Inhibition of cardiac L-type calcium channels by protein kinase C phosphorylation of two sites in the N-terminal domain
Inhibition of cardiac L-type calcium channels by protein kinase C phosphorylation of two sites in the N-terminal domain
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DOI：
10.1073/pnas.210384297
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发表时间：
2000-10-24
影响因子：
11.1
通讯作者：
Catterall, WA
中科院分区：
文献类型：
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作者：
McHugh, D;Sharp, EM;Catterall, WA
我们研究了蛋白激酶C(PKC)对心肌细胞L钙电流的调节机制。应用膜片钳技术，我们发现4-α-佛波酯(PMA)或Rac-1-油基-2-乙酰甘油(OAG)激活PKC可显著降低TSA-201细胞中由α(1)1.2、β(1b)和α(2)β(1)亚基组成的钙通道的Ba2+电流。相反，通过Ca(V)1.2通道(RBC-11)的克隆脑内异构体的Ba2+电流不受PKC激活的影响。在心脏形式的Ca(V)1.2中，有两个潜在的PKC磷酸化位点存在于第27和31位，而在脑形式中则不存在。N-末端残基2-46的缺失阻止了PKC的抑制。27位和31位的苏氨酸转化为丙氨酸也取消了Ca(V)1.2的PKC敏感性。苏氨酸单独转化为丙氨酸的突变Ca(V)1.2通道对PKC的调节也不敏感，这表明两个残基的磷酸化是PKC依赖的调节所必需的。与此一致的是，在单独的突变体中，将每个苏氨酸分别突变为天冬氨酸，恢复了Ca(V)1.2的PKC敏感性，表明通过两个位点的磷酸化改变净电荷是导致抑制的原因。我们的结果确定了PKC途径抑制心脏Ca(V)1.2通道的分子基础。
We have investigated the mechanism underlying the modulation of the cardiac L-type Ca2+ current by protein kinase C (PKC). Using the patch-clamp technique, we found that PKC activation by 4-alpha -phorbol 12-myristate 13-acetate (PMA) or rac-1-oleyl-2-acetyl-glycerol (OAG) caused a substantial reduction in Ba2+ current through Ca(v)1.2 channels composed of alpha (1)1.2, beta (1b), and alpha (2)delta (1) subunits expressed in tsA-201 cells. In contrast, Ba2+ current through a cloned brain isoform of the Ca(v)1.2 channel (rbC-11) was unaffected by PKC activation. Two potential sites of PKC phosphorylation are present at positions 27 and 31 in the cardiac form of Ca(v)1.2, but not in the brain form. Deletion of N-terminal residues 2-46 prevented PKC inhibition. Conversion of the threonines at positions 27 and 31 to alanine also abolished the PKC sensitivity of Ca(v)1.2. Mutant Ca(v)1.2 channels in which the threonines were converted singly to alanines were also insensitive to PKC modulation, suggesting that phosphorylation of both residues is required for PKC-dependent modulation. Consistent with this, mutating each of the threonines individually to aspartate in separate mutants restored the PKC sensitivity, of Ca(v)1.2, indicating that a change in net charge by phosphorylation of both sites is responsible for inhibition. Our results define the molecular basis for inhibition of cardiac Ca(v)1.2 channels by the PKC pathway.