Molecular Determinants of L-type Calcium Channel Gating

Ca2+内流通过单一的l型Ca2+通道提供了触发Ca2+从肌浆网（SR）储存的更大释放，导致肌肉收缩。因此，影响l型Ca2+通道门控的因素是心脏兴奋-收缩（EC）耦合的关键决定因素。这些因素中最主要的是通道的α 1和β亚基组成，以及蛋白激酶A （PKA）介导的磷酸化对通道活性的上调。在心力衰竭（HF）中观察到的α 1和β亚基的密度或补体的变化可能是l型Ca2+通道门控改变和对PKA调制敏感性降低的基础。因此，通道亚基表达的这种变化可能是观察到的EC偶联异常和β -肾上腺素能激动剂在HF中有益作用丧失的候选分子机制。然而，关于心脏l型通道的α - 1c （Cav1.2）和β亚基的确切身份和结构的基本模糊阻碍了严格评估它们的差异表达和失调如何有助于心脏病理生理的努力。这些知识上的差距严重限制了开发针对心衰的新治疗策略的机会。因此，本提案的长期目标是加深对心脏l型通道亚基的分子特性和结构的理解，并将这些基本知识与心脏中这些新通道的功能运作和调节的新认识联系起来。电生理学，重组Ca2+通道，和天然心脏Ca2+通道的分子操纵，以及心脏l型通道结构和功能的分子操纵。1. 阐明翻译后裂解的α - 1c c端片段在心脏l型通道门控中的作用。2. 阐明PKA调节心脏l型通道的分子决定因素和机制。3. 确定由选择性剪接产生的Ca2+通道β 2亚基的分子多样性的功能后果。4. 利用反义策略确定在心脏中表达的功能优势β 2剪接变体。

Ca2+ influx through single L-type Ca2+ channels provides the trigger for the larger release of Ca2+ from sarcoplasmic reticulum (SR) stores that leads to muscle contraction. As such, factors that influence L-type Ca2+ channel gating are key determinants of cardiac excitation- contraction (EC) coupling. Chief among such factors are the alpha1 and beta subunit composition of the channel, and up-regulation of channel activity by protein kinase A (PKA)-mediated phosphorylation. Observed changes in the density or complement of alpha1 and beta subunits in heart failure (HF) may underlie altered L-type Ca2+ channel gating, and reduced sensitivity to PKA modulation. Therefore, such changes in channel subunit expression loom as candidate molecular mechanisms for observed EC coupling abnormalities, and loss of beneficial effects of beta-adrenergic agonists in HF. However, fundamental ambiguities regarding the exact identity and configuration of alpha1C (Cav1.2) and beta subunits of cardiac L-type channels hinder efforts to rigorously assess how their differential expression and dysregulation may contribute to cardiac pathophysiology. These gaps in knowledge seriously limit opportunities for development of novel therapeutic strategies against HF. Hence, the long-term objective of this proposal is to deepen understanding of the molecular identity and configuration of cardiac L-type channel subunits, and to bridge this basic knowledge to a new appreciation of the functional operation and modulation of these new channels in heart. Electrophysiology, recombinant Ca2+ channels, and molecular manipulation of native heart Ca2+ channels, and molecular manipulation of cardiac L-type channel structure and function. 1. Clarify the role of a post-translationally cleaved C-terminus fragment of alpha1C in turning the gating of cardiac L-type channels. 2. Elucidate molecular determinants and mechanisms of PKA modulation of cardiac L-type channels. 3. Determine functional consequences of molecular diversity of Ca2+ channel beta2 subunits generated by alternative splicing. 4. Determine the functionally dominant beta2 splice variant expressed in heart utilizing an antisense strategy.