Regulation of ion channels in nerve cells

描述（由申请人提供）：我建议继续我们正在进行的与钙依赖性钾通道相关的调节蛋白复合物的研究。重点将放在Slob上，这是一种来自果蝇的蛋白质，它结合并调节由果蝇Slowpoke基因编码的大电导钙依赖性钾通道（dSlo）。果蝇、啮齿动物和人类体内的慢钾通道在调节神经元兴奋性、释放神经递质和神经激素方面发挥着重要作用。该提案将建立在当前资助期间的几个意想不到的发现之上，最值得注意的是：(1)通过选择性剪接，单个基因可以产生几种不同的Slob mRNA和蛋白质产物；(2) Slob蛋白水平以昼夜节律方式循环，Slob可能参与生物钟的输出；(3) Slob表达在果蝇大脑中一小部分已鉴定的神经元中尤为突出；(4) Slob表达减少的突变果蝇表现出运动节律缺陷，在食物剥夺中存活的时间比野生型果蝇长得多。我们现在将验证Slob通过其对dSlo的调节，影响关键的生理过程，如进食和运动活动的昼夜节律控制的假设。异种细胞表达将用于定义不同Slob剪接变体的结合相互作用和离子通道调节活性，并且将在体内研究Slob不同变体的表达模式和昼夜循环。通过构建转基因果蝇和使用生理和行为分析，将探索Slob在果蝇大脑神经元中dSlo电流的调节作用，以及在昼夜节律和摄食中的作用。我们很幸运地拥有分子、生化、遗传和电生理工具的独特组合，可以解决离子通道调节复合物在完整生物体行为控制中的作用这一基本问题。

DESCRIPTION (provided by applicant): I propose to continue our ongoing studies of regulatory protein complexes associated with calcium dependent potassium channels. The focus will be on Slob, a protein from the fruit fly Drosophila that binds to and modulates the large conductance calcium-dependent potassium channel (dSlo) encoded by the fly Slowpoke gene. Slowpoke potassium channels in flies, rodents and humans play a major role in the regulation of neuronal excitability, and in the release of neurotransmitters and neurohormones. This proposal will build on several unexpected findings during the current funding period, most notably that: (1) there are several different Slob mRNA and protein products produced from a single gene by alternative splicing; (2) Slob protein level cycles in a circadian manner, and Slob may participate in the output of the circadian clock; (3) Slob expression is particularly prominent in a small set of identified neurons in the fly brain; and (4) mutant flies in which Slob expression is decreased exhibit locomotor rhythm defects and survive food deprivation much longer than their wild type counterparts. We now will test the hypothesis that Slob, via its modulation of dSlo, influences critical physiological processes such as feeding and the circadian control of locomotor activity. Heterologous cell expression will be used to define the binding interactions and ion channel modulatory activity of different Slob splice variants, and the patterns of expression and circadian cycling of the different variants of Slob will be investigated in vivo. The role of Slob in the modulation of dSlo current in fly brain neurons in vivo, and in circadian rhythms and feeding, will be explored by the construction of transgenic flies and the use of physiological and behavioral assays. We are fortunate to have available a unique combination of molecular, biochemical, genetic and electrophysiological tools that can be brought to bear on this fundamental problem of the role of an ion channel regulatory complex in the control of behavior in an intact organism.