Student Theses and Dissertations

Date of Award

2012

Document Type

Thesis

RU Laboratory

Muir Laboratory

Abstract

Potassium channels are important for regulating the flow of potassium ions across semi-permeable cell membranes in an efficient and selective manner. Potassium channels form a conduction pore comprised of a selectivity filter responsible for the strong preference for potassium, and a water-filled central cavity that contributes to rapid conduction by lowering the energy barrier for potassium ions to cross the low dielectric membrane environment. In the high resolution structure of the potassium channel KcsA, a hydrated potassium ion was observed in the central cavity. It was proposed that some electrostatic stabilization for this potassium ion may come from the backbone of nearby α- helix C-termini, through the helix dipole effect. We studied the role of helix dipoles in KcsA using protein semisynthesis in order to modify the backbone of KcsA and reduce the dipoles of the implicated helix termini. The modified protein was studied by both X-ray crystallography and electrophysiology, demonstrating that the pore helix dipoles may play an important role in potassium conductance. In the course of these experiments, a new conformation for the KcsA cavity was discovered: a phenylalanine (Phe) from each subunit flipped into the center of the cavity, the cavity ion was no longer observed, and a new non-peptidic density extended into the cavity through lateral openings exposed by the conformational change of the Phe. Subsequent structural studies identified conditions that induce or prevent this conformational change. In particular, mutations were incorporated into KcsA that make the protein less likely to enter the alternative conformation, while not greatly affecting potassium conductance. Crystal structures of KcsA in complex with cavity blocking small molecules revealed that certain inhibitors bind to the cavity in its alternative conformation, and electrophysiology confirmed inhibition by one such molecule in the membrane environment. A sequence alignment between KcsA and several human potassium channels identified a subset of channels where this mode of cavity block may be conserved, including BK and HERG channels. Thus, this new conformation of block could have important implications for the pharmacology of human potassium channels. This work furthers our understanding of electrostatic interactions, structural plasticity, and a new mode of action for a family of inhibitors, within the cavity of KcsA. The study of helix dipoles has relevance for the function of a wide range of proteins, and characterization of conformational dependent cavity block has particular relevance to some pharmacologically relevant human potassium channels.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/527

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