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Ca 2+双重激活。BK通道由成孔、电压和Ca 2+敏感的α-亚基（BKα）单独或与组织特异性调节亚基（包括四种先前已知的β-亚基）联合组成。我们最近发现了一个新的BK通道辅助亚基，一个富含亮氨酸重复序列（LRR）的膜蛋白LRRC 26，它引起了前所未有的大的负移（~ -150 mV）的电压依赖性通道激活，通过大大增强电压传感器激活和通道的闭合-开放转换之间的变构偶联，允许BK通道在甚至接近静息电压和可兴奋和不可兴奋细胞中的钙水平下激活。我们还鉴定了三种LRRC 26样旁系同源蛋白，它们在不同程度上改变了BK通道激活的电压依赖性。LRRC 26及其旁系同源LRR蛋白在结构和功能上与β-亚基不同，因此统称为BK通道γ-亚基家族。本研究旨在确定这些辅助性γ亚基调节BK通道的生理相关性和分子机制：1）确定BK通道γ亚基在人体组织和细胞中的生理和功能表达; 2）确定γ亚基调节BK通道的生化机制; 3）确定γ亚基调节功能的翻译后调节。本研究将进行分子生物学、生物化学及电生理学实验以达成所提出的目标。总的来说，这项资助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LRRC 26及其旁系同源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普遍表达的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地操纵BK通道活性。

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 α-subunits (BKα) either alone or in association with the tissue-specific regulatory subunits including the four previously known ß-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 ß-subunits and are thus collectively designated as a family of BK channel γ-subunits. Three specific aims are designed to determine the physiological relevance and molecular mechanisms of BK channel regulation by these auxiliary γ-subunits: 1) determine the physiological and functional expression of the BK channel γ-subunits in human tissues and cells; 2) determine the biochemical mechanisms of BK channel modulation by the γ-subunits; 3) determine the posttranslational regulation of the γ-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.