Probing the Movement of the Ball and Chain during N-type Inactivation in Kv Channels

Abstract

N-type inactivation is a mechanism in certain Kv channels where the N-terminal peptide occludes the pore upon depolarization resulting in block of ionic currents (ball-and-chain). Numerous mutational studies have characterized N-type inactivation functionally, while X-ray crystal structures have yet to include the ball-and-chain structure. It still remains unknown how far the N-terminus travels during N-type inactivation and where it is located in the resting state. Does it reside in a fixed position near the T1 window or is it randomly floating in the cytosol? By incorporating a fluorescent unnatural amino acid (Anap) into the N-terminus and into receptor sites (T1 window and S4-S5 linker) we directly tracked the movement of the ball peptide using voltage clamp fluorometry in Xenopus oocytes. Voltage dependency and kinetics of ball-peptide movement correlated with N-type inactivating currents. Furthermore, we address the question as to whether the fluorescence changes are caused by polarity changes or quenching, by measuring the Anap emission spectra at both resting and activated state.