Bernard, Cheryl E.

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.

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.

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.

Anoctamin‐1 (Ano1) is a widely expressed protein responsible for endogenous Ca 2+ ‐activated Cl 2 currents. Ano1 is overexpressed in cancer. Differential expression of transcriptional variants is also found in other diseases. However, the mechanisms underlying regulation of Ano1 are unknown. This study identifies the Ano1 promoter and defines a mechanism for regulating its expression. Next‐generation RNA sequencing (RNA‐seq) analysis in human gastric muscle found a new exon upstream of the reported exon 1 and identified a promoter proximal to this new exon. Reporter assays in human embryonic kidney 293 cells showed a 6.7 ± 2.1fold increase in activity over empty vector. Treatment with a known regulator of Ano1 expression, IL‐4, increased promoter activity by 1.6 ± 0.02‐fold over untreated cells. The promoter region contained putative binding sites for multiple transcription factors including signal transducer and activator of transcription 6 (STAT6), a downstream effector of IL‐4. Chromatin immunoprecipitation (ChIP) experiments on T84 cells, which endogenously express Ano1, showed a 2.1 ± 0.12‐fold increase in binding of STAT6 to P0 after IL‐4 treatment. These results were confirmed by mutagenesis, expression, and RNA interference techniques. This work allows deeper understanding of the regulation of Ano1 in physiology and as a potential therapeutic target in a variety of diseases—Mazzone, A., Gibbons, S. J., Bernard, C. E., Nowsheen, S., Middha, S., Almada, L. L., Ordog, T., Kendrick, M. L., Reid Lombardo, KM., Shen, K. R., Galietta, L. J. V., Fernandez‐Zapico, M. E., and Farrugia, G., Identification and characterization of a novel promoter for the human ANO1 gene regulated by the transcription factor signal transducer and activator of transcription 6 (STAT6). FASEB J. 29, 152–163 (2015). www.fasebj.org

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.

Hydrogen sulfide (H2S) is produced endogenously by l-cysteine metabolism. H2S modulates several ion channels with an unclear mechanism of action. A possible mechanism is through reduction-oxidation reactions attributable to the redox potential of the sulfur moiety. The aims of this study were to determine the effects of the H2S donor NaHS on NaV1.5, a voltage-dependent sodium channel expressed in the gastrointestinal tract in human jejunum smooth muscle cells and interstitial cells of Cajal, and to elucidate whether H2S acts on NaV1.5 by redox reactions. Whole cell Na+ currents were recorded in freshly dissociated human jejunum circular myocytes and NaV1.5-transfected human embryonic kidney-293 cells. RT-PCR amplified mRNA for H2S enzymes cystathionine β-synthase and cystathionine γ-lyase from the human jejunum. NaHS increased native Na+ peak currents and shifted the half-point ( V1/2) of steady-state activation and inactivation by +21 ± 2 mV and +15 ± 3 mV, respectively. Similar effects were seen on the heterologously expressed NaV1.5 α subunit with EC50s in the 10−4 to 10−3 M range. The reducing agent dithiothreitol (DTT) mimicked in part the effects of NaHS by increasing peak current and positively shifting steady-state activation. DTT together with NaHS had an additive effect on steady-state activation but not on peak current, suggesting that the latter may be altered via reduction. Pretreatment with the Hg2+-conjugated oxidizer thimerosal or the alkylating agent N-ethylmaleimide inhibited or decreased NaHS induction of NaV1.5 peak current. These studies show that H2S activates the gastrointestinal Na+ channel, and the mechanism of action of H2S is partially redox independent.

Antispasmodics are used clinically to treat a variety of gastrointestinal disorders by inhibition of smooth muscle contraction. The main pathway for smooth muscle Ca2+entry is through L-type channels; however, there is increasing evidence that T-type Ca2+channels also play a role in regulating contractility. Otilonium bromide, an antispasmodic, has previously been shown to inhibit L-type Ca2+channels and colonic contractile activity. The objective of this study was to determine whether otilonium bromide also inhibits T-type Ca2+channels. Whole cell currents were recorded by patch-clamp technique from HEK293 cells transfected with cDNAs encoding the T-type Ca2+channels, CaV3.1 (α1G), CaV3.2 (α1H), or CaV3.3 (α1I) alpha subunits. Extracellular solution was exchanged with otilonium bromide (10−8to 10−5M). Otilonium bromide reversibly blocked all T-type Ca2+channels with a significantly greater affinity for CaV3.3 than CaV3.1 or CaV3.2. Additionally, the drug slowed inactivation in CaV3.1 and CaV3.3. Inhibition of T-type Ca2+channels may contribute to inhibition of contractility by otilonium bromide. This may represent a new mechanism of action for antispasmodics and may contribute to the observed increased clinical effectiveness of antispasmodics compared with selective L-type Ca2+channel blockers.