non-hydrolyzable ADPR analogue AMPCPR on nvTRPM2 channel gating. Compared to ADPR, AMPCPR showed a lower apparent affinity, and caused shorter channel openings, suggesting that it fits less well into the binding pocket. These results together suggest that nvTRPM2 is a true channel-enzyme, but its enzymatic activity is not coupled to channel gating.

Mechanism of BK Channel Inhibition by the Opioid Agonist Loperamide

Large conductance Ca2+-activated K+ (BK) channels are expressed in a variety of tissues and control a range of physiological functions, including action potential duration in some neurons and contractility in smooth muscle. Pharmacological BK channel modulators may be promising therapeutic options in human disease; however, the current library of effective BK-selective modulators is limited. Using a high-throughput screening approach, we observed that the diphenylpropylamine μ-opioid agonist, loperamide, could inhibit BK channel activity. Here we analyze the molecular mechanism of this inhibition using patch-clamp electrophysiology. In excised inside-out patch recordings (Ca2+ = 88 μM at the cytosolic side), loperamide reversibly inhibited BK current in a dose-dependent manner when applied to the cytosolic face of BK channels, with an IC50 of 1.0 ± 0.2 μM. Over a range of voltages at which BK channels were open nearly 100% of the time, the IC50 did not significantly increase or decrease as a function of voltage, suggesting that loperamide does not inhibit as an open-channel blocker. Single-channel recordings revealed that loperamide does not reduce unitary current amplitudes, and does not inhibit BK channels at voltages where voltage sensors were at rest (between −140 and −160 mV; Po = 0.0035 ± 0.0002 and Po = 0.0033 ± 0.0001 with 0 and 30 μM loperamide, respectively). Loperamide inhibited BK channels in the nominal absence of Ca2+, in channels co-expressed with the γ1 subunit (LRRC26), suggesting that inhibition does not strictly depend on the presence of Ca2+. Together, these data are consistent with the idea that loperamide inhibits BK channels either through directly inhibiting activation of the BK voltage sensor, or by inhibiting allosteric coupling between voltage sensor activation and pore opening. This work has been supported by NIH grant R01 GM126581 and AHA Grant-in-Aid to B.S.R.

Single Channel Studies of the Cation Permeation Pathway of the Shaker Kv Isolated Voltage-Sensing Domain (iVSD)

In the absence of a pore domain, the isolated voltage-sensing domain (iVSD) of Shaker Kv channel forms a cation-selective ion channel with a strong preference for protons. iVSDs produce hyperpolarization-activated ionic currents, but the location of the gating pore in the iVSD is unknown. Identifying the molecular determinants underlying iVSD gating would help in the understanding of VSD relaxation since iVSD gating and relaxation have been shown to be correlated. We studied the single channel behavior of iVSD ionic currents; the single channel recordings displayed two conformational sublevels, which can be effectively inhibited by the intracellular guanidine derivative 2GBI in inside-out patch configuration, or by the extracellular ZnCl2 in outside-out patch configuration. We also characterized the effects of a series of residue substitutions along the suspected gating pore of the Shaker channel on the IVSD single channels. Our study supports the notion that the cation permeation of Shaker IVSD follows the pathway through the gating pore like in HV1 channels and ω currents.

Phosphatidylinositol Inhibits TRPV1 via its Vanilloid Binding Site

Regulation of the heat- and capsaicin-activated Transient Receptor Potential Vanilloid 1 (TRPV1) channels by phosphoinositides is complex and controversial. In excised inside-out patches phosphatidylinositol 4,5-bisphosphate (PIP2) and its precursor phosphatidylinositol 4-phosphate (PIP) reproducibly potentiate TRPV1 activity. In a recent cryoEM structure a phosphoinositide was detected in the vanilloid binding site in the absence of agonists. Phosphoinositides were proposed to negatively regulate TRPV1 as competitive inhibitors of vanilloids, but the resolution of the structure was sufficient to determine the exact nature of the lipid. Here we used computational modeling, site directed mutagenesis and patch clamp electrophysiology to identify the phosphoinositide in the vanilloid binding site. We find that PIP2 diffuses away from the vanilloid binding site in Molecular Dynamics simulations, but phosphatidylinositol, the precursor of PIP and PIP2, remains there. We also find that mutating two residues predicted to interact with phosphatidylinositol, but not with capsaicin shifts the dose-response of capsaicin to the left. We also show that in excised inside-out patch clamp experiments phosphatidylinositol inhibited TRPV1 activity induced by PIP2 at low capsaicin concentrations. We conclude that phosphatidylinositol binds to the Vanilloid binding site of TRPV1 in resting conditions, and this may serve to set the basal sensitivity of the channel to vanilloids as well as heat and low pH.