Large-conductance, calcium- and voltage-activated potassium (BK) channels play a variety of physiologically important roles, are innovative drug targets for disorders of almost every organ system, and possess biophysical features that make them an ideal system for studying allosteric mechanisms of channel function (gating) by voltage and ligands and modulation by drugs. The BK channel is a unique member of the potassium channel family, characterized by exceptionally large single-channel conductance and dual activation by two physiological signals, membrane voltage and intracellular free calcium. A variety of experimental evidence indicates that BK channels lack the intracellular bundle-crossing gate that is present in many other potassium channels. Thus the opening and closing of the BK channel pore during activation must be controlled by other mechanisms. Recent determination of the 3D structure of the complete BK channel from Aplysia californica at a near-atomic resolution provides a new structural basis for understanding these mechanisms. The structures not only confirm that the lack of a bundle-crossing gate, but suggest novel mechanisms of BK channel activation mediated by state-dependent interaction among amino acids in the deep pore and selectivity filter regions. We hypothesize that the BK channel activation gate is located within the selectivity filter and/or deep-pore. We have made progress towards testing this hypothesis by establishing methods to determine the relationship between activation and selectivity filter inactivation and analyzing the structure-function relationship of BK channel pore residues. With the newly available structural information and novel tools that we have developed, including concatenated tandem subunit constructs to restrict mutations to individual BK channel subunits within the tetrameric channel, we are now poised to determine the pore gate localization and central channel pore gating mechanisms. We propose to pursue the following three specific aims to elucidate the pore-gating mechanisms of BK channels: 1) determine the properties and mechanisms of selectivity filter gating in BK channels; 2) determine the role of the deep-pore residues and their interactions in BK channel gating; and 3) define the location of the activation and inactivation gates by determining the state- dependent accessibility of the selectivity filter and deep pore to Cys-modifying reagents. Overall, the proposed research is designed to investigate systematically the central pore-gating mechanisms of BK channels. The findings from the proposed studies will deepen our understanding of molecular mechanisms of BK channel activation by voltage and calcium and facilitate development of novel therapeutic reagents targeting BK channels for the treatment or prevention of neurobiological, cardiovascular, and other types of disorders and diseases.