We will investigate the factors regulating the subcellular distribution of the high conductance, calcium- and voltage-gated BK channel, a critical ion channel that regulates neuronal firing output in health and disease. Despite its powerful role in modulating excitability, experimental evidence indicates that it is sparsely distributed at the plasma membrane, a phenomenon that is regulated via interactions with the brain-specific accessory subunit, ?4. Conventional methods to study membrane protein localization have relied heavily upon overexpression of tagged proteins, a method that can significantly alter protein distribution by changing the stoichiometry of the target with its regulatory factors. To accurately determine how BK channels are distributed across the cell, it is important to be able to determine the location of individual molecules at endogenous expression levels to preserve critical concentration-dependent interactions with regulatory partners. We have developed a novel protein/dye tag with high-fluorescence emission that enables single-molecule detection, for both high- and low-abundance proteins. To preserve normal channel expression levels, we will generate a transgenic mouse where this tag has been inserted into the endogenous BK channel gene. The localization of this channel in primary neurons derived from these animals will be evaluated, and its accessory subunit and activity-regulated surface distribution will be determined.
BK channels are important in the function of many different organs, including the vascular, renal, and nervous systems. In the CNS, BK channel activity can control neural output, and mutations in the BK channel or its regulatory subunit can lead to epilepsy. Despite their critical role in regulating firing, BK channels are sparsely and heterogeneously distributed in the plasma membrane. In order to study the factors that determine the biological role of BK channels, we will use new technologies for localizing single channels, expressed at normal levels in living neurons. Understanding how BK channels are targeted within the cell will enable the development of highly specific therapies for treatment of epilepsy and other neurological disorders.