The large conductance, calcium- and voltage-activated potassium (BK) channel is a unique member of the potassium channel family, which has the largest single channel conductance and is dually activated by voltage and cytosolic free Ca2+. BK channels consist of the pore-forming, voltage- and Ca2+-sensing ?-subunits (BK?) either alone or in association with the tissue-specific regulatory subunits including the four previously known -subunits. We recently identified a novel BK channel auxiliary subunit, a leucine-rich repeat (LRR) containing membrane protein LRRC26, which causes an unprecedented large negative shift (~ -150 mV) in voltage dependence of channel activation by greatly enhancing the allosteric coupling between the voltage-sensor activation and the channel's closed-open transition, allowing BK channel activation at even near resting voltages and calcium levels in excitable and non-excitable cells. We have additionally identified three LRRC26- like paralogous proteins that modify the BK channel's voltage dependence of activation to different extents. LRRC26 and its paralogous LRR proteins are structurally and functionally distinct from the -subunits and are thus collectively designated as a family of BK channel ?-subunits. Three specific aims are designed to determine the physiological relevance and molecular mechanisms of BK channel regulation by these auxiliary ?-subunits: 1) determine the physiological and functional expression of the BK channel ?-subunits in human tissues and cells; 2) determine the biochemical mechanisms of BK channel modulation by the ?-subunits; 3) determine the posttranslational regulation of the ?-subunits' modulatory functions. Molecular biological, biochemical and electrophysiological experiments will be performed to achieve the proposed aims. Overall, the proposed research in this grant application is designed to systematically investigate these auxiliary LRR proteins for their physiological relevance and the underlying molecular mechanisms of channel modulation. The findings from the proposed studies will establish the physiological relevance of a new family of BK channel auxiliary subunits, and provide an in-depth understanding of the molecular mechanisms governing the LRRC26 and its paralogs' unique capacity in shifting the voltage dependence of a voltage-gated ion channel. These studies will thus offer a new molecular basis for an understanding and exploration of the ubiquitously expressed BK channel's diverse physiological functions, and help in creation of novel reagents and therapeutics to rationally manipulate BK channel activity.