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.
Structural information on the pore-gate-domains of eukaryotic ion channels remains limited. Functional studies have indicated that the Ca2+- and voltage-regulated BK-type K+ channel exhibits a pore-gate-domain that may be unique among voltage-dependent K+ channels. This project seeks to obtain isolated BK-channel pore-gate-domain modules that will be used for biochemical characterization, functional studies using channel recordings in bilayers, and structural studies using solution NMR in lipid bicelles. This approach seeks to define a method that may have applicability to structural and dynamic studies of isolated ion channel pore-gate domains and promises to provide unique insights into dynamic regulation of BK channels and naturally occurring mutations implicated in human disease, such as asthma. Structural data will also facilitate the development of therapeutics to target asthma using structure-based drug design.
