Activity-induced gain-of-function in BK channels and beta4-dependent regulation of channel trafficking

Neuronal activity results in numerous modifications in the brain including changes in both the intrinsic and network properties of neurons. These changes can either be in response to physiological activity resulting in experience-dependent learning, or, they can be the result of a pathological experience such as seizure and can predispose the brain to further disease states. We look at seizure-induced changes in large conductance voltage- and calcium-dependent potassium channels (BK/slo/KCNMA1 channels) in cortical and hippocampal neurons in mice. These changes are manifested as changes in molecular, cell biological and electrophysiological properties of these channels resulting in an activity-induced gain-of-function in BK channels.;Taken together, these results illustrate how activity can modulate the network properties of a brain region by changing intrinsic properties of single neurons. In addition, changes at the level of gene expression and splicing lead to redistribution of ion channels on the cell surface and consequently, to a modulation of neuronal output and potential changes in animal behavior. Furthermore, this thesis proposes that a major function of the brain-specific beta4 subunit in neurons is not modulation of channel biophysical function, but rather control of subcellular localization. Thus, activity-modulated beta4 expression can be a pivotal control point for regulating BK channel current and neural excitability.;We observed a post-seizure increase in whole-cell potassium currents carried by BK channels in pyramidal neurons from superficial layers of the neocortex and CA3 hippocampal region. Furthermore, neocortical excitatory cells show a BK channel-dependent narrowing of action potential accompanied by an increase in intrinsic as well as network excitability following seizure activity. Although hippocampal neurons do not show an increase in excitability post-seizure, there is an increase in the contribution of BK channels to maintaining intrinsic excitability. Interestingly, this gain-of-function is not accompanied by an increase in the expression of the BK channel pore-forming alpha subunit. Instead, there is a seizure-induced downregulation of the regulatory beta4 subunit in cortical as well as hippocampal tissue. In heterologous cells, BK channels associated with the beta4 subunit have higher calcium and depolarization requirements for channel opening, slower activation kinetics and are resistant to the BK channel antagonist iberiotoxin. In control neurons exhibiting high beta4 expression, whole-cell BK channel currents do not display the electrophysiological signature of BKalpha+beta4 channels described in heterologous cells. Using a novel genetic tag to track the cellular location of the pore-forming BKalpha subunit in HEK293 cells, we show that beta4 expression profoundly reduces surface expression of BK channels via an ER retention sequence at the C-terminus. In agreement with these results, we see a seizure-induced change in the splicing of the beta4 subunit resulting in the introduction of a stop codon, which might contribute to the decrease in beta4 expression. We also observe an increase in inactivating BK channel currents post-seizure, suggesting that the downregulation of the beta4 subunit is accompanied by the compensatory upregulation of an inactivating beta subunit.