Identification of the Acid-Sensitive Site Critical for Chloral Hydrate (CH) Activation of the Proton-Activated Chloride Channel

Alignment of the extracellular domain of the PAC channels. A, Alignment of amino acid sequences of the PAC orthologs that encode extracellular domain between TM1 and TM2. Residues shown in red color are conservative negative charged residues. The hPAC represents human TMEM206 (RefSeq: NM_018252.2; UniProtKB: Q9H813), zPAC (zebrafish TMEM206, RefSeq: NM_001291762.1; UniProtKB: X1WG42), cPAC (chicken TMEM206, RefSeq: XM_419431.6; UniProtKB: A0A1D5PSZ0), rPAC (naked mole rat TMEM206, RefSeq: XM_004853543.3; UniProtKB: A0A0N8ETT8), gPAC (green anole TMEM206, RefSeq: XM_003216011.3; UniProtKB: G1KFB8). B, The extracellular domain and the location of the conservative negative charged residues of the hPAC channel.

E257Q and D289N mutants abolished pH-dependent currents of the hPAC channel. A–C, The typical whole-cell current traces of the hPAC channel (WT) were recorded in the TEVC configuration. The cells were bathed with solutions at indicated pH values. D–F, The typical whole-cell current traces of the D289N mutant in bath solutions at displayed pH values. G–I, The plotted G-V curves of the hPAC WT channel, E257Q, and D289N mutants. J, The ΔV_h values of the WT hPAC channel and mutants as listed. ΔV_h was calculated as the V_h values obtained at pH 5.5 minus the V_h values of the same construct at pH 4.6. Data are expressed as mean ± SEM, n ≧ 6. K, Relative conductance values of the WT hPAC channel and its mutants at 200-mV depolarization. Data are expressed as mean ± SEM, n ≧ 6. The V_h and relative conductance values of mutants are statistically lower than those of the WT hPAC channel labeled by *. The V_h and relative conductance values of the mutants are statistically higher than the value of the WT channel labeled by #. The pH dependence of the WT hPAC channel and some mutants at 200-mV depolarization are shown in Extended Data Figure 3-1.

E257 and D289 mutants remove the pH sensitivity of the hPAC channel but maintain temperature-dependent activity. A–C, Time courses of currents change of the WT, E257Q, and uninjected oocytes while the temperature of bath solution was enhanced from room temperature to 40°C. D–F, Plotted GV curves of E257D, D289K, and E107C at different pH values, respectively. G, H, The plotted V_h values of E257, D289, and E107 mutants at different pH, respectively. I, The relative conductance values of E107, E257, and D289 mutants at 200 mV. J, The relative conductance values of the mutants are statistically different from the WT channel are labeled by *. The membrane expression levels of the WT channel, E257Q, and D289N mutants were not substantially altered (Extended Data Fig. 4-1).

The disulfide bond formed between E257C and D289C stabilizes the opening conformation of the hPAC channel protein without proton binding. A, The conformation model of the hPAC channel based on the cryo-EM structure at pH 4 (left top) and pH 8 (left bottom) were shown, respectively. The enlarged local conformation of E257 (blue) on one subunit (green), D289 (yellow), and E107 (red) on its adjacent subunit (silver) were shown. The spatial distances among them were measured at pH 4 (right-top) and 8 (right-bottom). B, C, The Western blot results of the hPAC channel and its mutants using the plasma membrane protein in transfected HEK293T cells. D, E, The expression level of the hPAC channel and its mutants in the plasma membrane of Xenopus oocytes. F, The plotted GV curves of the double mutant E257C/D289C bathed in solutions at different pH and in solution at pH 4.6 applied with 50 mm DTT (red). G, H, The plotted GV curves of the single mutants E257C and D289C at different pH values. I, Time course of currents decline of E257C/D289C double mutant in solution at pH 4.6 response to 50 mm DTT application. J, K, The plotted GV curves of the double mutants H98C/E107C and E257D/D289E in bath solutions at indicated pH values.

In the background of the A81C mutant, the E261Q removes the pH-dependent activity rather than E257Q and D289N mutants. A, B, TEVC recorded the sample current traces of A81C mutant in a solution at indicated pH values. C–F, The plotted GV curves of the mutants A81C, A81C/E257Q, A81C/D289N, and A81C/E261Q. G, H, TEVC recorded the typical current traces of the A81C/E261Q mutant in the solution at indicated pH values. I, The plotted GV curves of the mutants A81C/E224Q. J, The plotted V_h values of the WT hPAC, A81C, and A81C/E261Q mutants at different pH. K, The ΔV_h of the indicated mutants from pH 7.3 to pH 4.6. The relative conductance values of mutants A81C, A81C/E224Q, A81C/E257Q, A81C/E261Q, and A81C/D289N at 200 mV. The relative conductance of mutants was compared with the conductance of wild-type channels by t test; *p < 0.05.

Chloral hydrate activates the hPAC channel by stabilizing the pH-dependent activation conformation. A, B, The sample current traces of the WT hPAC in the perfusion solution at pH 5.5 were perfused with 0 (A) or 50 (B) mm chloral hydrate as indicated. C, D, The sample traces of the uninjected oocytes with 0 (C) or 100 (D) mm chloral hydrate. E, The plotted GV curves of the hPAC channel at different concentrations of chloral hydrate. The conductance values are normalized to maximal conductance (G_max) at 200 mV with 50 mm chloral hydrate application. F, The Hill equation fit of the activation of chloral hydrate in 200 mV (EC₅₀ = 22.09 ± 4.79 mm, coefficient factor = 1.9 ± 0.9). G, EC₅₀ values show weak voltage dependence from 20- to 200-mV depolarization. H, The activation time course of the hPAC channel response to different concentrations of chloral hydrate at pH 5.5. I–K, The plotted GV curves of E257C/D289C, A81C, and E257D/D289E mutants in solution with different concentrations of chloral hydrate. L, The Hill equation fit of the activation effect of chloral hydrate on E257D/D289E mutants at 200 mV (EC₅₀ = 20.79 ± 1.69 mm, coefficient factor = 2.78 ± 0.68).

The activation effect of chloral hydrate on the hPAC channel at pH 4.6 and 7.3. A, The plotted GV curves of the hPAC channel at pH 4.6 with different concentrations of chloral hydrate. The conductance values are normalized to maximal conductance (G_max) at 200 mV with 20 mm chloral hydrate. B, The Hill equation fit of the activation of chloral hydrate at 200 mV (EC₅₀ = 1.4 ± 0.3 mm, coefficient factor = 0.9 ± 0.2). C, EC₅₀ values show weak voltage dependence from 20- to 200-mV depolarization. D, The plotted GV curves of the hPAC channel at different concentrations of chloral hydrate at pH 7.3.