Electrophysiological analysis of Kv2 channel regulation by non-canonical and canonical mechanisms

Maverick, Emily E., author
Tamkun, Michael, advisor
Amberg, Gregory, committee member
Krapf, Diego, committee member
Tsunoda, Susan, committee member
Vigh, Jozsef, committee member
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Kv2 channels are the most abundant voltage-gated potassium channels in the mammalian nervous system and entire body. These channels regulate action potential firing and apoptosis via their canonical conducting functions. However, Kv2 channels also play a non-conducting role in the cells in which they are expressed. Specifically, they form junctions between the endoplasmic reticulum and plasma membranes, and these junctions regulate a myriad of cellular process. Several studies have now shown that many Kv2.1 channels expressed on the plasma membranes of mammalian cells do not respond canonically to changes in membrane voltage. Instead of opening to allow potassium efflux, the pores of these non-canonical channels are locked in a non-conducting state. This state has likely evolved to prevent electrical paralysis that would otherwise be conferred upon cells expressing high levels of completely functional Kv2 channels. The mechanism bringing about the non-conducting state of Kv2.1 channels is unknown. The work described in the first part of this dissertation was carried out with the ultimate goal of revealing the mechanism of the Kv2.1 channel non-conducting state. I describe an improved, all-electrophysiological method to quantify the numbers of nonconducting Kv channels expressed in heterologous systems. I validate this approach by measuring the fraction of non-conducting Kv2.1 channels that arise when expressed in HEK293 cells. I go on to use this approach to show evidence for a non-conducting state in the second Kv2 isoform, Kv2.2, for the first time. I find that like Kv2.1, the Kv2.2 nonconducting state is dependent on the density of channels in the membrane. Surprisingly, I also find that two Shaker-related channels, Kv1.4 and Kv1.5 also show density dependence in the fraction of channels that conduct. These results suggest that the mechanism underlying the non-conducting state is more common than we thought, and I discuss hypotheses that should be tested in the future. In the last part of this dissertation I describe the effects of the assembly of Kv2 channels with a newly discovered family of Kv β subunits, the AMIGOs. The experiments in this portion of the dissertation focus on each AMIGO's ability to modulate canonical, conducting Kv2 channels, as well as Kv2's ability to alter AMIGO trafficking and localization. I find that both Kv2.1 and Kv2.2 promote AMIGO trafficking to the plasma membrane and alter their localization there. I also find that while all three AMIGO isoforms promote Kv2 channel opening, AMIGO2 confers an additional stabilizing effect on the open state by slowing inactivation and deactivation. In all, the work in this dissertation expands on our current understanding of Kv channel function. These findings should guide future experiments to probe both canonical and non-canonical functions of Kv channels.
2020 Fall.
Includes bibliographical references.
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ion channel
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