A novel exon in the human Ca^2+-activated Cl^- channel Ano1 imparts greater sensitivity to intracellular Ca²⁺

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 T16A_inh-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.