Regulation of Membrane Excitability

该项目的长期目标是确定骨骼型兴奋-收缩(EC)偶联背后的分子相互作用，EC偶联是将电兴奋与肌肉收缩联系起来的过程。EC偶联依赖于位于肌浆网(SR)的钙释放通道Ryanodine受体(RyR)和位于质膜的电压门控钙通道二氢吡啶受体(DHPR)之间的功能相互作用。许多实验方法将被用来探索DHPR和RyR之间的相互作用，包括使用膜片钳、钙指示染料、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靶向于质膜和肌浆网之间的连接的决定因素。这将通过在绿色荧光蛋白(GFP)标记的Alpha1S和Alpha1H(一种遥远的相关钙通道)嵌合体的异常(Alpha1S-空)肌管中表达，以及通过使用潜在的Alpha1S靶向结构域作为诱饵的酵母双杂交筛选肌肉文库。第二个目的是利用异常肌管中表达的α1S/α1H嵌合体作为一种手段来测试DHPR的β亚基是否是骨架型偶联所必需的，确定II-III环外的细胞质结构域的初级序列是否对偶联至关重要，并鉴定导致DHPR被组织成四分体的α1S序列(S)。第三个目标是测试EC耦合是否取决于Alpha1S II-III环的构象变化，这将通过将结构扰动引入环“临界结构域”周围的环区域来检查。需要测试的一个扰动是生物素受体结构域的引入，该结构域规定了生物素与含有这种基本辅因子的少量天然酶的代谢加成。第四个目标是试图通过在非肌肉细胞中表达最少量的肌肉蛋白来重建骨骼类型的偶联。

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.