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

描述（由申请人提供）：Ca 2+激活的BK型K+通道广泛表达，在细胞兴奋性调节中具有不同的作用。天然存在的多态性与包括哮喘在内的多种人类病理学相关，并且作为治疗哮喘的潜在治疗靶点而受到关注。BK通道表现出许多不寻常的功能特征，这些特征将它们与它们的远亲同源物电压依赖性K+通道（Kv通道）区分开。首先，根据与Kv序列的同源性预测的BK通道中的孔衬残基可能是不正确的。第二，BK中央腔似乎比Kv通道宽得多，允许排除在Kv通道之外的化合物进入。第三，BK中央腔似乎含有辅助B亚基调节肽段的特异性结合位点。所有这些特征表明BK通道的孔门域（PGD）可能表现出不同于Kv通道的独特结构特征。不幸的是，关于BK通道的直接结构信息还不可用，并且仍然难以获得。鉴于最近成功的各种团体在获得生物化学上易处理的PGD的，在这里，我们概述了一个发展的建议，旨在确定生物化学上易处理的PGD的组成完全的BK通道序列或富含BK通道序列。这项工作的目的是获得PGD的，形成稳定的四聚体，表现出生物化学和功能特性与适当的四聚体组装一致，并有可能适用于NMR研究，这将允许测试不仅BK PGD结构域的结构，但也探针的BK PGD与其他调控分子的相互作用。如果成功的话，由于NMR的优势，揭示有关蛋白质行为的动态信息，这种方法有望提供一个一般的方法，调查孤立的真核PGD的。

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.