Two-step voltage-sensor activation of the human K _V 7.4 channel and effect of a deafness-associated mutation

Voltage-gated, potassium-selective K _V 7.4 channels are expressed in the inner ear and are crucial for hair-cell function and survival. Loss-of-function variants of KCNQ4 , the gene encoding K _V 7.4-channel subunits, cause non-syndromic progressive hearing loss (DFNA2). K _V 7.4 opening requires a voltage-dependent conformational change (activation) of the charged voltage-sensor domains (VSDs), and its transduction to the pore. Previously, fast charge displacement was reported during VSD activation at negative potentials, but it is unclear how this is coupled to slow channel opening occurring at more depolarized potentials. Here, we optically tracked K _V 7.4 VSD activation with voltage-clamp fluorometry, leveraging two different fluorophores and pulsed excitation, to thoroughly characterize VSD movements. We found that VSD activation comprises several voltage-dependent transitions, some of which had kinetics and voltage-dependence matching those of channel opening and closing. The deafness-associated mutation R216H, which substitutes a charged amino-acid in the VSD, impaired both VSD movements and channel opening, shifting them towards more depolarized potentials. This suggested that R216H impaired K _V 7.4 function by destabilizing VSD activation. Using molecular dynamics, we found that H216 reduced intramolecular interactions, thus decreasing the stability of an active VSD conformation. We propose that the K _V 7.4 VSD activates in two steps: a fast movement at negative voltages that represents a first transition to an intermediate state of activation; and this is followed by slower, depolarized component that represents subsequent full VSD activation, which drives channel opening.