PDF(Pubmed)
Abstract：
L-type CaV1.2 current (ICa,L) links electrical excitation to contraction in cardiac myocytes. ICa,L is tightly regulated to control cardiac output. Rad is a Ras-related, monomeric protein that binds to L-type calcium channel β subunits (CaVβ) to promote inhibition of ICa,L. In addition to CaVβ interaction conferred by the Rad core motif, the highly conserved Rad C-terminus can direct membrane association in vitro and inhibition of ICa,L in immortalized cell lines. In this work, we test the hypothesis that in cardiomyocytes the polybasic C-terminus of Rad confers t-tubular localization, and that membrane targeting is required for Rad-dependent ICa,L regulation. We introduced a 3xFlag epitope to the N-terminus of the endogenous mouse Rrad gene to facilitate analysis of subcellular localization. Full-length 3xFlag-Rad (Flag-Rad) mice were compared with a second transgenic mouse model, in which the extended polybasic C-termini of 3xFlag-Rad was truncated at alanine 277 (Flag-RadΔCT). Ventricular cardiomyocytes were isolated for anti-Flag-Rad immunocytochemistry and ex vivo electrophysiology. Full-length Flag-Rad showed a repeating t-tubular pattern whereas Flag-RadΔCT failed to display membrane association. ICa,L in Flag-RadΔCT cardiomyocytes showed a hyperpolarized activation midpoint and an increase in maximal conductance. Additionally, current decay was faster in Flag-RadΔCT cells. Myocardial ICa,L in a Rad C-terminal deletion model phenocopies ICa,L modulated in response to β-AR stimulation. Mechanistically, the polybasic Rad C-terminus confers CaV1.2 regulation via membrane association. Interfering with Rad membrane association constitutes a specific target for boosting heart function as a treatment for heart failure with reduced ejection fraction.
摘要：
L型CaV1.2电流(ICa,L）将电激发与心肌细胞收缩联系起来。ICa,严格调节L以控制心输出量。Rad是与Ras相关的,与L型钙通道β亚基（CaVβ）结合以促进ICa抑制的单体蛋白，L.除了Rad核心基序赋予的CaVβ相互作用外，高度保守的RadC端可以在体外指导膜缔合和抑制ICa，L在永生化细胞系中。在这项工作中,我们检验了这样一个假设，即在心肌细胞中，Rad的多碱性C端赋予了t-小管定位，Rad依赖性ICa需要膜靶向，L调节。我们将3xFlag表位引入内源性小鼠Rrad基因的N末端以促进亚细胞定位的分析。将全长3xFlag-Rad（Flag-Rad）小鼠与第二个转基因小鼠模型进行比较，其中3xFlag-Rad的延伸的多碱性C-末端在丙氨酸277处被截短(Flag-RadΔCT)。分离心室心肌细胞用于抗Flag-Rad免疫细胞化学和离体电生理学。全长Flag-Rad显示重复的t-管状模式，而Flag-RadΔCT未能显示膜缔合。ICa,Flag-RadΔCT心肌细胞中的L显示超极化激活中点，最大电导增加。此外,Flag-RadΔCT细胞的电流衰减更快。心肌ICa,RadC端缺失模型中的L表型ICa，响应于β-AR刺激而调制L。机械上,多元RadC末端通过膜缔合赋予CaV1.2调节。干扰Rad膜关联构成了增强心脏功能的特定目标，可作为射血分数降低的心力衰竭的治疗方法。
