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Insights into Kv1.2 activation and deactivation using voltage clamp fluorimetry Horne, Andrew James

Abstract

Voltage-gated potassium (Kv) channels are essential membrane proteins in modulating membrane excitability and related cellular processes. Many details associated with the voltage response are unclear, particularly the complete role of the voltage sensing domain, and not just the densely charged S4 helix. Based on its crystal structure, the Kv1.2 channel represents an ideal model in which to study these questions. This thesis investigates Kv1.2 activation and deactivation gating, using the voltage clamp fluorimetry technique. This technique utilizes an environmentally sensitive fluorophore introduced at locations of interest in order to visualize conformational changes in protein structure. Labelling of Kv1.2 channels at the extracellular end of S4 reports a fast quenching of fluorescence emission upon depolarization that correlates extremely well with gating current measurements, suggesting it is a report of voltage-dependent S4 translocation. In addition, a slow quenching component is observed with a very negative voltage-dependence (V₁/₂ = -73.9 mV ± 1.4 mV), not seen in any other Kv channels studied to date, that involves regions of the voltage sensing domain in S1 and S2. This slow quenching is selectively removed from the fluorescence report with transfer of extracellular S1-S2 or S3-S4 linkers from the homologous Shaker potassium channel, suggesting that it arises from channel-specific interactions between the Kv1.2 linker segments. However, transfer of Kv1.2 linker segments into Shaker fail to recapitulate this quenching component, suggesting that these linker interactions likely underlie further differences in voltage sensor domain gating and/or structure. This slow quenching component correlates with deactivation of ionic current, and is prolonged with co-expression of the N-type inactivation-conferring Kvβ1.2 subunit. In the presence of the beta subunit, this likely reflects unbinding of the inactivation moiety from the pore domain, allowing deactivation and S4 return, but in the α-subunit alone we suggest that this may be a report of a voltage-dependent rearrangement in the voltage sensor domain that stabilizes the S4 in an activated conformation. Such interactions have been reported in other voltage-gated proteins, and provides further evidence that we must consider more than just S4 translocation when it comes to understanding the complete potassium channel voltage response, Kv1.2 or otherwise.

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