External protons accelerate the deactivation rate of cardiac hERG channels by an unknown mechanism. Recently, we showed that the hERG voltage sensor displays pronounced mode-shift behaviour due to relaxation, which stabilizes the activated voltage sensor position and limits its return upon repolarization1. We hypothesized that protons accelerate deactivation by destabilizing the relaxed state of the voltage sensor. To test this, we used cut-open voltage clamp and voltage clamp fluorimetry in Xenopus oocytes to measure voltage sensor movement, in addition to measurements of pore gating using two-electrode voltage clamp. Using durations that capture the majority of charge movement, the voltage-dependence of off-gating charge movement at pH 7.4 (V1/2 = −75.5 ± 2.3 mV, n=4) was found to be −34 mV left-shifted from that of on-gating charge (V1/2 = −41.4 ± 3.5 mV, n=5) indicative of relaxation-induced stabilization of the activated voltage sensor. At pH 6.5 this mode-shift was reduced to −19 mV (p<0.002; off-gating charge V1/2 = −51.8 ± 3.3 mV, n=5; on-gating charge V1/2=-32.7 ± 4.2 mV, n=5). This finding was corroborated by fluorescence reports of voltage sensor movement, which showed that the mode-shift was reduced from −43 mV at pH 7.4 (n=5) to −23 mV at pH 6.5 (n=5). In both of these reports of voltage sensor mode-shift, acidic pH induced a large depolarizing shift in the voltage-dependence of voltage sensor return upon hyperpolarization. These data suggest that the relaxed state of the voltage sensor is destabilized by external protons and that this contributes to faster deactivation gating in hERG channels. 1Thouta et al., Biophys J.112:300-312.2017