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)患者的失神经支配或肌管 在没有神经的情况下培养，骨骼肌是高度兴奋的，因为 一系列动作电位通常是在一种诱发的 收缩。这些情况的细胞特征是出现 多肽毒素阿帕明的受体，一种有效的小分子阻滞剂 电导钙激活钾(SK)通道。事实上，应用程序 阿帕明对失神经或强直性营养不良骨骼肌的作用 抑制超兴奋性，表明SK通道是 极度兴奋的状态。我们已经克隆了阿帕明敏感的SK通道 来自骨骼肌的SK3，并发现在失神经时或之后 肌肉细胞系L6分化过程中SK3基因的表达 在正常神经支配的肌肉中，它不表达。既不是 SK通道在高兴奋性骨骼肌中的生理作用 控制SK3基因表达的分子线索目前还不清楚。在这 建议，我们将检验假设：(1)。SK3渠道驻留在 失神经骨骼肌细胞的横管。膜片钳 测量将使用去神经正常和拔管进行 培养的肌管。SK3通道特异性免疫组织化学 将进行抗体和I125-阿帕明结合研究。(2)。斯凯 通道活动会导致过度兴奋。骨骼肌肌管和 神经细胞将进行共培养。SK频道将是异质的 通过感染重组逆转录病毒和细胞表达 进行了电生理检测。(3)。SK3启动器被激活 在培养的L6成肌细胞分化后。A)SK3 启动子/荧光素酶的构建将被引入L6成肌细胞，并 分化前后荧光素酶活性测定；b)凝胶漂移 足迹分析将使用前和后的核提取液进行 分化后的L6细胞；c)以前未鉴定的序列 SK3启动子是成肌细胞激活所必需的 分化将用于筛选分化的L6骨骼肌 CDNA表达文库。(4)。强直性肌营养不良(DMAHP) 同源域蛋白或DMPK(强直性肌营养不良蛋白激酶)调节SK 渠道表达式。A)DMAHP和/或DMPK将异位表达为 L6成肌细胞和SK3基因表达及通道活性的检测 差异化。B)将进行凝胶移位分析和足迹分析 SK基因启动子和重组DMAHP；c)SK3启动子/荧光素酶 将在L6中引入带或不带DMAHP和/或DMPK的构造 成肌细胞和负责调节的启动子元件将是 下定决心。这些研究将建立一个框架，以了解 过度兴奋的分子、细胞和生理异常 骨骼肌与SK基因表达的协调调控 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.