Independent activation of distinct pores in dimeric TMEM16A channels

Abstract

The TMEM16 family encompasses Ca²⁺-activated Cl^- channels (CaCCs) and lipid scramblases. These proteins are formed by two identical subunits, as confirmed by the recently solved crystal structure of a TMEM16 lipid scramblase. However, the high-resolution structure did not provide definitive information regarding the pore architecture of the TMEM16 channels. In this study, we express TMEM16A channels constituting two covalently linked subunits with different Ca²⁺ affinities. The dose-response curve of the heterodimer appears to be a weighted sum of two dose-response curves-one corresponding to the high-affinity subunit and the other to the low-affinity subunit. However, fluorescence resonance energy transfer experiments suggest that the covalently linked heterodimeric proteins fold and assemble as one molecule. Together these results suggest that activation of the two TMEM16A subunits likely activate independently of each other. The Ca²⁺ activation curve for the heterodimer at a low Ca²⁺ concentration range ([Ca²⁺] < 5 µM) is similar to that of the wild-type channel-the Hill coefficients in both cases are significantly greater than one. This suggests that Ca²⁺ binding to one subunit of TMEM16A is sufficient to activate the channel and that each subunit contains more than one Ca²⁺-binding site. We also take advantage of the I-V curve rectification that results from mutation of a pore residue to address the pore architecture of the channel. By introducing the pore mutation and the mutation that alters Ca²⁺ affinity in the same or different subunits, we demonstrate that activation of different subunits appears to be associated with the opening of different pores. These results suggest that the TMEM16A CaCC may also adopt a "double-barrel" pore architecture, similar to that found in CLC channels and transporters.

Figure 1.

Constructing a covalently linked tandem dimer of the TMEM16A channel. (A) Structure of the nhTMEM16 molecule, showing the positions of corresponding amino acid residues, the mutations of which alter the Ca²⁺ affinity (E698 and E701) and current rectification (K584) of the TMEM16A channel. The nhTMEM16 residues corresponding to K584, E698, and E701 of TMEM16A are N378, E503, and E506, respectively. (B) Schematic diagrams and the nomenclature of various homodimeric and heterodimeric TMEM16A constructs. Red stars represent the E698C mutation.

Figure 2.

Recording traces of wild-type TMEM16A and tandemly linked dimers. Channel currents were induced by a test [Ca²⁺] pulse (the middle pulse, indicated by red horizontal lines) and two saturating [Ca²⁺] pulses (indicated by blue horizontal lines) before and after the test pulse for inducing maximal current. Red traces: 40 mV; black traces: −40 mV. (A) Wild-type TMEM16A (namely, WT∙WT). (B) E698C∙E698C. (C) E698C-E698C. (D) E698C-WT.

Figure 3.

Dose–response curves of various wild-type and mutant TMEM16A channels. (A) Comparison of the dose–response curves of WT∙WT, E698C∙E698C, E698C-E698C, and E698C-WT obtained at −40 mV (top) and 40 mV (bottom). The dose–response curve of the WT∙WT channel was obtained from Ni et al. (2014). Values of K_1/2 (µM) and h for WT∙WT, E698C∙E698C, and E698C-E698C are shown. The curves for the tandem heterodimeric channel (E698C-WT) in two regions were scaled from the dose–response curves of the WT∙WT channel (I = a × I_WT∙WT, black) and the E698C∙E698C channel (I = a + (1 − a) × I_{E698C∙E698C}, red), where the values of a (0.39 and 0.44 in the top and bottom panel, respectively) were determined by averaging the values of I_norm at [Ca²⁺] = 3.13 µM and [Ca²⁺] = 5 µM. (B) Comparison of the dose–response curves of WT∙WT, E701C∙E701C, and E701C-WT obtained at −40 mV (top) and 40 mV (bottom). Values of the K_1/2 and h for E701C∙E701C are not depicted because of the lack of current saturation in the dose–response curve. The curves for E701C-WT were scaled from those of the WT∙WT channel (I = b × I_WT∙WT, black) and the E701C∙E701C channel (I = b + (1 − b) × I_{E701C∙E701C}, red). The values of b (0.542 and 0.566 in the top and bottom panel, respectively) were obtained by averaging the values of I_norm at [Ca²⁺] = 3.13 µM and [Ca²⁺] = 5 µM. All data points in A and B are depicted as mean ± SEM. The fitted values of K_1/2 and Hill coefficient are shown next to the fitted curves.

Figure 4.

Comparison of the apparent FRET efficiencies (E_app) between C/Y-tagged monomeric WT constructs (black) or C/Y-tagged tandem E698C-WT dimeric constructs (red).

Figure 5.

Dose–response curves of the WT-WT linked homodimer and the E698C-WT linked heterodimer at [Ca²⁺] < 5 µM. All current was normalized to that obtained with 5 µM [Ca²⁺]. Error bars represent SEM. The fitted values of K_1/2 and Hill coefficient are shown next to the fitted curves.

Figure 6.

Comparison of Na⁺ and Cl⁻ permeation properties through the pores of the WT∙WT channel (abbreviated as WT) and the K584Q mutant. (A) I-V curves of the wild-type channel (left) and the K584Q mutant (right) in three [NaCl]_i conditions: 140 mM (black traces), 84 mM (red traces), and 42 mM (blue traces). The reversal potentials (E_rev) in each recording are better shown in the inset of each panel. (B) Averaged values of E_rev of WT∙WT (black), K584Q mutant (red), E698C-WT(K584Q) (green), and E698C(K584Q)-WT (blue) in 140, 84, and 42 mM [NaCl]_i. Values of E_rev of various channels in the same ionic conditions (Table S1) were not significantly different from one another (P > 0.05, one-way ANOVA).

Figure 7.

Pore properties of the wild-type TMEM16A channel, K584Q mutant, and the MT-WT(K584Q) and MT(K584Q)-WT heterodimeric channels, where MT refers to the E698C subunit. WT(K584Q) and MT(K584Q), respectively, represent the WT and E698C subunits harboring K584Q mutation. [Cl⁻]_i = [Cl⁻]_o = 140 mM in all recordings. (A) Recordings of the K584Q mutant in 20 µM [Ca²⁺] (left) in response to a voltage step family from −80 to 80 mV (right). Leak current has been subtracted. (B) I-V relationships of the K584Q mutant obtained from a ramp voltage protocol (−80 to 80 mV) in 20 mM [Ca²⁺] (black) and 20 µM [Ca²⁺] (red) and from a voltage step protocol like that shown in A (red squares, n = 5). For the two ramp I-V curves, the width of each curve represents the SEM of the averaged current of each digitized data point in the I-V curve. RI = 0.45 ± 0.01 (n = 7) and 0.48 ± 0.01 (n = 5) for the I-V curves in 20 µM [Ca²⁺] and 20 mM [Ca²⁺], respectively. (C) Comparison of the I-V curve rectifications between the wild-type channel and K584Q mutant (left) and between E698C-WT (abbreviated as MT-WT; blue) and MT(K584Q)-WT (red, middle) or MT-WT(K584Q) (red, right). All I-V curves were obtained with 20 µM [Ca²⁺]. Four to seven normalized I-V curves were averaged. The width of each I-V curve represents the SEM of the averaged values. RIs for WT, K584Q, MT-WT, MT(K584Q)-WT, and MT-WT(K584Q) were 1.00 ± 0.02, 0.45 ± 0.01, 0.78 ± 0.06, 0.78 ± 0.05, and 0.47 ± 0.04, respectively.