Strege, Peter R.

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 Ca 2+ 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.

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.

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 Na V 1.5. 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 Na V 1.5. Within this set, a novel Na V 1.5 regulator, let-7f, resulted in decreased Na V 1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest Na V 1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.

ABSTRACT The Ca 2+ ‐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 Ca 2+ ‐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 Ca 2+ ‐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. FASEB J. 33, 6632–6642 (2019). www.fasebj.org

Significance Mechanical forces are important for normal gastrointestinal tract function. The enterochromaffin cells in the gastrointestinal epithelium have been proposed, but not previously shown, to be specialized sensors that convert forces into serotonin release, and serotonin released from these cells is important for normal gastrointestinal secretion and motility. The findings in this study show that some enterochromaffin cells are indeed mechanosensitive, and that they use mechanosensitive Piezo2 channels to generate an ionic current that is critical for the intracellular Ca 2+ increase, serotonin release, and epithelial fluid secretion.

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.

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.

AbstractBackgroundThe SCN5A‐encoded voltage‐gated sodium channel NaV1.5 is expressed in human jejunum and colon. Mutations in NaV1.5 are associated with gastrointestinal motility disorders. The rat gastrointestinal tract expresses voltage‐gated sodium channels, but their molecular identity and role in rat gastrointestinal electrophysiology are unknown.MethodsThe presence and distribution of Scn5a‐encoded NaV1.5 was examined by PCR, Western blotting and immunohistochemistry in rat jejunum. Freshly dissociated smooth muscle cells were examined by whole cell electrophysiology. Zinc finger nuclease was used to target Scn5a in rats. Lentiviral‐mediated transduction with shRNA was used to target Scn5a in rat jejunum smooth muscle organotypic cultures. Organotypic cultures were examined by sharp electrode electrophysiology and RT‐PCR.Key ResultsWe found NaV1.5 in rat jejunum and colon smooth muscle by Western blot. Immunohistochemistry using two other antibodies of different portions of NaV1.5 revealed the presence of the ion channel in rat jejunum. Whole cell voltage‐clamp in dissociated smooth muscle cells from rat jejunum showed fast activating and inactivating voltage‐dependent inward current that was eliminated by Na+ replacement by NMDG+. Constitutive rat Scn5a knockout resulted in death in utero. NaV1.5 shRNA delivered by lentivirus into rat jejunum smooth muscle organotypic culture resulted in 57% loss of Scn5a mRNA and several significant changes in slow waves, namely 40% decrease in peak amplitude, 30% decrease in half‐width, and 7 mV hyperpolarization of the membrane potential at peak amplitude.Conclusions & InferencesScn5a‐encoded NaV1.5 is expressed in rat gastrointestinal smooth muscle and it contributes to smooth muscle electrophysiology.

Anoctamin 1 (Ano1; TMEM16A) is a Ca2+-activated Cl− channel (CACC) expressed in interstitial cells of Cajal. The mechanisms by which Ca2+ 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 Ca2+ 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 Ca2+, 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 Ca2+. 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 Ca2+ and CACC inhibitors.