BK channel regulation by auxiliary LRR proteins

描述（由申请人提供）：大电导，钙和电压激活钾（BK）通道是钾通道家族的独特成员，具有最大的单通道电导，并由电压和胞质游离Ca2+双重激活。BK通道由孔隙形成、电压和Ca2+感应亚基（BK）单独或与组织特异性调节亚基（包括先前已知的四种-亚基）联合组成。我们最近发现了一种新的BK通道辅助亚基，一种含有膜蛋白LRRC26的富含亮氨酸的重复序列（LRR），它通过极大地增强电压传感器激活和通道的闭开转换之间的变构耦合，导致通道激活的电压依赖性出现前所未有的大负位移（~ -150 mV），允许BK通道在接近静息电压和钙水平时激活，在可兴奋和不可兴奋的细胞中。我们还发现了三种LRRC26样的旁系蛋白，它们在不同程度上改变了BK通道的电压依赖性。LRRC26及其相似的LRR蛋白在结构和功能上都不同于？-亚基，因此统称为BK通道-亚基族。设计了三个特定的目标来确定这些辅助因子调控BK通道的生理相关性和分子机制。-亚单位：1)决定BK通道的生理和功能表达？-人体组织和细胞中的亚基；2)确定-亚基调控BK通道的生化机制；3)确定翻译后的调控？-亚单位的调节功能。分子生物学，生物化学和电生理实验将进行，以实现所提出的目标。总的来说，这项拨款申请中的拟议研究旨在系统地研究这些辅助LRR蛋白的生理相关性和通道调节的潜在分子机制。这些研究结果将建立一个新的BK通道辅助亚基家族的生理相关性，并深入了解控制LRRC26及其类似物在改变电压门控离子通道的电压依赖性方面的独特能力的分子机制。这些研究将为理解和探索无处不在表达的BK通道的多种生理功能提供新的分子基础，并有助于创造新的试剂和治疗方法来合理操纵BK通道的活性。

DESCRIPTION (provided by applicant): The large conductance, calcium- and voltage-activated potassium (BK) channel is a unique member of the potassium channel family, which has the largest single channel conductance and is dually activated by voltage and cytosolic free Ca2+. BK channels consist of the pore-forming, voltage- and Ca2+-sensing -subunits (BK) either alone or in association with the tissue-specific regulatory subunits including the four previously known ?-subunits. We recently identified a novel BK channel auxiliary subunit, a leucine-rich repeat (LRR) containing membrane protein LRRC26, which causes an unprecedented large negative shift (~ -150 mV) in voltage dependence of channel activation by greatly enhancing the allosteric coupling between the voltage-sensor activation and the channel's closed-open transition, allowing BK channel activation at even near resting voltages and calcium levels in excitable and non-excitable cells. We have additionally identified three LRRC26- like paralogous proteins that modify the BK channel's voltage dependence of activation to different extents. LRRC26 and its paralogous LRR proteins are structurally and functionally distinct from the ?-subunits and are thus collectively designated as a family of BK channel ?-subunits. Three specific aims are designed to determine the physiological relevance and molecular mechanisms of BK channel regulation by these auxiliary ?-subunits: 1) determine the physiological and functional expression of the BK channel ?-subunits in human tissues and cells; 2) determine the biochemical mechanisms of BK channel modulation by the ?-subunits; 3) determine the posttranslational regulation of the ?-subunits' modulatory functions. Molecular biological, biochemical and electrophysiological experiments will be performed to achieve the proposed aims. Overall, the proposed research in this grant application is designed to systematically investigate these auxiliary LRR proteins for their physiological relevance and the underlying molecular mechanisms of channel modulation. The findings from the proposed studies will establish the physiological relevance of a new family of BK channel auxiliary subunits, and provide an in-depth understanding of the molecular mechanisms governing the LRRC26 and its paralogs' unique capacity in shifting the voltage dependence of a voltage-gated ion channel. These studies will thus offer a new molecular basis for an understanding and exploration of the ubiquitously expressed BK channel's diverse physiological functions, and help in creation of novel reagents and therapeutics to rationally manipulate BK channel activity. PUBLIC HEALTH RELEVANCE: The findings from the proposed research will provide a new molecular basis for an understanding and exploration of the ubiquitously expressed BK channel's diverse physiological functions, and help in creation of novel reagents and therapeutics to rationally manipulate BK channel activity.