Laboratorio de Electrofisiologia, Instituto de Cardiologia y Cirugia Cardiovascular, Havana, Cuba.
Pflugers Arch. 2010 Sep;460(4):719-30. doi: 10.1007/s00424-010-0853-x. Epub 2010 Jul 7.
Ahnak1, a giant 700 kDa protein, has been implicated in Ca(2+) signalling in various cells. Previous work suggested that the interaction between ahnak1 and Cavbeta(2) subunit plays a role in L-type Ca(2+) current (I (CaL)) regulation. Here, we performed structure-function studies with the most C-terminal domain of ahnak1 (188 amino acids) containing a PxxP consensus motif (designated as 188-PSTP) using ventricular cardiomyocytes isolated from rats, wild-type (WT) mice and ahnak1-deficient mice. In vitro binding studies revealed that 188-PSTP conferred high-affinity binding to Cavbeta(2) (K (d) approximately 60 nM). Replacement of proline residues by alanines (188-ASTA) decreased Cavbeta(2) affinity about 20-fold. Both 188-PSTP and 188-ASTA were functional in ahnak1-expressing rat and mouse cardiomyocytes during whole-cell patch clamp. Upon intracellular application, they increased the net Ca(2+) influx by enhancing I (CaL) density and/or increasing I (CaL) inactivation time course without altering voltage dependency. Specifically, 188-ASTA, which failed to affect I (CaL) density, markedly slowed I (CaL) inactivation resulting in a 50-70% increase in transported Ca(2+) during a 0 mV depolarising pulse. Both ahnak1 fragments also slowed current inactivation with Ba(2+) as charge carrier. By contrast, neither 188-PSTP nor 188-ASTA affected any I (CaL) characteristics in ahnak1-deficient mouse cardiomyocytes. Our results indicate that the presence of endogenous ahnak1 is required for tuning the voltage-dependent component of I (CaL) inactivation by ahnak1 fragments. We suggest that ahnak1 modulates the accessibility of molecular determinants in Cavbeta(2) and/or scaffolds selectively different beta-subunit isoforms in the heart.
Ahnak1 是一种 700 kDa 的巨型蛋白，已被牵连到各种细胞中的 Ca(2+)信号转导中。先前的研究表明，ahnak1 与 Cavbeta(2)亚基之间的相互作用在 L 型 Ca(2+)电流 (I (CaL))调节中发挥作用。在这里，我们使用从大鼠、野生型 (WT) 小鼠和 ahnak1 缺陷型小鼠分离的心室心肌细胞，对包含 PxxP 基序的 ahnak1 的最 C 端结构域（指定为 188-PSTP）进行了结构功能研究。体外结合研究表明，188-PSTP 与 Cavbeta(2)具有高亲和力结合（K (d)约 60 nM）。脯氨酸残基被丙氨酸取代（188-ASTA）使 Cavbeta(2)亲和力降低约 20 倍。在整个细胞贴片钳中，188-PSTP 和 188-ASTA 在表达 ahnak1 的大鼠和小鼠心肌细胞中均具有功能。在细胞内应用时，它们通过增加 I (CaL)密度和/或增加 I (CaL)失活时程来增加净 Ca(2+)内流，而不改变电压依赖性。具体而言，188-ASTA 不影响 I (CaL)密度，但显著减慢 I (CaL)失活，导致在 0 mV 去极化脉冲期间转运的 Ca(2+)增加 50-70%。两种 ahnak1 片段也以 Ba(2+)为载流子减慢电流失活。相比之下，在 ahnak1 缺陷型小鼠心肌细胞中，既没有 188-PSTP 也没有 188-ASTA 影响任何 I (CaL)特性。我们的结果表明，内源性 ahnak1 的存在是 ahnak1 片段调节 I (CaL)失活的电压依赖性成分所必需的。我们认为 ahnak1 调节 Cavbeta(2)中的分子决定因素的可及性，并/或在心脏中支架选择性地不同的β亚基同工型。
