GENERATION OF BK CHANNEL PORE-GATE-DOMAIN PEPTIDES FOR FUNCTIONAL AND STRUCTURAL

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DESCRIPTION (provided by applicant): Ca2+-activated, BK-type K+ channels are widely expressed and have diverse roles in the regulation of cellular excitability. Naturally occurring polymorphisms are associated with a variety of human pathologies, including asthma, and have received attention as potential therapeutic targets for the treatment of asthma. BK channels exhibit a number of unusual functional features that distinguish them from their distantly related homologues, the voltage- dependent K+ channels (Kv channels). First, pore-lining residues in BK channels, predicted based on homology to Kv sequences may be incorrect. Second, the BK central cavity appears to be much broader than in Kv channels, allowing accessibility of compounds excluded from the Kv channels. Third, the BK central cavity appears to contain specific binding sites for regulatory peptide segments of auxiliary b subunits. All of these features suggest that the pore-gate- domain (PGD) of BK channels may exhibit unique structural features distinct from those of Kv channels. Unfortunately, direct structural informatio regarding the BK channel is not yet available and remains difficult to obtain. Given recent success of various groups in obtaining biochemically tractable PGD's, here we outline a developmental proposal that seeks to identify biochemically tractable PGD's either composed entirely of BK channel sequence or enriched in BK channel sequence. The aims of the work are to obtain PGD's that form stable tetramers, exhibit biochemical and functional properties consistent with appropriate tetrameric assembly, and are potentially suitable for NMR studies that would allow tests not only of BK PGD domain structure, but also probes of interactions of the BK PGD with other regulatory molecules. If successful, given the advantages of NMR for revealing dynamic information regarding protein behavior, this approach promises to offer a general method of investigation of isolated eukaryotic PGD's.