INVOLVEMENT OF THE CARBOXYL-TERMINAL REGION OF THE ALPHA(1) SUBUNIT IN VOLTAGE-DEPENDENT INACTIVATION OF CARDIAC CALCIUM CHANNELS
INVOLVEMENT OF THE CARBOXYL-TERMINAL REGION OF THE ALPHA(1) SUBUNIT IN VOLTAGE-DEPENDENT INACTIVATION OF CARDIAC CALCIUM CHANNELS
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DOI：
10.1074/jbc.270.29.17306
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发表时间：
1995-07-21
影响因子：
4.8
通讯作者：
SCHWARTZ, A
中科院分区：
文献类型：
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作者：
KLOCKNER, U;MIKALA, G;SCHWARTZ, A
细胞内应用蛋白酶可使心脏钙电流增加至与β-肾上腺素能刺激相似的水平。利用瞬时转染的HEK 293细胞，我们研究了蛋白水解处理刺激钙通道的分子机制。用1 mg/ml胰蛋白酶或羧肽酶A灌注共表达人心脏（hHT）α（1）、α（2）和β（3）亚基的HEK细胞，可使钙通道电流的峰值幅度增加3-4倍，而不影响电压依赖性。在用hHT α（1）和α（2）共转染或单独用α（1）共转染的HEK细胞中获得了类似的结果，表明α（1）亚基本身的修饰是通过蛋白水解增强电流的原因。为了进一步表征胰蛋白酶对α（1）亚基的修饰，我们表达了一种缺失突变体，其中羧基末端尾的一部分被去除，直到氨基酸1673。表达的钙通道电流不再响应于蛋白酶的细胞内应用;然而，与野生型相比，观察到3倍更高的电流密度以及更快的失活。结果提供的证据表明，心脏α（1）亚基的羧基末端尾部的特定区域是一个重要的调节片段，可能作为门控机制的关键组成部分，影响失活特性以及通道的可用性。
Intracellular application of proteases increases cardiac calcium current to a level similar to beta-adrenergic stimulation. Using transiently transfected HEK 293 cells, we studied the molecular mechanism underlying calcium channel stimulation by proteolytic treatment. Perfusion of HEK cells, coexpressing the human cardiac (hHT) alpha(1), alpha(2), and beta(3) subunits, with 1 mg/ml of trypsin or carboxypeptidase A, increased the peak amplitude of the calcium channel current 3-4-fold without affecting the voltage dependence. Similar results were obtained in HEK cells cotransfected with hHT alpha(1) and alpha(2) or with alpha(1) alone, suggesting that modification of the alpha(1) subunit itself is responsible for the current enhancement by proteolysis. To further characterize the modification of the alpha(1) subunit by trypsin, we expressed a deletion mutant in which part of the carboxyl-terminal tail up to amino acid 1673 was removed. The expressed calcium channel currents no longer responded to intracellular application of the proteases; however, a 3-fold higher current density as well as faster inactivation compared with the wild type was observed. The results provide evidence that a specific region of the carboxyl-terminal tail of the cardiac alpha(1) subunit is an important regulatory segment that may serve as a critical component of the gating machinery that influences both inactivation properties as well as channel availability.