UBC Theses and Dissertations
Moment-to-moment regulation of voltage-gated potassium channel function Baronas, Victoria
Kv1.2 channels are prominently expressed in neurons where they help to set the threshold of action potential firing. While we have a good understanding of the mechanism of voltage sensing and gating, we have comparatively little information on the compendium of regulatory molecules that can impact Kv1.2 expression and function. Kv1.2 channels are subject to a unique mechanism of regulation whereby a train of brief, repetitive depolarizations elicit increasing amounts of current, a phenotype we term ‘use-dependent activation’. In heterologous cells expressing Kv1.2 and primary hippocampal cultures from rats, there is remarkable diversity in this phenotype. While use-dependent activation is absent in all other Kv1 channels, it persists in heteromeric channels containing at least one Kv1.2 subunit. Exposing cells expressing Kv1.2 to reducing conditions causes a dramatic shift in use-dependent activation where there is very little or no current elicited by the first pulse, but over the course of the train there is a hundred-fold or more increase in current. Additionally, reducing conditions cause a depolarizing shift in the activation curve of Kv1.2 by +64 mV. Taken together, we postulate that use-dependence arises from an extrinsic, redox-sensitive inhibitory regulator that associates with Kv1.2 preferentially in the closed, reduced state. We have identified a new regulator of Kv1.2 function, Slc7a5, an amino acid transporter. Co-expression of these two proteins decreases Kv1.2 expression and produces a hyperpolarizing shift of the activation and inactivation curves. Together these effects result in Kv1.2 channels being caught in an ‘inactivation trap’. These effects of Slc7a5 can be rescued by co-expressing a third protein, Slc3a2, which is known to heterodimerize with the Slc7a5 channel. Using BRET we show that Slc7a5 and Kv1.2 can be within 10 nm of each other. Other Kv1 channels we have tested (Kv1.1 and Kv1.5) are insensitive to the activation shift produced by Slc7a5, however Kv1.1 channels are exquisitely sensitive to current inhibition. Overall, the work in this thesis expands our knowledge of how Kv1.2 channels are regulated and opens the door to examining how these interactions contribute to normal neuronal function.
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