Supplementary Figure 7: S1 and S4 interact during gating.
From: A specialized molecular motion opens the Hv1 voltage-gated proton channel
(a-b) Voltage-dependence of channel conductance and ΔFs of 175C* and 249C* channels containing different combinations of D1 and R3 neutralizations. (a) G-V (closed circles) and F-V (open circles) of 175C* mutants. Note the good match between the G-V and F-V curves of each mutant. No current was observed for D1N-175C* channels, so only the F-V curve is provided for this construct. Curves were fitted with single Boltzmann. 175C* (n = 28 oocytes): V1/2 = 53 ± 3 mV, kT/ze0 = 19.6 ± 0.7 mV (G-V), V1/2 = 44 ± 3 mV, kT/ze0 = 23.0 ± 0.6 mV (F-V); R3S 175C* (n = 11 oocytes): V1/2 = 68 ± 2 mV, kT/ze0 = 27 ± 1 mV (G-V), V1/2 = 75 ± 6 mV, kT/ze0 = 26 ± 2 mV (F-V); D1N 175C* (n = 10 oocytes): V1/2 = 52 ± 3 mV, kT/ze0 = 15 ± 1 mV (F-V); and D1N R3S 175C* (n = 5 oocytes): V1/2 = 66 ± 11 mV, kT/ze0 = 25 ± 3 mV (G-V), V1/2 = 72 ± 9 mV, kT/ze0 = 21 ± 2 mV (F-V). (b) G-V (closed circles) and F-V (open circles) of 249C* mutants. No current was observed for D1N G249C* channels, so only the F-V curve is provided for this construct. Curves were fitted with single Boltzmann. 249C* (n = 10 oocytes): V1/2 = 55 ± 4 mV, kT/ze0 = 24 ± 1 mV (G-V), V1/2 = 3 ± 3 mV, kT/ze0 = 26 ± 2 mV (F-V); D1N 249C* (n = 1 oocyte): V1/2 = 21 mV, kT/ze0 = 15 mV (F-V); and D1N R3S 249C* (n = 6 oocytes): V1/2 = 97 ± 5 mV, kT/ze0 = 40 ± 3 mV (G-V), V1/2 = −18 ± 8 mV, kT/ze0 = 45 ± 2 mV (F-V). (c,d) Superposition of current (black) to inverted fluorescence (purple) or fluorescence (red) following repolarization from +100 to −80 mV for 175C* and D1N R3S 175C* channels (c), and 249C* and D1N R3S 249C* channels (d). (e,f) S4’s third arginine (R261, referred to as R3) influences S1 movement. (e) VCF recordings of 175C* (black trace) and 175C* with the R3S (red trace) or D1N (blue trace) mutation, with superimposed normalized fluorescence tails of 175C*, R3S 175C* and D1N 175C* following repolarization from +100 to −80 mV on the right. (f) Time constants of fluorescence recovery (τoff) of 175C* (n = 14 oocytes), R3S 175C* (n = 10 oocytes) and D1N 175C* (n = 10 oocytes) following repolarization from +100 to −80 mV. **, p < 0.01, two-tailed Student’s t-test. (g,h) S1’s aspartate D1 influences S4 movement. (g) VCF recordings of 249C* with (blue trace) or without (black trace) the D1N mutation, with superimposed normalized fluorescence tails of G249C* and D1N G249C* following repolarization from +100 to −80 mV on the right. (h) Time constants of the fast component of fluorescence recovery (fast τoff) of G249C* (n = 9 oocytes) and D1N G249C* (n = 7 oocytes) following repolarization from +100 to −80 mV. p < 0.01, two-tailed Student’s t-test. Error bars, s.e.m.
