Role of Calsequestrin in Ca2+ Storage & Cardiac Disease

描述（由申请人提供）：我在本基金中提出的研究将剖析CSQ的C-末端在Ca 2+缓冲中的作用。CSQ是连接肌浆网（jSR）中的Ca 2+储存蛋白，其在兴奋-收缩偶联期间的Ca 2+处理中起重要作用。介导Ca 2+缓冲的具体结构元件尚不清楚。对于本研究提案的特定目的，我的第一个具体目标是确定是否存在于C-末端区域的天冬氨酸残基簇在CSQ的Ca 2+缓冲能力中具有积极作用。为了实现这一点，突变CSQ，其中其天冬氨酸簇存在于C-末端将突变为丙氨酸，并通过腺病毒基因转移在培养的大鼠心肌细胞中表达。在表达这些突变体后，我将通过测量Ca 2+瞬变和SR Ca 2+负荷来评估它们对SR Ca 2+储存的影响。其次，我想确定CSQ中天然存在的突变如何影响体外和体内的Ca 2+缓冲。该突变是D307->H氨基酸改变，与胆碱能多态性室性心动过速（CPVT）相关。为了确定突变是否影响Ca 2+缓冲，我将产生D307->H突变，并通过腺病毒基因转移在培养的大鼠心肌细胞中表达。对Ca 2+缓冲的影响将通过测量Ca 2+瞬变和SR Ca 2+负荷在体内测量，这将有助于确定突变体CSQ是否确实具有较低的Ca 2+缓冲能力。在体外，D307->H CSQ突变体的Ca 2+缓冲能力将使用先前建立的平衡透析技术测定。我相信，对Ca 2+缓冲的详细研究将为Ca 2+处理和CSQ在兴奋-收缩偶联中的作用提供新的有用的见解。

DESCRIPTION (provided by applicant): The studies I propose in this grant will dissect the role of the C-terminus of CSQ in Ca2+ buffering. CSQ is a Ca2+ storage protein in the junctional sarcoplasmic reticulum (jSR) that plays an important in Ca2+ handling during excitation-contraction coupling. The specific structural elements that mediate Ca2+ buffering are unknown. For specific purposes of this research proposal, my first specific aim is to establish whether the clusters of aspartate residues present in the C-terminal region have an active role in Ca2+ buffering capacity of CSQ. To accomplish this, mutant CSQ where its aspartate clusters present in the C-terminus will be mutated to alanine and expressed in cultured rat cardiac myocytes by adenoviral gene transfer. After expression of these mutants, I will assess the impact they have on the SR Ca2+ stores by measuring Ca2+ transients and SR Ca2+ load. Second, I want to determine how a naturally occurring mutation found in CSQ affects Ca2+ buffering in vitro and in vivo. The mutation is a D307->H amino acid change and was correlated with Chatecolaminergic Polymorphic Ventricular Tachycardia (CPVT). To determine whether the mutation affect Ca2+ buffering, I will generate the D307->H mutation and express it in cultured rat cardiac myocytes by adenoviral gene transfer. The effect on Ca2+ buffering will be measured in vivo by measuring Ca2+ transients and SR Ca2+ load, which will help to determine if the mutant CSQ has indeed a lower Ca2+ buffering capacity. In vitro, the Ca2+ buffering capacity of the D307->H CSQ mutant will be determined using the previously established equilibrium dialysis technique. I believe that a detailed study of Ca2+ buffering will provide new and useful insights in Ca2+ handling and the role of CSQ in excitation-contraction coupling.