STRUCTURE/FUNCTION OF SARCOPLASMIC RETICULUM

肌肉生理学中最重要的未解决的问题之一是 详细了解以下顺序事件：（一） 横小管系统（T-小管）的去极化，（ii）Ca 2 + 从肌浆网（SR）释放导致收缩，（iii） 通过SR Ca 2+泵释放的Ca 2+的再积累，导致 放松. 我们的长期目标是解决这些过程中的每一个， 在正常和患病肌肉中的分子水平。 我们目前的工作 假设如下。 在T-小管中激发的兴奋信号 通过二氢吡啶（DHP）受体传递，可能与 借助于几种其它蛋白质，到足蛋白（FP）。 这导致 FP的构象变化，并反过来激活Ca 2+通道 位于FP。 信号进一步传递到SR中的蛋白质 管腔钙螯合蛋白（CSQ），从CSQ中解离结合的钙。 的 游离的Ca 2+，最终释放到细胞质中 通过激活的通道，激活Ca 2+泵，导致提示 释放的Ca 2+的重新积聚。 我们的具体目标是解决 假设的链式反应的各个步骤， 纯化的能进行生理性兴奋-收缩偶联的三联体 蛋白质成分和重组系统。 所涉及的各种步骤 在DHP受体介导的信号传递途径将得到解决 通过使用化学拮抗剂和抗体。 其他参与 T-小管- SR通讯中的蛋白质也将通过使用 针对它们的抗体（和Fab亚片段）， 重构实验 诱导的FP构象变化 Ca 2+释放的各种效应，尤其是构象变化 诱导的T-小管去极化，将使用 荧光探针掺入到FP部分的各个结构域中， 现场指导的时尚。 关于FP - CSQ沟通的问题 将通过关联FP和CSQ的构象变化来研究， 以及在Ca 2+释放反应期间CSQ结合的钙的解离。 到 测试CSQ控制SR Ca 2+释放的假设， 瞬时增加内腔Ca 2+和随后的Ca 2+释放 SR将是相关的。 SR钙泵在Ca ~（2+）过程中的动力学行为 释放后将检查Ca 2+泵的再激活和 CSQ在重新激活过程中的假定作用。

One of the most important unsolved questions in muscle physiology is a detailed understanding of the following sequential events: (i) depolarization of the transverse tubular system (T-tubule), (ii) Ca2+ release from sarcoplasmic reticulum (SR) leading to contraction, (iii) reaccumulation of the released Ca2+ by the SR Ca2+ pump leading to relaxation. Our long range goal is to resolve each of these processes at a molecular level in normal and diseased muscles. Our current working hypothesis is as follows. The excitation signal elicited in the T-tubule is transmitted via the dihydropyridine (DHP) receptor, probably with the aid of several other proteins, to the foot protein (FP). This leads to conformational changes in the FP, and in turn activates the Ca2+ channel located in FP. The signal is further transmitted to a protein in the SR lumen calsequestrin (CSQ), dissociating the bound calcium from CSQ. The dissociated Ca2+, which will eventually be released to the cytoplasm through the activated channel, activates the Ca2+ pump leading to a prompt reaccumulation of the released Ca2+. Our specific aims are to resolve individual steps of the hypothetical chain reactions using the isolated triads capable of physiological excitation-contraction coupling, purified protein components, and the reconstituted system. Various steps involved in the DHP receptor-mediated signal transmission pathway will be resolved by using chemical antagonists and antibodies. Involvement of other proteins in the T-tubule - SR communication will also be examined by using antibodies (and Fab subfragments) directed against them, and by reconstitution experiments. Conformational changes in the FP induced by various effectors of Ca2+ release, especially the conformational changes induced by the T-tubule depolarization, will be investigated using the fluorescent probe incorporated into various domains of the FP moiety in a site-directed fashion. The question regarding the FP - CSQ communication will be investigated by correlating conformational changes of FP and CSQ, and dissociation of the CSQ-bound calcium during Ca2+ release reaction. To test the hypothesis that CSQ controls SR Ca2+ release, the kinetics of the transient increase in the lumenal Ca2+ and the subsequent Ca2+ release from SR will be correlated. Kinetic behavior of the SR Ca2+ pump during Ca2+ release will be followed to examine reactivation of Ca2+ pumping and the postulated role of CSQ in the reactivation process.