EPR approaches to ion channel structure and function.

Abstract

The fundamental processes that underlie ion channel function are permeation/ selectivity and gating. In an effort to understand ion channel gating, we have used an approach that combines reporter-group spectroscopic techniques (spin labelling/ electron paramagnetic resonance, EPR) and electrophysiological methods with classical biochemical and molecular biological procedures. As an ideal test channel, we have focused our attention on the K+ channel from Streptomyces lividans, KcsA. Through site-directed spin labelling, cysteine chemistry was used to introduce nitroxide radicals into specific sites within KcsA with high reactivity and specificity. EPR spectroscopy analysis of the spin labelled mutants yields two types of structural information: (1) mobility and solvent accessibility of the attached nitroxide through collisional relaxation methods and (2) distances between pairs of nitroxides through dipole-dipole interactions. Using this approach, we analysed the correlation between KcsA crystal structure and the EPR data, extend it to derive low-resolution folds of full-length KcsA and apply it in the determination of the molecular rearrangements responsible for pH-dependent gating.