Long-lasting effect of membrane stretch on Hv1 activation does not require membrane depolarization and is slowly reversible. (A) Hv1 proton current elicited by consecutive depolarization steps R (reference) and T (test) in the absence of mechanical stimulation (pH_i and pH_o 6.0). Changes in kinetics of activation between the first and second depolarization are quantified by the ratio τ_a,T/τ_a,R reported in E. (B) Hv1 current measured as in A with mechanical stimulation (negative pressure) delivered at holding membrane potential between T and R pulses. (C) Hv1 current measured as in A with an additional depolarization step (P) between the reference and test pulses. (D) Hv1 current measured as in C with mechanical stimulation delivered during the depolarization step (P) between T and R pulses. (E) Box plot comparing τ_a,T/τ_a,R values measured under the voltage and pressure protocols shown in A–D. τ_a,T/τ_a,R < 1 indicates accelerated activation kinetics during the T pulse compared with the R pulse. Boxes indicate median ± SD. Whiskers show 5th and 95th percentiles. Asterisks indicate statistically significant difference: ***, P < 0.0001. (F) Recovery from membrane stretch–induced acceleration of Hv1 activation. Hv1 was first activated with a combined voltage/pressure protocol as in D. (left) τ_a,R, τ_a,P, and τ_a,T parameters were measured and normalized, using τ_a,R as reference. After the time interval Δt, the combined voltage/pressure protocol was applied again, and the normalized τ_a values were recalculated. The protocol was applied only once per patch. Light gray circles (Δt = 1 s): n = 4. Gray squares (Δt = 60 s): n = 5. Black diamonds (Δt = 300 s): n = 7. Error bars are SEM. (right) Bar chart showing the recovery of τ_a,R over time after mechanical stimulation (black columns). The reference values corresponding to full recovery are shown as purple columns. Error bars are SEM. Asterisks indicate statistically significant difference: *, P < 0.01. The normalized τ_a,P and τ_a,T values in the left panel corresponding to a particular Δt (τ_a,P(Δt)/τ_a,R(Δt), and τ_a,T(Δt)/τ_a,R(Δt)) can be renormalized relative to τ_a,R(control) using the corresponding τ_a,R(Δt)/τ_a,R(control) ratios from the right panel as multiplication factors.