Regulation of Membrane Excitability

该项目的长期目标是确定神经元型兴奋-收缩（EC）偶联的分子相互作用，该过程将电兴奋与肌肉收缩联系起来。 EC偶联依赖于Ryanodine受体（RyR）（位于肌浆网（SR）中的Ca 2+释放通道）和二氢吡啶受体（DHPR）（位于质膜中的电压门控Ca 2+通道，包含α 1 S作为其主要亚基）之间的功能性相互作用。 许多实验方法将用于探测DHPR和RyR之间的相互作用，包括使用膜片钳，Ca 2+指示染料，电子显微镜和分子生物学。 该提案的第一个具体目标是确定导致DHPR靶向质膜和肌浆网之间的连接的决定因素。 这将通过在发育不良的（α 1 S-无效）肌管中表达α 1 S和α 1 H（一种远亲Ca 2+通道）的绿色荧光蛋白（GFP）标记的嵌合体，以及通过使用α 1 S的潜在靶向结构域作为诱饵的肌肉文库的酵母双杂交筛选来实现。第二个目的是使用在发育不良的肌管中表达的α 1 S/α 1H嵌合体作为测试DHPR的β亚基是否是双链型偶联所需的手段，以确定II-III环外的胞质结构域的一级序列是否是偶联的关键，并鉴定导致DHPR组织成“四分体”的α 1 S序列。 第三个目的是测试EC耦合是否依赖于α 1 S II-III环的构象变化，这将通过引入结构扰动到环的区域中来检查，该区域围绕环的“关键域”。 待测试的一个扰动是引入生物素受体结构域，其指定生物素代谢添加到含有该必需辅因子的少量天然酶中。 第四个目标是试图通过在非肌肉细胞中表达最小的一组肌肉蛋白来重建肌肉型偶联。

The long-term objective of this project is to identify the molecular interactions underlying skeletal-type excitation- contraction (EC) coupling, the process which links electrical excitation to muscular contraction. EC coupling hinges upon a functional interaction between the ryanodine receptor (RyR), a Ca2+-release channel located in the sarcoplasmic reticulum (SR), and the dihydropyridine receptor (DHPR), a voltage-gated Ca2+ channel which is located in the plasma membrane and contains alpha1S as its principal subunit. A number of experimental approaches will be used to probe the interaction between the DHPR and RyR, including the use of patch clamping, Ca2+ indicator dyes, electron microscopy and molecular biology. The proposal's first specific aim is to establish the determinants that cause DHPRs to target to junctions between the plasma membrane and sarcoplasmic reticulum. This will be accomplished by expression in dysgenic (alpha1S-null) myotubes of green fluorescent protein (GFP) tagged chimeras of alpha1S and alpha1H (a distantly related Ca2+ channel), and by a yeast two-hybrid screen of a muscle library using a potential targeting domains of alpha1S as baits. The second aim is to use alpha1S/alpha1H chimeras expressed in dysgenic myotubes as a means of testing whether the beta subunit of the DHPR is required for skeletal-type coupling, to determine whether the primary sequence of cytoplasmic domains outside the II-III loop are critical for coupling, and to identify the sequence(s) of alpha1S that cause DHPRs to be organized into "tetrads". The third aim is to test whether EC coupling depends upon conformational changes of the alpha1S II-III loop, which will be examined by means of introducing structural perturbations into the regions of the loop which surround the "critical domain" of the loop. One perturbation to be tested is the introduction of the biotin acceptor domain, which specifies the metabolic addition of biotin to a small number of native enzymes containing this essential cofactor. The fourth aim is to attempt to reconstitute skeletal-type coupling by expressing a minimal set of muscle proteins in a non-muscle cell.