Probing the S4-S5 Linker Movement During Activation in KV Channels

Abstract

Numerous electrophysiological and fluorescence studies have been performed on the voltage sensor (VS) movement in Kv channels, while less have focused on the S4-S5 linker, mainly due to limitations in fluorophore labeling. Electromechanical coupling between the VS movement and pore opening in Kv channels is, in part, controlled by the S4-S5 linker and it is therefore crucial to understand the mechanism underlying S4-S5 linker movement. The question remains which part of the S4-S5 linker moves during independent voltage sensor activation and which part during cooperative pore opening. We also wanted to address with which of the major charge movements S4-S5 linker movements is associated. In order to address the movement of the S4-S5 linker, we have incorporated a fluorescent unnatural amino acid (Anap) at four positions in the linker of a Shaker Kv channel (L382, R387, K390 and A391) to probe conformational changes. Using voltage clamp fluorometry, we found that the entire linker moves during voltage sensor activation. Only the C-terminal part of the linker also moves during pore opening and is thus associated with pore opening. To separate the 1st and 2nd major gating charge movements, we inserted the F290A mutation. We found that the entire linker moves during both major charge movements. Based on these results, we suggest a model where the linker undergoes distinct conformational changes during each S4 movement throughout voltage sensor activation. The energy generated during voltage sensor activation is stored in the C-terminal S4-S5 linker that finally relaxes during pore opening.