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

描述(由申请人提供)：钙激活的BK型K+通道广泛表达，并在调节细胞兴奋性方面具有不同的作用。自然发生的基因多态性与包括哮喘在内的多种人类疾病相关，并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，它们有别于它们的远亲同系物--电压依赖性K+通道(Kv通道)。首先，根据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，允许从Kv通道中排除的化合物的可访问性。第三，BK中央空腔似乎包含辅助b亚基的调节肽片段的特定结合位点。所有这些特征表明，BK通道的孔门结构域(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没有得到，而且仍然很难获得。鉴于最近不同的小组在获得生化易处理的PGD方面取得的成功，我们在这里概述了一项开发计划，试图鉴定完全由BK通道序列组成的或富含BK通道序列的生化易处理的PGD。这项工作的目的是获得形成稳定的四聚体的PGD，表现出与适当的四聚体组装一致的生化和功能特性，并可能适合于核磁共振研究，这不仅允许测试BK PGD结构域的结构，也允许探测BK PGD与其他调节分子的相互作用。如果成功，鉴于核磁共振在揭示蛋白质行为的动态信息方面的优势，这种方法有望为分离的真核PGD的研究提供一种通用的方法。

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.