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Voltage dependent gating and modulation in Shaker-type potassium channels Peters, Christian Joseph

Abstract

The voltage-gated potassium channels of the Kv1 (Shaker-type) family are proteins found in many cell types throughout the body, and are critical for regulating membrane excitability. Ion channel proteins are dynamic by nature, and undergo structural reorientations in response to voltage and other external stimuli. In this thesis, I will describe the results of experiments using the voltage clamp fluorimetry technique, which can relate movements within protein domains to associated electrical behaviour. In Kv1.2 channels, fluorescent emissions from a fluorophore attached to the S4 helix faithfully report the movement of gating charge during depolarization. However, a second phase of fluorescence is also observed which is unique to Kv1.2. Using chimaeras where the external linkers were exchanged between Kv1 homologues, we determined that this phase tracks an interaction between the external linkers which slows channel deactivation. When fluorescence was recorded from Kv1.2 in the presence of the Kvβ1.2 subunit possessing a channel blocking N-terminus, fluorescence was unchanged during activation but slowed during deactivation, suggesting that the blocker sterically hinders activation gate closure and prevents the return of the gating charge. While Kv1.2 requires a Kvβ1 subunit to inactivate, the Drosophila homologue Shaker possesses an N-type inactivation domain on its own N-terminus. Shaker can also inactivate through conformational changes in its selectivity filter, so-called C-type inactivation. This process is structurally linked to activation gate opening, and is accelerated by N-terminal block. We have found that the conformations of the activation gate and the selectivity filter are allosterically linked, and that the N-terminus accelerates C-type inactivation by expelling potassium from a selectivity filter binding site known to inhibit its conformational change. Acceleration of C-type inactivation was also implicated as the mechanism by which an inherited genetic mutation near the Kv1.1 activation gate causes episodic ataxia type-1. However, results from experiments using voltage clamp fluorimetry and single channel patch clamp suggest that accelerated current decay observed in those mutants is more likely due to destabilization of the open state of the activation gate. Taken together, the results of this thesis demonstrate how structural variability between channel homologues leads to their broad functional diversity.

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