Movement of the S4-S5 Linker of KvAP during Gating

Abstract

Voltage-gated potassium channels are formed by four voltage sensor domains (VSD) and a central pore domain. The voltage sensors are activated during membrane depolarization, leading to pore opening through a process called electromechanical coupling. Functional and structural evidence suggests that the covalent link between the VSD and the pore, the α-helical S4-S5 linker, plays a crucial role in the electromechanical coupling. However, no data on the rearrangements at the cytosolic face of the channel is presently available leaving the molecular mechanism of coupling subject for speculation. In this study, we determined the position of the S4-S5 linker in the open and closed state of the bacterial KvAP channel. To this end, we utilized LRET (Lanthanide Resonance Energy Transfer) to measure the cross pore distances of various positions in the S4-S5 linker. A KvAP dimer with a single cysteine has been constructed resulting in a tetramer with two symmetric cysteines, which we label with a Tb-chelate and an organic fluorophore, respectively. The labeled protein is reconstituted in lipid vesicles. We have measured distances in both closed and open state for 8 positions along the 10 amino acid linker. Based on the results we aim to create a 3D model of the movement of the S4-S5 linker during the opening of the pore.