Alvarez Julio, Hamplova Jana, Hohaus Annette, Morano Ingo, Haase Hannelore, Vassort Guy
Physiopathologie Cardiovasculaire, INSERM U-390, CHU Arnaud de Villeneuve, F-34295 Montpellier Cedex 5, France.
J Biol Chem. 2004 Mar 26;279(13):12456-61. doi: 10.1074/jbc.M312177200. Epub 2004 Jan 12.
Ahnak, a protein of 5643 amino acids, interacts with the regulatory beta-subunit of cardiac calcium channels and with F-actin. Recently, we defined the binding sites among the protein partners in the carboxyl-terminal domain of ahnak. Here we further narrowed down the beta(2)-interaction sites to the carboxyl-terminal 188 amino acids of ahnak by the recombinant ahnak protein fragments P3 (amino acids 5456-5556) and P4 (amino acids 5556-5643). The effects of these P3 and P4 fragments on the calcium current were investigated under whole-cell patch clamp conditions on rat ventricular cardiomyocytes. P4 but not P3 increased significantly the current amplitude by 22.7 +/- 5% without affecting its voltage dependence. The slow component of calcium current inactivation was slowed down by both P3 and P4, whereas only P3 slowed significantly the fast one. The composite recombinant protein fragment P3-P4 induced similar modifications to the ones induced by each of the ahnak fragments. In the presence of carboxyl-terminal ahnak protein fragments, isoprenaline induced a similar relative increase in current amplitude and shift in current kinetics. The actin-stabilizing agents, phalloidin and jasplakinolide, did not modify the effects of these ahnak protein fragments on calcium current in control conditions nor in the presence of isoprenaline. Hence, our results suggest that the functional effects of P3, P4, and P3-P4 on calcium current are mediated by targeting the ahnak-beta(2)-subunit interaction rather than by targeting the ahnak-F-actin interaction. More specifically they suggest that binding of the beta(2)-subunit to the endogenous subsarcolemmal giant ahnak protein re-primes the alpha(1C)/beta(2)-subunit interaction and that the ahnak-derived proteins relieve the beta(2)-subunit from this inhibition.
安克蛋白由5643个氨基酸组成，它与心肌钙通道的调节β亚基以及F - 肌动蛋白相互作用。最近，我们确定了安克蛋白羧基末端结构域中蛋白质伙伴之间的结合位点。在此，我们通过重组安克蛋白片段P3（氨基酸5456 - 5556）和P4（氨基酸5556 - 5643）进一步将β(2)相互作用位点缩小至安克蛋白的羧基末端188个氨基酸。在大鼠心室心肌细胞的全细胞膜片钳条件下，研究了这些P3和P4片段对钙电流的影响。P4而非P3显著增加了电流幅度，增幅为22.7±5%，且不影响其电压依赖性。P3和P4都减缓了钙电流失活的慢成分，而只有P3显著减缓了快成分。复合重组蛋白片段P3 - P4诱导的修饰与每个安克蛋白片段诱导的修饰相似。在存在羧基末端安克蛋白片段的情况下，异丙肾上腺素诱导电流幅度有相似的相对增加以及电流动力学的改变。肌动蛋白稳定剂鬼笔环肽和贾斯普拉金诺尔在对照条件下以及存在异丙肾上腺素的情况下，均未改变这些安克蛋白片段对钙电流的影响。因此，我们的结果表明，P3、P4和P3 - P4对钙电流的功能作用是通过靶向安克蛋白 - β(2)亚基相互作用介导的，而非通过靶向安克蛋白 - F - 肌动蛋白相互作用。更具体地说，它们表明β(2)亚基与内源性肌膜下巨大安克蛋白的结合重新启动了α(1C)/β(2)亚基相互作用，并且安克蛋白衍生的蛋白质解除了β(2)亚基的这种抑制作用。
