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Acidic Amino Acids in the First Intracellular Loop Contribute to Voltage- And Calcium- Dependent Gating of anoctamin1/TMEM16A

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Acidic Amino Acids in the First Intracellular Loop Contribute to Voltage- And Calcium- Dependent Gating of anoctamin1/TMEM16A

Qinghuan Xiao et al. PLoS One.

Erratum in

  • PLoS One. 2014;9(9):e107343

Abstract

Anoctamin1 (Ano1, or TMEM16A) is a Ca2+-activated chloride channel that is gated by both voltage and Ca2+. We have previously identified that the first intracellular loop that contains a high density of acidic residues mediates voltage- and calcium-dependent gating of Ano1. Mutation of the four consecutive glutamates (444EEEE447) inhibits the voltage-dependent activation of Ano1, whereas deletion of these residues decreases apparent Ca2+ sensitivity. In the present study, we further found that deletion of 444EEEEEAVKD452 produced a more than 40-fold decrease in the apparent Ca2+ sensitivity with altered activation kinetics. We then systematically mutated each acidic residue into alanine, and analyzed the voltage- and calcium dependent activation of each mutation. Activation kinetics of wild type Ano1 consisted of a fast component (τfast) that represented voltage-dependent mode, and a slow component (τslow) that reflected the Ca2+-dependent modal gating. E444A, E445A, E446A, E447A, E448A, and E457A mutations showed a decrease in the τfast, significantly inhibited voltage-dependent activation of Ano1 in the absence of Ca2+, and greatly shifted the G-V curve to the right, suggesting that these glutamates are involved in voltage-gating of Ano1. Furthermore, D452A, E464A, E470A, and E475A mutations that did not alter voltage-dependent activation of the channel, significantly decreased Ca2+ dependence of G-V curve, exhibited an increase in the τslow, and produced a 2-3 fold decrease in the apparent Ca2+ sensitivity, suggesting that these acidic residues are involved in Ca2+-dependent gating of the channel. Our data show that acidic residues in the first intracellular loop are the important structural determinant that couples the voltage and calcium dependent gating of Ano1.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Activation of wild type and Δ444EEEEEAVKD452 Ano1 by Ca2+ in the whole-cell recordings.
A–F. Representative traces of wild type (A–C) and Δ444EEEEEAVKD452 (D–F) Ano1 activated by Ca2+ concentrations ranging from 180 nM to 25 µM. Cells were voltage clamped from a holding potential of 0 mV to various potentials between −100 mV to +100 mV in 20 mV increments for 700 ms, followed by a 100-ms step to −100 mV. G.H. The steady state current densities at +100 were plotted versus Ca2+ concentrations from wild type (G) and Δ444EEEEEAVKD452 (H). The plots were fitted to Hill equations. n = 4–9 cells.
Figure 2
Figure 2. Activation kinetics of Ano1 by Ca2+ and voltage.
A. Representative currents of wild type Ano1, activated by 180 µM Ca2+ at +100 mV from a holding potential of 0 mV (voltage protocol shown above). The currents were fitted to single exponentials (superimposed in green lines) and to two exponentials (superimposed in red lines). B. The relationship of the fast (τfast) (dash lines) and slow (τslow) (solid lines) components of the activation time constant of wild type Ano1 activated by 180 nM (blue triangle), 360 nM (green square), and 1 µM (red circle) Ca2+ at voltages between +20 mV to +100 mV from a holding potential of 0 mV (n = 4–6 cells). C. Representative currents of wild type, 444EEEE/AAAA447, ΔEAVK, and Δ444EEEEEAVKD452 activated by 1 µM Ca2+ at +100 mV from a holding potential of 0 mV. The currents were fitted to single exponentials (superimpose in green lines) and to two exponentials (superimposed in red lines). D.E. The time constant of the slow (D) and fast (E) components of wild type, 444EEEE/AAAA447, ΔEAVK, and Δ444EEEEEAVKD452 activated by 1 µM Ca2+ at +100 mV from a holding potential of 0 mV. n = 4–8 cells; *p<0.05 vs wild type.
Figure 3
Figure 3. Activation kinetics of single alanine substituted mutations.
A. Representative currents of WT, E446A, D452A, E464A, E470A, and E475A Ano1, activated by 1802+ at +100 mV from a holding potential of 0 mV. The currents were fitted to two exponentials (superimposed in red lines). B.C. The time constant of the slow (B) and fast (C) components of wild type and each single alanine substituted mutation activated by 180 nM Ca2+ at +100 mV from a holding potential of 0 mV. n = 4–8 cells.
Figure 4
Figure 4. Voltage-dependent activation of Ano1.
A–G. Representative traces of Ano1 activated by nominal “0” Ca2+ for wild type (A), E446A (B), E448A (C), D452A (D), E457A (E), and E464A (F). Cells were voltage clamped by stepping from a holding potential of 0 mV to various potentials between −100 mV to +200 mV in 20 mV increment for 50 ms, following by a step to −100 mV (voltage protocol is shown above B). G. Effects of single alanine-substituted mutations on currents activated by depolarization in the absence of Ca2+. The steady state outward currents at +200 mV were normalized to the maximal currents activated by 25 µM Ca2+. n = 4–11 cell; *p<0.05 vs wild type.
Figure 5
Figure 5. Activation of single alanine-substituted mutations by Ca2+ in excised patches.
A. Representative currents of wild type, E446A, D452A, and E464A Ano1 activated by 1 µM and 2 µM Ca2+. Patches were voltage clamped with 300-ms voltage steps from −160 to +200 mV in 20-mV increments, following by a 300-ms step to −100 mV (voltage protocol shown above). B–G. Normalized G-V relations of wild type (black), as well as E446A (red, A), E448A (red, B), and D452A (red, C), E457A (red, D), E459A (red, E), and E464A (red, F) activated by 1 µM and 2 µM Ca2+. Each G-V curve was fitted to a Boltzmann function, and then normalized to the maximum of the fit. n = 5–11 cells. H.I. The V0.5 (H) and equivalent gating charge z (I) obtained from the G-V curve of each single alanine-substituted mutation. n = 5–11 cells; *p<0.05 vs wild type.
Figure 6
Figure 6. D452A, E464A, E470A, and E475A mutations decreased the calcium sensitivity.
The steady state current densities at +100 were plotted versus Ca2+ concentrations from wild type (black), D452A (red, A), E464A (red, B), E470A (red, C), and E475A (red, D). The plots were fitted to Hill equations. n = 4–9 cells.

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