Stretch response of the mechano-gated channel TMEM63A in membrane patches and single cells.

The recently discovered ion channel TMEM63A has biophysical features distinctive for mechano-gated cation channels, activating at high pressures with slow kinetics while not inactivating. However, some biophysical properties are less clear, including no information on its function in whole cells. The aim of this study is to expand the TMEM63A biophysical characterization and examine the function in whole cells. Piezo1-knockout HEK293T cells were cotransfected with human TMEM63A and green fluorescent protein (GFP), and macroscopic currents in cell-attached patches were recorded by high-speed pressure clamp at holding voltages from -120 to -20 mV with 0-100 mmHg patch suction for 1 s. HEK293 cells cotransfected with TMEM63A and GCaMP5 were seeded onto polydimethylsiloxane (PDMS) membrane, and the response to 3-12 s of 1%-15% whole cell isotropic (equi-biaxial) stretch induced by an IsoStretcher was measured by the change in intracellular calcium ([Ca2+]i) and presented as (ΔF/F0 > 1). Increasing patch pressures activated TMEM63A currents with accelerating activation kinetics and current amplitudes that were pressure dependent but voltage independent. TMEM63A currents were plateaued within 2 s, recovered quickly, and were sensitive to Gd3+. In whole cells stretched on flexible membranes, radial stretch increased the [Ca2+]i responses in a larger proportion of cells cotransfected with TMEM63A and GCaMP5 than GCaMP5-only controls. TMEM63A currents are force activated and voltage insensitive, have a high threshold for pressure activation with slow activation and deactivation, and lack inactivation over 5 s. TMEM63A has the net polarity and kinetics that would depolarize plasma membranes and increase inward currents, contributing to a sustained [Ca2+]i increase in response to high stretch.NEW & NOTEWORTHY TMEM63A has biophysical features distinctive for mechano-gated cation channels, but some properties are less clear, including no functional information in whole cells. We report that pressure-dependent yet voltage-independent TMEM63A currents in cell membrane patches correlated with cell size. In addition, radial stretch of whole cells on flexible membranes increased the [Ca2+]i responses more in TMEM63A-transfected cells. Inward TMEM63A currents in response to high stretch can depolarize plasma membranes and contribute to a sustained [Ca2+]i increase.

Mechanosensitive pore opening of a prokaryotic voltage-gated sodium channel.

Voltage-gated ion channels (VGICs) orchestrate electrical activities that drive mechanical functions in contractile tissues such as the heart and gut. In turn, contractions change membrane tension and impact ion channels. VGICs are mechanosensitive, but the mechanisms of mechanosensitivity remain poorly understood. Here, we leverage the relative simplicity of NaChBac, a prokaryotic voltage-gated sodium channel from Bacillus halodurans, to investigate mechanosensitivity. In whole-cell experiments on heterologously transfected HEK293 cells, shear stress reversibly altered the kinetic properties of NaChBac and increased its maximum current, comparably to the mechanosensitive eukaryotic sodium channel NaV1.5. In single-channel experiments, patch suction reversibly increased the open probability of a NaChBac mutant with inactivation removed. A simple kinetic mechanism featuring a mechanosensitive pore opening transition explained the overall response to force, whereas an alternative model with mechanosensitive voltage sensor activation diverged from the data. Structural analysis of NaChBac identified a large displacement of the hinged intracellular gate, and mutagenesis near the hinge diminished NaChBac mechanosensitivity, further supporting the proposed mechanism. Our results suggest that NaChBac is overall mechanosensitive due to the mechanosensitivity of a voltage-insensitive gating step associated with the pore opening. This mechanism may apply to eukaryotic VGICs, including NaV1.5.

Capsaicin as an amphipathic modulator of NaV1.5 mechanosensitivity.

SCN5A-encoded NaV1.5 is a voltage-gated Na+ channel that drives the electrical excitability of cardiac myocytes and contributes to slow waves of the human gastrointestinal smooth muscle cells. NaV1.5 is mechanosensitive: mechanical force modulates several facets of NaV1.5's voltage-gated function, and some NaV1.5 channelopathies are associated with abnormal NaV1.5 mechanosensitivity (MS). A class of membrane-active drugs, known as amphiphiles, therapeutically target NaV1.5's voltage-gated function and produce off-target effects including alteration of MS. Amphiphiles may provide a novel option for therapeutic modulation of NaV1.5's mechanosensitive operation. To more selectively target NaV1.5 MS, we searched for a membrane-partitioning amphipathic agent that would inhibit MS with minimal closed-state inhibition of voltage-gated currents. Among the amphiphiles tested, we selected capsaicin for further study. We used two methods to assess the effects of capsaicin on NaV1.5 MS: (1) membrane suction in cell-attached macroscopic patches and (2) fluid shear stress on whole cells. We tested the effect of capsaicin on NaV1.5 MS by examining macro-patch and whole-cell Na+ current parameters with and without force. Capsaicin abolished the pressure- and shear-mediated peak current increase and acceleration; and the mechanosensitive shifts in the voltage-dependence of activation (shear) and inactivation (pressure and shear). Exploring the recovery from inactivation and use-dependent entry into inactivation, we found divergent stimulus-dependent effects that could potentiate or mitigate the effect of capsaicin, suggesting that mechanical stimuli may differentially modulate NaV1.5 MS. We conclude that selective modulation of NaV1.5 MS makes capsaicin a promising candidate for therapeutic interventions targeting MS.

Genome-wide analysis of 944 133 individuals provides insights into the etiology of haemorrhoidal disease.

OBJECTIVE: Haemorrhoidal disease (HEM) affects a large and silently suffering fraction of the population but its aetiology, including suspected genetic predisposition, is poorly understood. We report the first genome-wide association study (GWAS) meta-analysis to identify genetic risk factors for HEM to date. DESIGN: We conducted a GWAS meta-analysis of 218 920 patients with HEM and 725 213 controls of European ancestry. Using GWAS summary statistics, we performed multiple genetic correlation analyses between HEM and other traits as well as calculated HEM polygenic risk scores (PRS) and evaluated their translational potential in independent datasets. Using functional annotation of GWAS results, we identified HEM candidate genes, which differential expression and coexpression in HEM tissues were evaluated employing RNA-seq analyses. The localisation of expressed proteins at selected loci was investigated by immunohistochemistry. RESULTS: We demonstrate modest heritability and genetic correlation of HEM with several other diseases from the GI, neuroaffective and cardiovascular domains. HEM PRS validated in 180 435 individuals from independent datasets allowed the identification of those at risk and correlated with younger age of onset and recurrent surgery. We identified 102 independent HEM risk loci harbouring genes whose expression is enriched in blood vessels and GI tissues, and in pathways associated with smooth muscles, epithelial and endothelial development and morphogenesis. Network transcriptomic analyses highlighted HEM gene coexpression modules that are relevant to the development and integrity of the musculoskeletal and epidermal systems, and the organisation of the extracellular matrix. CONCLUSION: HEM has a genetic component that predisposes to smooth muscle, epithelial and connective tissue dysfunction.

Mechanotransduction in gastrointestinal smooth muscle cells: role of mechanosensitive ion channels.

Mechanosensation, the ability to properly sense mechanical stimuli and transduce them into physiologic responses, is an essential determinant of gastrointestinal (GI) function. Abnormalities in this process result in highly prevalent GI functional and motility disorders. In the GI tract, several cell types sense mechanical forces and transduce them into electrical signals, which elicit specific cellular responses. Some mechanosensitive cells like sensory neurons act as specialized mechanosensitive cells that detect forces and transduce signals into tissue-level physiological reactions. Nonspecialized mechanosensitive cells like smooth muscle cells (SMCs) adjust their function in response to forces. Mechanosensitive cells use various mechanoreceptors and mechanotransducers. Mechanoreceptors detect and convert force into electrical and biochemical signals, and mechanotransducers amplify and direct mechanoreceptor responses. Mechanoreceptors and mechanotransducers include ion channels, specialized cytoskeletal proteins, cell junction molecules, and G protein-coupled receptors. SMCs are particularly important due to their role as final effectors for motor function. Myogenic reflex-the ability of smooth muscle to contract in response to stretch rapidly-is a critical smooth muscle function. Such rapid mechanotransduction responses rely on mechano-gated and mechanosensitive ion channels, which alter their ion pores' opening in response to force, allowing fast electrical and Ca2+ responses. Although GI SMCs express a variety of such ion channels, their identities remain unknown. Recent advancements in electrophysiological, genetic, in vivo imaging, and multi-omic technologies broaden our understanding of how SMC mechano-gated and mechanosensitive ion channels regulate GI functions. This review discusses GI SMC mechanosensitivity's current developments with a particular emphasis on mechano-gated and mechanosensitive ion channels.

Expression of the regulated isoform of the electrogenic Na+/HCO3- cotransporter, NBCe1, is enriched in pacemaker interstitial cells of Cajal.

Interstitial cells of Cajal (ICCs) generate electrical slow waves, which are required for normal gastrointestinal motility. The mechanisms for generation of normal pacemaking are not fully understood. Normal gastrointestinal contractility- and electrical slow-wave activity depend on the presence of extracellular HCO3-. Previous transcriptional analysis identified enrichment of mRNA encoding the electrogenic Na+/HCO3- cotransporter (NBCe1) gene (Slc4a4) in pacemaker myenteric ICCs in mouse small intestine. We aimed to determine the distribution of NBCe1 protein in ICCs of the mouse gastrointestinal tract and to identify the transcripts of the Slc4a4 gene in mouse and human small intestinal tunica muscularis. We determined the distribution of NBCe1 immunoreactivity (NBCe1-IR) by immunofluorescent labeling in mouse and human tissues. In mice, NBCe1-IR was restricted to Kit-positive myenteric ICCs of the stomach and small intestine and submuscular ICCs of the large intestine, that is, the slow wave generating subset of ICCs. Other subtypes of ICCs were NBCe1-negative. Quantitative real-time PCR identified >500-fold enrichment of Slc4a4-207 and Slc4a4-208 transcripts ["IP3-receptor-binding protein released by IP3" (IRBIT)-regulated isoforms] in Kit-expressing cells isolated from KitcreERT2/+, Rpl22tm1.1Psam/Sj mice and from single GFP-positive ICCs from Kittm1Rosay mice. Human jejunal tunica muscularis ICCs were also NBCe1-positive, and SLC4A4-201 and SLC4A4-204 RNAs were >300-fold enriched relative to SLC4A4-202. In summary, NBCe1 protein expressed in ICCs with electrical pacemaker function is encoded by Slc4a4 gene transcripts that generate IRBIT-regulated isoforms of NBCe1. In conclusion, Na+/HCO3- cotransport through NBCe1 contributes to the generation of pacemaker activity in subsets of ICCs.NEW & NOTEWORTHY In this study, we show that the electrogenic Na+/HCO3- cotransporter, NBCe1/Slc4a4, is expressed in subtypes of interstitial cells of Cajal (ICCs) responsible for electrical slow wave generation throughout the mouse gastrointestinal tract and is absent in other types of ICCs. The transcripts of Slc4a4 expressed in mouse ICCs and human gastrointestinal smooth muscle are the regulated isoforms. This indicates a key role for HCO3- transport in generation of gastrointestinal motility patterns.

microRNA overexpression in slow transit constipation leads to reduced NaV1.5 current and altered smooth muscle contractility.

OBJECTIVE: This study was designed to evaluate the roles of microRNAs (miRNAs) in slow transit constipation (STC). DESIGN: All human tissue samples were from the muscularis externa of the colon. Expression of 372 miRNAs was examined in a discovery cohort of four patients with STC versus three age/sex-matched controls by a quantitative PCR array. Upregulated miRNAs were examined by quantitative reverse transcription qPCR (RT-qPCR) in a validation cohort of seven patients with STC and age/sex-matched controls. The effect of a highly differentially expressed miRNA on a custom human smooth muscle cell line was examined in vitro by RT-qPCR, electrophysiology, traction force microscopy, and ex vivo by lentiviral transduction in rat muscularis externa organotypic cultures. RESULTS: The expression of 13 miRNAs was increased in STC samples. Of those miRNAs, four were predicted to target SCN5A, the gene that encodes the Na+ channel NaV1.5. The expression of SCN5A mRNA was decreased in STC samples. Let-7f significantly decreased Na+ current density in vitro in human smooth muscle cells. In rat muscularis externa organotypic cultures, overexpression of let-7f resulted in reduced frequency and amplitude of contraction. CONCLUSIONS: A small group of miRNAs is upregulated in STC, and many of these miRNAs target the SCN5A-encoded Na+ channel NaV1.5. Within this set, a novel NaV1.5 regulator, let-7f, resulted in decreased NaV1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest NaV1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.

SCN5A mutation G615E results in NaV1.5 voltage-gated sodium channels with normal voltage-dependent function yet loss of mechanosensitivity.

SCN5A is expressed in cardiomyocytes and gastrointestinal (GI) smooth muscle cells (SMCs) as the voltage-gated mechanosensitive sodium channel NaV1.5. The influx of Na+ through NaV1.5 produces a fast depolarization in membrane potential, indispensable for electrical excitability in cardiomyocytes and important for electrical slow waves in GI smooth muscle. As such, abnormal NaV1.5 voltage gating or mechanosensitivity may result in channelopathies. SCN5A mutation G615E - found separately in cases of acquired long-QT syndrome, sudden cardiac death, and irritable bowel syndrome - has a relatively minor effect on NaV1.5 voltage gating. The aim of this study was to test whether G615E impacts mechanosensitivity. Mechanosensitivity of wild-type (WT) or G615E-NaV1.5 in HEK-293 cells was examined by shear stress on voltage- or current-clamped whole cells or pressure on macroscopic patches. Unlike WT, voltage-clamped G615E-NaV1.5 showed a loss in shear- and pressure-sensitivity of peak current yet a normal leftward shift in the voltage-dependence of activation. In current-clamp, shear stress led to a significant increase in firing spike frequency with a decrease in firing threshold for WT but not G615E-NaV1.5. Our results show that the G615E mutation leads to functionally abnormal NaV1.5 channels, which cause disruptions in mechanosensitivity and mechano-electrical feedback and suggest a potential contribution to smooth muscle pathophysiology.

Direct repression of anoctamin 1 (ANO1) gene transcription by Gli proteins.

The Ca2+-activated Cl- channel, anoctamin 1 (Ano1, also known as transmembrane protein 16A) contributes to intestinal pacemaking, fluid secretion, cellular excitability, and tissue development. The human ANO1 promoter contains binding sites for the glioma-associated oncogene (Gli) proteins. We investigated regulation of ANO1 transcription by Gli. ANO1 promoter activity was determined using a luciferase reporter system. Binding and functional effects of Glis on ANO1 transcription and expression were demonstrated by chromatin immunoprecipitation, small interfering RNA knockdown, PCR, immunolabeling, and recordings of Ca2+-activated Cl- currents in human embryonic kidney 293 (HEK293) cells. Results from previous genome-wide association studies were used to test ANO1 promoter polymorphisms for association with disease. Gli1 and Gli2 bound to the promoter and repressed ANO1 transcription. Repression depended on Gli binding to a site close to the ANO1 transcriptional start site. Mutation of this site prevented Gli binding and transcriptional repression. Knockdown of Gli expression and inhibition of Gli activity increased expression of ANO1 RNA and Ca2+-activated Cl- currents in HEK293 cells. A single-nucleotide polymorphism prevented Gli binding and showed association with irritable bowel syndrome. We conclude that Gli1 and Gli2 repress ANO1 by a novel mechanism that is independent of Gli cleavage and that has a role in gastrointestinal function.-Mazzone, A., Gibbons, S. J., Eisenman, S. T., Strege, P. R., Zheng, T., D'Amato, M., Ordog, T., Fernandez-Zapico, M. E., Farrugia, G. Direct repression of anoctamin 1 (ANO1) gene transcription by Gli proteins.

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells.

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory cells, EC cells sense luminal stimuli and convert them into serotonin (5-hyroxytryptamine, 5-HT) release events. However, the electrophysiology of these cells is poorly understood because they are difficult to culture and to identify. The method presented in this paper outlines primary EC cell cultures optimized for single cell electrophysiology. This protocol utilizes a transgenic cyan fluorescent protein (CFP) reporter to identify mouse EC cells in mixed primary cultures, advancing the approach to obtaining high-quality recordings of whole cell electrophysiology in voltage- and current-clamp modes.

A population of gut epithelial enterochromaffin cells is mechanosensitive and requires Piezo2 to convert force into serotonin release.

Enterochromaffin (EC) cells constitute the largest population of intestinal epithelial enteroendocrine (EE) cells. EC cells are proposed to be specialized mechanosensory cells that release serotonin in response to epithelial forces, and thereby regulate intestinal fluid secretion. However, it is unknown whether EE and EC cells are directly mechanosensitive, and if so, what the molecular mechanism of their mechanosensitivity is. Consequently, the role of EE and EC cells in gastrointestinal mechanobiology is unclear. Piezo2 mechanosensitive ion channels are important for some specialized epithelial mechanosensors, and they are expressed in mouse and human EC cells. Here, we use EC and EE cell lineage tracing in multiple mouse models to show that Piezo2 is expressed in a subset of murine EE and EC cells, and it is distributed near serotonin vesicles by superresolution microscopy. Mechanical stimulation of a subset of isolated EE cells leads to a rapid inward ionic current, which is diminished by Piezo2 knockdown and channel inhibitors. In these mechanosensitive EE cells force leads to Piezo2-dependent intracellular Ca2+ increase in isolated cells as well as in EE cells within intestinal organoids, and Piezo2-dependent mechanosensitive serotonin release in EC cells. Conditional knockout of intestinal epithelial Piezo2 results in a significant decrease in mechanically stimulated epithelial secretion. This study shows that a subset of primary EE and EC cells is mechanosensitive, uncovers Piezo2 as their primary mechanotransducer, defines the molecular mechanism of their mechanotransduction and mechanosensitive serotonin release, and establishes the role of epithelial Piezo2 mechanosensitive ion channels in regulation of intestinal physiology.

Irritable bowel syndrome patients have SCN5A channelopathies that lead to decreased NaV1.5 current and mechanosensitivity.

The SCN5A-encoded voltage-gated mechanosensitive Na+ channel NaV1.5 is expressed in human gastrointestinal smooth muscle cells and interstitial cells of Cajal. NaV1.5 contributes to smooth muscle electrical slow waves and mechanical sensitivity. In predominantly Caucasian irritable bowel syndrome (IBS) patient cohorts, 2-3% of patients have SCN5A missense mutations that alter NaV1.5 function and may contribute to IBS pathophysiology. In this study we examined a racially and ethnically diverse cohort of IBS patients for SCN5A missense mutations, compared them with IBS-negative controls, and determined the resulting NaV1.5 voltage-dependent and mechanosensitive properties. All SCN5A exons were sequenced from somatic DNA of 252 Rome III IBS patients with diverse ethnic and racial backgrounds. Missense mutations were introduced into wild-type SCN5A by site-directed mutagenesis and cotransfected with green fluorescent protein into HEK-293 cells. NaV1.5 voltage-dependent and mechanosensitive functions were studied by whole cell electrophysiology with and without shear force. Five of 252 (2.0%) IBS patients had six rare SCN5A mutations that were absent in 377 IBS-negative controls. Six of six (100%) IBS-associated NaV1.5 mutations had voltage-dependent gating abnormalities [current density reduction (R225W, R433C, R986Q, and F1293S) and altered voltage dependence (R225W, R433C, R986Q, G1037V, and F1293S)], and at least one kinetic parameter was altered in all mutations. Four of six (67%) IBS-associated SCN5A mutations (R225W, R433C, R986Q, and F1293S) resulted in altered NaV1.5 mechanosensitivity. In this racially and ethnically diverse cohort of IBS patients, we show that 2% of IBS patients harbor SCN5A mutations that are absent in IBS-negative controls and result in NaV1.5 channels with abnormal voltage-dependent and mechanosensitive function. NEW & NOTEWORTHY The voltage-gated Na+ channel NaV1.5 contributes to smooth muscle physiology and electrical slow waves. In a racially and ethnically mixed irritable bowel syndrome cohort, 2% had mutations in the NaV1.5 gene SCN5A. These mutations were absent in irritable bowel syndrome-negative controls. Most mutant NaV1.5 channels were loss of function in voltage dependence or mechanosensitivity.

Sodium channel NaV1.3 is important for enterochromaffin cell excitability and serotonin release.

In the gastrointestinal (GI) epithelium, enterochromaffin (EC) cells are enteroendocrine cells responsible for producing >90% of the body's serotonin (5-hydroxytryptamine, 5-HT). However, the molecular mechanisms of EC cell function are poorly understood. Here, we found that EC cells in mouse primary cultures fired spontaneous bursts of action potentials. We examined the repertoire of voltage-gated sodium channels (NaV) in fluorescence-sorted mouse EC cells and found that Scn3a was highly expressed. Scn3a-encoded NaV1.3 was specifically and densely expressed at the basal side of both human and mouse EC cells. Using electrophysiology, we found that EC cells expressed robust NaV1.3 currents, as determined by their biophysical and pharmacologic properties. NaV1.3 was not only critical for generating action potentials in EC cells, but it was also important for regulating 5-HT release by these cells. Therefore, EC cells use Scn3a-encoded voltage-gated sodium channel NaV1.3 for electrical excitability and 5-HT release. NaV1.3-dependent electrical excitability and its contribution to 5-HT release is a novel mechanism of EC cell function.

EAVK segment "c" sequence confers Ca2+-dependent changes to the kinetics of full-length human Ano1.

Anoctamin1 (Ano1 and TMEM16A) is a calcium-activated chloride channel specifically expressed in the interstitial cells of Cajal (ICC) of the gastrointestinal tract muscularis propria. Ano1 is necessary for normal electrical slow waves and ICC proliferation. The full-length human Ano1 sequence includes an additional exon, exon "0," at the NH2 terminus. Ano1 with exon 0 [Ano1(0)] had a lower EC50 for intracellular calcium ([Ca2+]i) and faster chloride current (ICl) kinetics. The Ano1 alternative splice variant with segment "c" encoding exon 13 expresses on the first intracellular loop four additional amino acid residues, EAVK, which alter ICl at low [Ca2+]i Exon 13 is expressed in 75-100% of Ano1 transcripts in most human tissues but only 25% in the human stomach. Our aim was to determine the effect of EAVK deletion on Ano1(0)ICl parameters. By voltage-clamp electrophysiology, we examined ICl in HEK293 cells transiently expressing Ano1(0) with or without the EAVK sequence [Ano1(0)ΔEAVK]. The EC50 values of activating and deactivating ICl for [Ca2+]i were 438 ± 7 and 493 ± 9 nM for Ano1(0) but higher for Ano1(0)ΔEAVK at 746 ± 47 and 761 ± 26 nM, respectively. Meanwhile, the EC50 values for the ratio of instantaneous to steady-state ICl were not different between variants. Congruently, the time constant of activation was slower for Ano1(0)ΔEAVK than Ano1(0) currents at intermediate [Ca2+]i These results suggest that EAVK decreases the calcium sensitivity of Ano1(0) current activation and deactivation by slowing activation kinetics. Differential expression of EAVK in the human stomach may function as a switch to increase sensitivity to [Ca2+]i via faster gating of Ano1.NEW & NOTEWORTHY Calcium-activated chloride channel anoctamin1 (Ano1) is necessary for normal slow waves in the gastrointestinal interstitial cells of Cajal. Exon 0 encodes the NH2 terminus of full-length human Ano1 [Ano1(0)], while exon 13 encodes residues EAVK on its first intracellular loop. Splice variants lack EAVK more often in the stomach than other tissues. Ano1(0) without EAVK [Ano1(0)ΔEAVK] has reduced sensitivity for intracellular calcium, attributable to slower kinetics. Differential expression of EAVK may function as a calcium-sensitive switch in the human stomach.

A novel exon in the human Ca2+-activated Cl- channel Ano1 imparts greater sensitivity to intracellular Ca2.

Anoctamin 1 (Ano1; TMEM16A) is a Ca(2+)-activated Cl(-) channel (CACC) expressed in interstitial cells of Cajal. The mechanisms by which Ca(2+) regulates Ano1 are incompletely understood. In the gastrointestinal tract, Ano1 is required for normal slow wave activity and is involved in regulating cell proliferation. Splice variants of Ano1 have varying electrophysiological properties and altered expression in disease states. Recently, we identified a transcript for human Ano1 containing a novel exon-"exon 0" upstream of and in frame with exon 1. The electrophysiological properties of this longer Ano1 isoform are unknown. Our aim was to determine the functional contribution of the newly identified exon to the Ca(2+) sensitivity and electrophysiological properties of Ano1. Constructs with [Ano1(+0)] or without [Ano1(-0)] the newly identified exon were transfected into human embryonic kidney-293 cells. Voltage-clamp electrophysiology was used to determine voltage- and time-dependent parameters of whole cell Cl(-) currents between isoforms with varying concentrations of intracellular Ca(2+), extracellular anions, or Cl(-) channel inhibitors. We found that exon 0 did not change voltage sensitivity and had no impact on the relative permeability of Ano1 to most anions. Ano1(+0) exhibited greater changes in current density but lesser changes in kinetics than Ano1(-0) in response to varying intracellular Ca(2+). The CACC inhibitor niflumic acid inhibited current with greater efficacy and higher potency against Ano1(+0) compared with Ano1(-0). Likewise, the Ano1 inhibitor T16Ainh-A01 reduced Ano1(+0) more than Ano1(-0). In conclusion, human Ano1 containing exon 0 imparts its Cl(-) current with greater sensitivity to intracellular Ca(2+) and CACC inhibitors.

Ranolazine inhibits voltage-gated mechanosensitive sodium channels in human colon circular smooth muscle cells.

Human jejunum smooth muscle cells (SMCs) and interstitial cells of Cajal (ICCs) express the SCN5A-encoded voltage-gated, mechanosensitive sodium channel NaV1.5. NaV1.5 contributes to small bowel excitability, and NaV1.5 inhibitor ranolazine produces constipation by an unknown mechanism. We aimed to determine the presence and molecular identity of Na(+) current in the human colon smooth muscle and to examine the effects of ranolazine on Na(+) current, mechanosensitivity, and smooth muscle contractility. Inward currents were recorded by whole cell voltage clamp from freshly dissociated human colon SMCs at rest and with shear stress. SCN5A mRNA and NaV1.5 protein were examined by RT-PCR and Western blots, respectively. Ascending human colon strip contractility was examined in a muscle bath preparation. SCN5A mRNA and NaV1.5 protein were identified in human colon circular muscle. Freshly dissociated human colon SMCs had Na(+) currents (-1.36 ± 0.36 pA/pF), shear stress increased Na(+) peaks by 17.8 ± 1.8% and accelerated the time to peak activation by 0.7 ± 0.3 ms. Ranolazine (50 µM) blocked peak Na(+) current by 43.2 ± 9.3% and inhibited shear sensitivity by 25.2 ± 3.2%. In human ascending colon strips, ranolazine decreased resting tension (31%), reduced the frequency of spontaneous events (68%), and decreased the response to smooth muscle electrical field stimulation (61%). In conclusion, SCN5A-encoded NaV1.5 is found in human colonic circular smooth muscle. Ranolazine blocks both peak amplitude and mechanosensitivity of Na(+) current in human colon SMCs and decreases contractility of human colon muscle strips. Our data provide a likely mechanistic explanation for constipation induced by ranolazine.

Loss-of-function of the voltage-gated sodium channel NaV1.5 (channelopathies) in patients with irritable bowel syndrome.

BACKGROUND & AIMS: SCN5A encodes the α-subunit of the voltage-gated sodium channel NaV1.5. Many patients with cardiac arrhythmias caused by mutations in SCN5A also have symptoms of irritable bowel syndrome (IBS). We investigated whether patients with IBS have SCN5A variants that affect the function of NaV1.5. METHODS: We performed genotype analysis of SCN5A in 584 persons with IBS and 1380 without IBS (controls). Mutant forms of SCN5A were expressed in human embryonic kidney-293 cells, and functions were assessed by voltage clamp analysis. A genome-wide association study was analyzed for an association signal for the SCN5A gene, and replicated in 1745 patients in 4 independent cohorts of IBS patients and controls. RESULTS: Missense mutations were found in SCN5A in 13 of 584 patients (2.2%, probands). Diarrhea-predominant IBS was the most prevalent form of IBS in the overall study population (25%). However, a greater percentage of individuals with SCN5A mutations had constipation-predominant IBS (31%) than diarrhea-predominant IBS (10%; P < .05). Electrophysiologic analysis showed that 10 of 13 detected mutations disrupted NaV1.5 function (9 loss-of-function and 1 gain-of-function function). The p. A997T-NaV1.5 had the greatest effect in reducing NaV1.5 function. Incubation of cells that expressed this variant with mexiletine restored their sodium current and administration of mexiletine to 1 carrier of this mutation (who had constipation-predominant IBS) normalized their bowel habits. In the genome-wide association study and 4 replicated studies, the SCN5A locus was strongly associated with IBS. CONCLUSIONS: About 2% of patients with IBS carry mutations in SCN5A. Most of these are loss-of-function mutations that disrupt NaV1.5 channel function. These findings provide a new pathogenic mechanism for IBS and possible treatment options.

Inhibition of cell proliferation by a selective inhibitor of the Ca(2+)-activated Cl(-) channel, Ano1.

BACKGROUND: Ion channels play important roles in regulation of cellular proliferation. Ano1 (TMEM16A) is a Ca(2+)-activated Cl(-) channel expressed in several tumors and cell types. In the muscle layers of the gastrointestinal tract Ano1 is selectively expressed in interstitial cells of Cajal (ICC) and appears to be required for normal gastrointestinal slow wave electrical activity. However, Ano1 is expressed in all classes of ICC, including those that do not generate slow waves suggesting that Ano1 may have other functions. Indeed, a role for Ano1 in regulating proliferation of tumors and ICC has been recently suggested. Recently, a high-throughput screen identified a small molecule, T16A(inh)-A01 as a specific inhibitor of Ano1. AIM: To investigate the effect of the T16A(inh)-A01 inhibitor on proliferation in ICC and in the Ano1-expressing human pancreatic cancer cell line CFPAC-1. METHODS: Inhibition of Ano1 was demonstrated by whole cell voltage clamp recordings of currents in cells transfected with full-length human Ano1. The effect of T16A(inh)-A01 on ICC proliferation was examined in situ in organotypic cultures of intact mouse small intestinal smooth muscle strips and in primary cell cultures prepared from these tissues. ICC were identified by Kit immunoreactivity. Proliferating ICC and CFPAC-1 cells were identified by immunoreactivity for the nuclear antigen Ki67 or EdU incorporation, respectively. RESULTS: T16A(inh)-A01 inhibited Ca(2+)-activated Cl(-) currents by 60% at 10µM in a voltage-independent fashion. Proliferation of ICC was significantly reduced in primary cultures from BALB/c mice following treatment with T16A(inh)-A01. Proliferation of the CFPAC-1 human cell-line was also reduced by T16A(inh)-A01. In organotypic cultures of smooth muscle strips from mouse jejunum, the proliferation of ICC was reduced but the total number of proliferating cells/confocal stack was not affected, suggesting that the inhibitory effect was specific for ICC. CONCLUSIONS: The selective Ano1 inhibitor T16A(inh)-A01 inhibited Ca(2+)-activated Cl(-) currents, reduced the number of proliferating ICC in culture and inhibited proliferation in the pancreatic cancer cell line CFPAC-1. These data support the notion that chloride channels in general and Ano1 in particular are involved in the regulation of proliferation.

Membrane permeable local anesthetics modulate Na(V)1.5 mechanosensitivity.

Voltage-gated sodium selective ion channel Na(V)1.5 is expressed in the heart and the gastrointestinal tract, which are mechanically active organs. Na(V)1.5 is mechanosensitive at stimuli that gate other mechanosensitive ion channels. Local anesthetic and antiarrhythmic drugs act upon Na(V)1.5 to modulate activity by multiple mechanisms. This study examined whether Na(V)1.5 mechanosensitivity is modulated by local anesthetics. Na(V)1.5 channels were expressed in HEK-293 cells, and mechanosensitivity was tested in cell-attached and excised inside-out configurations. Using a novel protocol with paired voltage ladders and short pressure pulses, negative patch pressure (-30 mmHg) in both configurations produced a hyperpolarizing shift in the half-point of the voltage-dependence of activation (V(1/2a)) and inactivation (V(1/2i)) by about -10 mV. Lidocaine (50 µM) inhibited the pressure-induced shift of V(1/2a) but not V(1/2i). Lidocaine inhibited the tonic increase in pressure-induced peak current in a use-dependence protocol, but it did not otherwise affect use-dependent block. The local anesthetic benzocaine, which does not show use-dependent block, also effectively blocked a pressure-induced shift in V(1/2a). Lidocaine inhibited mechanosensitivity in Na(V)1.5 at the local anesthetic binding site mutated (F1760A). However, a membrane impermeable lidocaine analog QX-314 did not affect mechanosensitivity of F1760A Na(V)1.5 when applied from either side of the membrane. These data suggest that the mechanism of lidocaine inhibition of the pressure-induced shift in the half-point of voltage-dependence of activation is separate from the mechanisms of use-dependent block. Modulation of Na(V)1.5 mechanosensitivity by the membrane permeable local anesthetics may require hydrophobic access and may involve membrane-protein interactions.

Ranolazine decreases mechanosensitivity of the voltage-gated sodium ion channel Na(v)1.5: a novel mechanism of drug action.

BACKGROUND: Na(V)1.5 is a mechanosensitive voltage-gated sodium-selective ion channel responsible for the depolarizing current and maintenance of the action potential plateau in the heart. Ranolazine is a Na(V)1.5 antagonist with antianginal and antiarrhythmic properties. METHODS AND RESULTS: Mechanosensitivity of Na(V)1.5 was tested in voltage-clamped whole cells and cell-attached patches by bath flow and patch pressure, respectively. In whole cells, bath flow increased peak inward current in both murine ventricular cardiac myocytes (24±8%) and human embryonic kidney 293 cells heterologously expressing Na(V)1.5 (18±3%). The flow-induced increases in peak current were blocked by ranolazine. In cell-attached patches from cardiac myocytes and Na(V)1.5-expressing human embryonic kidney 293 cells, negative pressure increased Na(V) peak currents by 27±18% and 18±4% and hyperpolarized voltage dependence of activation by -11 mV and -10 mV, respectively. In human embryonic kidney 293 cells, negative pressure also increased the window current (250%) and increased late open channel events (250%). Ranolazine decreased pressure-induced shift in the voltage dependence (IC(50) 54 µmol/L) and eliminated the pressure-induced increases in window current and late current event numbers. Block of Na(V)1.5 mechanosensitivity by ranolazine was not due to the known binding site on DIVS6 (F1760). The effect of ranolazine on mechanosensitivity of Na(V)1.5 was approximated by lidocaine. However, ionized ranolazine and charged lidocaine analog (QX-314) failed to block mechanosensitivity. CONCLUSIONS: Ranolazine effectively inhibits mechanosensitivity of Na(V)1.5. The block of Na(V)1.5 mechanosensitivity by ranolazine does not utilize the established binding site and may require bilayer partitioning. Ranolazine block of Na(V)1.5 mechanosensitivity may be relevant in disorders of mechanoelectric dysfunction.