SK CHANNELS IN HYPEREXCITABLE SKELETAL MUSCLE

描述：骨骼肌兴奋通常由 神经支配的影响。 然而，在神经支配之前， 强直性肌营养不良（DM）患者的去神经支配或肌管 在没有神经的情况下培养，骨骼肌是过度兴奋的， 一系列动作电位通常是在诱发的 收缩。 这些疾病的细胞标志是 肽毒素apamin的受体，一种有效的小分子 传导钙激活钾（SK）通道。 实际上，应用 apamin对失神经或肌强直性营养不良骨骼肌的作用 抑制过度兴奋，表明SK通道是中枢神经系统的 超兴奋状态 我们已经克隆了apamin敏感的SK通道 从骨骼肌，SK 3，并发现，在去神经支配或后， 在肌细胞系L 6的分化中，SK 3基因表达， 在正常受神经支配的肌肉中不表达。 无论是 SK通道在过度兴奋骨骼肌中的生理作用， 控制SK 3基因表达的分子线索尚未被了解。 在这 我们将检验以下假设：（1）。 SK 3通道位于 去神经骨骼肌细胞的横小管。 膜片钳 测量将使用去神经正常和去管 培养的肌管。 使用SK 3通道特异性 抗体和I125-apamin结合研究。 （二）、 SK 通道活性诱导过度兴奋。 骨骼肌肌管和 神经细胞将被共培养。 SK通道将是异源的 通过重组逆转录病毒感染表达， 电生理测定。 （三）、 SK 3启动子被激活 在培养的L 6成肌细胞分化后。 （a）SK3 启动子/荧光素酶构建体将被引入L 6成肌细胞， 在分化之前和之后评估荧光素酶活性; B）凝胶位移 和足迹分析将进行与核提取物从前和 分化后的L 6细胞; c）先前未表征的序列， SK 3启动子被证明是成肌细胞活化所必需的 分化将用于筛选分化的L 6骨骼肌 cDNA表达文库。 （四）、 肌强直性营养不良相关 同源结构域蛋白或DMPK（强直性肌营养不良蛋白激酶）调节SK 渠道表达。 a）DMAHP和/或DMPK将异位表达于 L 6成肌细胞和SK 3 mRNA和通道活性评估前后 分化 B）凝胶迁移试验和足迹将使用 SK基因启动子和重组DMAHP; c）SK 3启动子/荧光素酶 将在有或没有DMAHP和/或DMPK的情况下将构建体引入L 6 成肌细胞和负责调控的启动子元件将被 测定 这些研究将建立一个框架， 高兴奋性的分子、细胞和生理异常 以及SK基因表达的协调调节 在肌肉组织中。

DESCRIPTION: Skeletal muscle excitation is normally controlled by the influence of innervating nerve. However, prior to innervation, upon denervation, in patients with myotonic muscular dystrophy (DM), or myotubes cultured in the absence of nerve, skeletal muscle is hyperexcitable, in that a train of action potentials is often induced following an evoked contraction. The cellular hallmark of these conditions is the appearance of receptors for the peptide toxin apamin, a potent blocker of small conductance calcium-activated potassium (SK) channels. Indeed, application of apamin to denervated or myotonic dystrophic skeletal muscle dramatically repress the hyperexcitability, demonstrating that SK channels are central to the hyperexcitable state. We have cloned the apamin sensitive SK channels from skeletal muscle, SK3, and found that upon denervation or after differentiation of the muscle cell line, L6, the SK3 gene is expressed while in normally innervated muscle it is not expressed. Neither the physiological role of SK channels in hyperexcitable skeletal muscle nor the molecular cues controlling SK3 gene expression are yet understood. In this proposal, we will test the hypothesis that: (1). SK3 channels reside in the transverse tubules of denervated skeletal muscle cells. Patch clamp measurements will be performed using denervated normal and detubulated cultured myotubes. Immunohistochemistry using SK3 channel-specific antibodies, and I125-apamin binding studies will be performed. (2). SK channel activity induces hyperexcitability. Skeletal muscle myotubes and nerve cells will be co-cultured. SK channels will be heterologously expressed by infection with recombinant retroviruses and the cells electrophysiologically assayed. (3). The SK3 promotor is activated following differentiation of cultured L6 myoblasts. a) SK3 promotor/luciferase constructs will be introduced into L6 myoblasts, and luciferase activity assessed before and after differentiation; b) gel-shift and footprint assays will be performed with nuclear extracts from pre- and post-differentiated L6 cells; c) previously uncharacterized sequences in the SK3 promotor shown to be necessary for activation following myoblast differentiation will be used to screen a differentiated L6 skeletal muscle cDNA expression library. (4). DMAHP (myotonic dystrophy associated homeodomain protein or DMPK (myotonic dystrophy protein kinase) regulates SK channel expression. a) DMAHP and/or DMPK will be ectopically expressed in L6 myoblasts and SK3 mRNA and channel activity assessed before and after differentiation. b) gel-shift assays and footprints will be performed with the SK gene promotor and recombinant DMAHP; c) SK3 promoter/luciferase constructs will be introduced with or without DMAHP and/or DMPK into L6 myoblasts and the promoter elements responsible for regulation will be determined. These studies will establish a framework for understanding the molecular, cellular and physiological abnormalities of hyperexcitable skeletal muscle as well as the coordinate regulation of SK gene expression in muscle tissue.