Fig. 4: MFaN enhanced the proton gating of the mOtop1 channel without affecting its inhibition by Zn²⁺, activation rate, and ion selectivity.

A Typical traces (left panels) and statistics (right panel) illustrating similar inhibitions by 1 mM Zn²⁺ on mOtop1 currents activated by pH 5.5 alone (left upper panel) and pH 5.5 with 10 μM MFaN (left lower panel), currents were elicited using the provided solution perfusion protocols and recorded at -80 mV (n = 5). B Dose-response relationships of Zn²⁺ inhibiting mOtop1 currents activated by pH 5.5 alone or pH 5.5 with 10 μM MFaN, with the IC₅₀s determined as 72 ± 9 μM and 58 ± 13 μM, respectively (n = 5– 6). C Representative fitting analysis of the activation phase of mOtop1 currents activated by pH 5.5 alone (left panel) or pH 5.5 with 10 μM MFaN (either concurrently administrated or preincubated; middle and right panels) (n = 5). D Summary activation time constants from (C), showing that MFaN did not affect the activation rate of mOtop1 channels (n = 5). E mOtop1 currents were elicited by pH 5.5 alone (upper panel) or by pH 5.5 with 10 μM MFaN (lower panel) and recorded at the holding potential of -80 mV with 1 Hz voltage ramp from −80 mV to +80 mV (1 mV/ms). Purple and red arrows indicate the maximum voltage ramp-activated currents which were used to determine the channel’s reversal potential (V_rev), and gray arrows indicate the background voltage ramp-activated currents at pH 7.4. Note that the bath solution contains NMDG⁺ as the main cation. F Arrow-labeled currents in (E) were normalized to the cell capacitance, showing the averaged current density (MEAN ± SEM)-voltage relationships of mOtop1 channels, with MFaN robustly potentiating mOtop1 currents without affecting the channel’s V_rev (n = 9–13). G Summary V_rev comparison of mOtop1 channels activated by pH 5.5 alone or pH 5.5 with 10 μM MFaN in bath solutions containing the NMDG⁺, Na⁺, K⁺, or Cs⁺ (all at a concentration of 160 mM) as the main cation (n = 5–13). Data are presented as MEAN ± SEM. In (A, D, G), significant differences between groups were evaluated using unpaired t-test (***p < 0.001; ns, not significant).