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Comparative Study
. 2015 Nov 1;309(9):G743-9.
doi: 10.1152/ajpgi.00074.2015. Epub 2015 Sep 10.

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

Affiliations
Comparative Study

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

Peter R Strege et al. Am J Physiol Gastrointest Liver Physiol. .

Abstract

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) VSports手机版. 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. .

Keywords: T16Ainh-A01; TMEM16A; anoctamin 1; electrophysiology; ion channels; niflumic acid V体育安卓版. .

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Figures

Fig. 1.
Fig. 1.
Human anoctamin 1 (Ano1) isoform with the novel 5′ “exon 0” [Ano1(+0)] has a greater Cl current density yet similar voltage dependence than the isoform lacking this exon Ano1 without exon 0 [Ano1(−0)]. A–E: Ano1(−0) (dark gray), Ano1(+0) (black), or no construct (light gray) were transfected with green fluorescent protein (GFP) into human embryonic kidney (HEK)-293 cells, and Ca2+-activated Cl currents were then recorded by whole cell electrophysiology with symmetrical extracellular Cl concentraion ([Cl]o)/intracellular Cl concentration ([Cl]i) and 500 nM free intracellular Ca2+ concentration ([Ca2+]i). A: representative currents elicited by serial depolarizations from −100 mV. The dotted line indicates 0 pA/pF. B and C: mean current density (I/C; B) and I/C normalized to maximum (C) versus voltage stimulus. D and E: half-maximal voltage (V1/2) of I/C (D) and I/C at maximal conductance (max I/C; E) at 500 nM free [Ca2+]i. n = 9–16 cells. *P < 0.05 vs. Ano(−0) and †P < 0.05 vs. untransfected (UT) cells by an unpaired two-tailed Student's t-test.
Fig. 2.
Fig. 2.
Anion permeabilities of Ano1(+0) and Ano1(−0). A and B: Ano1(−0) (gray) or Ano1(+0) (black) were transfected with GFP into HEK-293 cells, and anion currents were then recorded by whole cell electrophysiology with the extracellular-to-intracellular anion ratio set to 130:0 for the extracellular X concentration-to-intracellular X concentration ratio and 30:150 for the [Cl]o-to-[Cl]i ratio, where anion X was CH3SO3 (MS), F, Cl, Br, or I. A: anion currents elicited by steps from −100 to 0 mV, the expected reversal potential of Cl. The dotted line indicates 0 pA/pF. B: relative permeabilities (PX/PCl) of Ano1(−0) or Ano1(+0) were derived from the Goldman-Hodgkin-Katz equation applied to reversal potentials from current-voltage curves. The current density at 0 mV I0/C (A) visually correlates with PX/PCl (B) because Cl potential ≈ 0 mV. n = 5 cells. *P < 0.05 vs. Ano1(−0) by a paired two-tailed Student's t-test.
Fig. 3.
Fig. 3.
Ano1(+0) has an increased [Ca2+]i sensitivity compared with Ano1(−0). A–H: Ano1(−0) or Ano1(+0) were transfected into HEK-293 cells, and Ca2+-activated Cl currents were then recorded by whole cell electrophysiology with symmetrical [Cl]o/[Cl]i and free [Ca2+]i buffered at 30-3,000 nM. A and B: representative current traces of Ano1(−0) (A) and Ano1(+0) (B) elicited by depolarizations from −100 to +100 mV with 30-3,000 nM [Ca2+]i. The dotted line indicates 0 pA/pF. C and D: mean I/C of Ano1(−0) (C) and Ano1(+0) (D) versus voltage stimulus with 30-3,000 nM [Ca2+]i. E: max I/C versus free [Ca2+]i. Upper asymptote: 641 pA/pF for Ano1(−0) and 763 pA/pF for Ano(+0). Lower asymptote: 60 pA/pF for Ano1(−0) and 85 pA/pF for Ano1(+0). Hill slope: 1.84 for Ano1(−0) and 2.54 for Ano1(+0). F: max I/C normalized to the asymptotes of E. EC50: 1.56 μM for Ano1(−0) and 0.71 for Ano1(+0). G and H: time constants (τ) of activation (τACT; G) and deactivation (τDEACT; H) of Ano1(−0) (gray) or Ano1(+0) (black) versus [Ca2+]i. n = 5 cells/concentration. *P < 0.05 vs. Ano1(−0) by an unpaired two-tailed Student's t-test.
Fig. 4.
Fig. 4.
Niflumic acid (NFA) exhibits greater block of and a lower IC50 against Ano1(+0) than Ano1(−0). A–C: Ano1(−0) (top) or Ano1(+0) (bottom) were transfected into HEK-293 cells, and Ca2+-activated Cl currents were then recorded by whole cell electrophysiology with symmetrical [Cl]o/[Cl]i and free [Ca2+]i buffered at 500 nM. A: representative currents elicited by steps from −100 to +100 mV with 0 (control, black trace) or 10−6–10−4 M NFA (gray traces). The dotted line indicates 0 pA/pF. B and C: mean I/C (B) and I/C normalized to maximum (C) versus voltage stimulus. D–F: τ of activation (D), V1/2 of I/C (E), and max I/C (F) versus the extracellular concentration of NFA ([NFA]o; in mol/l) of Ca2+-activated Cl channels from HEK-293 cells coexpressing GFP with the Ano1(−0) (gray) or Ano1(+0) isoform (black). n = 5 cells/concentration. *P < 0.05 by an unpaired two-tailed Student's t-test.
Fig. 5.
Fig. 5.
T16Ainh-A01 blocks the Ano1(+0) isoform. A–C: Ano1(−0) (top) or Ano1(+0) (bottom) were transfected into HEK-293 cells, and Ca2+-activated Cl currents were then recorded by whole cell electrophysiology with symmetrical [Cl]o/[Cl]i and free [Ca2+]i buffered at 500 nM. A: representative currents elicited by steps from −100 to +100 mV with 0 (control, black trace) or 10−6-10−4 M T16Ainh-A01 (gray traces). The dotted line indicates 0 pA/pF. B and C: mean I/C (B) and I/C normalized to maximum (C) versus voltage stimulus. D–F: τ of activation (D), V1/2 of I/C (E), and max I/C (F) versus the concentration of T16Ainh-A01 ([T16Ainh]o; in mol/l), of Ca2+-activated Cl channels from HEK-293 cells coexpressing GFP with the Ano1(−0) (gray) or Ano1(+0) isoform (black). n = 5 cells/concentration. *P < 0.05 by an unpaired two-tailed Student's t-test.

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