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, 108 (21), 8891-6

Voltage- And Calcium-Dependent Gating of TMEM16A/Ano1 Chloride Channels Are Physically Coupled by the First Intracellular Loop

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Voltage- And Calcium-Dependent Gating of TMEM16A/Ano1 Chloride Channels Are Physically Coupled by the First Intracellular Loop

Qinghuan Xiao et al. Proc Natl Acad Sci U S A.

Abstract

Ca(2+)-activated Cl(-) channels (CaCCs) are exceptionally well adapted to subserve diverse physiological roles, from epithelial fluid transport to sensory transduction, because their gating is cooperatively controlled by the interplay between ionotropic and metabotropic signals. A molecular understanding of the dual regulation of CaCCs by voltage and Ca(2+) has recently become possible with the discovery that Ano1 (TMEM16a) is an essential subunit of CaCCs. Ano1 can be gated by Ca(2+) or by voltage in the absence of Ca(2+), but Ca(2+)- and voltage-dependent gating are very closely coupled. Here we identify a region in the first intracellular loop that is crucial for both Ca(2+) and voltage sensing. Deleting (448)EAVK in the first intracellular loop dramatically decreases apparent Ca(2+) affinity. In contrast, mutating the adjacent amino acids (444)EEEE abolishes intrinsic voltage dependence without altering the apparent Ca(2+)affinity. Voltage-dependent gating of Ano1 measured in the presence of intracellular Ca(2+) was facilitated by anions with high permeability or by an increase in [Cl(-)](e). Our data show that the transition between closed and open states is governed by Ca(2+) in a voltage-dependent manner and suggest that anions allosterically modulate Ca(2+)-binding affinity. This mechanism provides a unified explanation of CaCC channel gating by voltage and ligand that has long been enigmatic.

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig. 1.
Fig. 1.
WT Ano1 gating by voltage and Ca2+. (A–E) Representative ICl,Ano1 in transfected HEK293 cells at the indicated free [Ca2+]. Voltage protocol is shown above A. (F) Steady-state current–voltage relationships (n = 5–9). (G) G/Gmax vs. Vm curves for Ano1 in excised patches activated by 0.6 μM (▲), 1 μM (▪), and 2 μM (●) Ca2+. (H) Instantaneous tail current density at −100 mV after prepulses to Vm plotted vs. free [Ca2+] and fitted to the Hill equation. (I) EC50 (▪) and nH (●) values from fits in H plotted vs. Vm.
Fig. 2.
Fig. 2.
G/Gmax vs. Vm curves for mutant Ano1 in excised patches at the indicated [Ca2+]i. (A and B) Solid lines without symbols: WT from Fig. 1G at 1 μM and 2 μM Ca2+. (A) ΔEAVK with 25 μM (▽), 2 μM (□), and 1 μM (●) Ca2+. (B) 444EEEE/AAAA447 with 25 μM (◇), 2 μM (□), and 1 μM (●) Ca2+. (C) V0.5 vs. [Ca2+] for WT (▪), ΔEAVK (formula image), and 444EEEE/AAAA447 (△).
Fig. 3.
Fig. 3.
Activation and deactivation kinetics of Ano1 with rapid Ca2+ and Ba2+ perfusion. (A–D) Representative traces of ICl,Ano1 in response to application (A and C) and washout (B and D) of 20 μM Ca2+ at the indicated holding potentials. (A and B) WT Ano1. (C and D) ΔEAVK. (E–G) Vm dependence of τon, τoff, and EC50 for WT Ano1 (○), 444EEEE/AAAA447 (▲), and ΔEAVK (●). (H and I) Representative traces of increase in current on application (H, arrowhead) and washout (I) of 1 mM Ba2+ (1 mM Ba2+, 100 μM EGTA) for WT Ano1. (J) τoff for Ba2+.
Fig. 4.
Fig. 4.
Deactivation of ICl,Ano1. (A–C) Vm and [Ca2+] dependence of tail current deactivation. Currents were activated using a +100-mV pulse, and tail currents were measured at the indicated potentials on the x-axis. (A) WT Ano1. (B) ΔEAVK. (C) 444EEEE/AAAA447. n = 4–6.
Fig. 5.
Fig. 5.
Activation of Ano1 in zero intracellular Ca2+. (A–C) Representative traces of WT Ano1 (A), 444EEEE/AAAA447 (B), and ΔEAVK (C) activated by Vm in nominally zero Ca2+. (D) Mean current density at +200 mV with zero intracellular Ca2+ plus 5 mM EGTA, zero intracellular Ca2+ and Mg2+ plus 5 mM EGTA (EGTA + 0 Mg), or zero Ca2+ plus 5 mM EGTA plus 1 mM BAPTA in both intracellular and extracellular solutions (EGTA + BAPTA). n = 5–11. (E) Steady-state I-V curve from cells dialyzed with zero Ca2+: nontransfected cells (control; □), WT Ano1 (formula image), Δ444EEEEEAVKD452 (△), ΔEAVK (▽), and 444EEEE/AAAA447 (♢). (n = 4–11).
Fig. 6.
Fig. 6.
WT Ano1 gating is dependent on permeant anions. (A and B) Current traces from a cell dialyzed with 180 nM Ca2+ and bathed in symmetrical 150 mM Cl (A) or 150 mM NO3 (B). (C) Fraction of instantaneous current relative to total current at the end of a 700-ms pulse to +100 mV. (D) Normalized (at +100 mV) I-V curves with Cl (●) or NO3 (▪) as permeant ions. (E) G/Gmax vs. Vm curves for WT Ano1 activated at different [Ca2+]i with Cl (open symbols) or SCN (filled symbols) as permeant anions. Circles represent 360 nM Ca2+; squares, 600 nM Ca2+; triangles, 1 μM; diamonds, 2 μM. (F) V0.5 determined from G/Gmax vs. Vm curves for Cl, NO3, and SCN. Red bars represent 0.6 μM Ca2+; green bars, 1 μM Ca2+. n = 4–11. (G) Cl dependence of G/Gmax vs. Vm curves. [Cl]e: 1 mM (green), 10 mM (red), 72 mM (blue), and 136 mM (black). (H) Anomalous mole fraction behavior of WT Ano1. The change in Erev is plotted as a function of the mole fraction of iodide (with the balance Cl). The red line represents the prediction from the Goldman–Hodgkin–Katz equation.

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