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

Abstract

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.

Regulation of TMEM16A/ANO1 and TMEM16F/ANO6 ion currents and phospholipid scrambling by Ca2+ and plasma membrane lipid

“…; Strege et al . ). Thus, other molecular regions within TMEM16 proteins as well as plasma membrane phospholipids control Cl − current and scramblase activity, in addition to the well‐described Ca 2+ binding sites (Fig.…”

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

“…3). The recorded currents were biophysically similar to heterologously expressed ANO1(0) currents (29, 30) with comparable time constants of activation (τ ACT at +100 mV: 664 ± 223 ms, I ANO1(0) ; 330 ± 50 ms, I gant61 ; n = 6–7, P > 0.05, by a 2‐tailed Student's t test). We conclude from these studies that blocking the binding of Gli1 and Gli2 to the ANO1 promoter DNA resulted in an increase in ANO1 protein expression and channel function.…”

“…3F). The recorded currents were biophysically similar to heterologously expressed ANO1(0) currents (29,30) with comparable time constants of activation (t ACT at +100 mV: 664 6 223 ms, I ANO1(0) ; 330 6 50 ms, I GANT61 ; n = 6-7, P . 0.05, by a 2-tailed Student's t test).…”

“…The abundance and variety of Ano1 proteins derive from inclusion or skipping of exons because of alternative splicing or the use of alternative promoters in the ANO1 gene (1, 18, 26–28). Alternative isoforms not only have altered kinetics and changes in their sensitivity to Ca 2+ (29, 30) but their expression is tissue dependent and has pathophysiological implications in disorders such as cancer, pain, and gastroparesis (18, 27, 31–33).…”

“…ABBREVIATIONS: Ano1, anoctamin 1; ChIP, chromatin immunoprecipitation; GANT61, Gli antagonist 61; Gli, glioma-associated oncogene protein; HEK293, human embryonic kidney 293; IBS, irritable bowel syndrome; rs7940681, reference SNP 7940681; siRNA, small interfering RNA; SNP, single-nucleotide polymorphism; T16A inh -A01, transmembrane protein 16A-ANO1 inhibitor; TSS, transcriptional start site The abundance and variety of Ano1 proteins derive from inclusion or skipping of exons because of alternative splicing or the use of alternative promoters in the ANO1 gene (1,18,(26)(27)(28). Alternative isoforms not only have altered kinetics and changes in their sensitivity to Ca 2+ (29,30) but their expression is tissue dependent and has pathophysiological implications in disorders such as cancer, pain, and gastroparesis (18,27,(31)(32)(33).…”