Abstract
The regulation of ion channels is critical to numerous physiological processes, especially neuronal function. The large conductance voltage and calcium-activated potassium (BK) channel can modulate neuronal excitability, neurotransmitter release, and may be involved in the development of epilepsy; following a seizure, hippocampal BK channels are more highly trafficked to the plasma membrane. In addition to the pore-forming alpha subunits, which are sufficient to confer channel function, Beta subunits can be incorporated to alter gating and trafficking. Studies of the brain-specific Beta4 subunit effects on surface expression have produced conflicting results, likely due to alternative splicing in the alpha subunit. Fluorogen-activating peptides (FAPs) developed in our lab are well-suited to study protein trafficking; consisting of a ScFv-derived fusion tag and fluorogen dyes added to the cellular media, the association of these two cognate parts results in bright, specific fluorescence. By using a FAP-tagged BKα construct and a pair of fluorogen dyes, we have developed a method for two-color labeling of surface-resident and intracellular BK channels in live cells, allowing for rapid analysis of protein localization by flow cytometry and direct imaging. Using this system in stably expressing HEK293 cells, we found that modulation of the C-terminal region by kinase activity exerted strong changes in surface expression. Taken together with previous observations that these same kinases alter channel gating, this provides a synergistic model for BK-mediated hyperexcitability. A common confound in BK channel research is due to the vast number of splicing isoforms, each having different sensitivities to post-translational modifications. To that end, we are currently generating a knock-in mouse model in which our FAP-tagged BKα is included in all splice variants to examine trafficking changes in Vivo as well as analysis of the behavior of single channels.
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