Ion channels are intimately involved in the generation and modulation of electrical events in excitable tissues like nerve and muscle. Differential ion channel activity underlies the resting potential, the generation and propagation of action potentials, neurotransmitter release, excitation-contraction coupling, etc. Recently, it has become clear that several human neuromuscular disorders are ion channel diseases: Hyperkalemic periodic paralysis, paramyotonia congenita, myotonia, congenita, episodic ataxia, and some forms of cardiac arrhythmias, like long QT syndromes. These findings make ion channels not only a subject of academic interest but also underline their great importance a potential targets for intervention by therapeutic drugs. Despite this central role in tissue excitability and an abundance of functional information, relatively little is known about the structure of ion channels. If we ever want to understand the mechanisms of channel function in molecular details, e.g., ion selectivity, voltage- or ligand-dependent gating, interaction with therapeutic drugs, we need to know the relationship between channel structure and function. Knowledge of the channel topology and secondary structural motifs and the eventual determination of the three dimensional structure will represent major advances in our understanding of voltage sensing and selective ion permeation. This knowledge will be extremely valuable in understanding interactions between the ion-conduction pathway and the mechanisms involved in voltage sensing and/or channel gating. It will also lay the foundation for molecular modelling studies which help us to understand channel-drug interactions, a topic of great therapeutic potential. The proposed research is entirely directed towards structural aspects of the K+ channels. The investigator proposes to use cysteine-substituted K+ channel mutants to measure distances in the vestibule and the ion conduction pathway of the channel applying homo- and heterobifunctional crosslinkers with spacers of known dimensions. The investigator also proposes to conduct systematic studies leading to overexpression of functional K+ and channel modules in E. coli suitable for purification, functional characterization and reconstitution, and structural studies including X-ray crystallography and NMR spectroscopy.
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