Peppiatt-Wildman C M, Albert A P, Saleh S N, Large W A
Ion Channel and Cell Signalling, Division of Basic Medical Sciences, St George's, University of London, Cranmer Terrace, London SW17 ORE, UK.
J Physiol. 2007 May 1;580(Pt.3):755-64. doi: 10.1113/jphysiol.2006.126656. Epub 2007 Feb 15.
In the present work we used patch pipette techniques to study the properties of a novel Ca(2+)-permeable cation channel activated by the potent coronary vasoconstrictor endothelin-1 (ET-1) in freshly dispersed rabbit coronary artery myocytes. With cell-attached recording bath application of 10 nm ET-1 evoked cation channel currents (I(cat)) with subconductance states of about 18, 34 and 51 and 68 pS, and a reversal potential of 0 mV. ET-1 evoked channel activity when extracellular Ca(2+) was the charge carrier, illustrating significant Ca(2+) permeability. ET-1-induced responses were inhibited by the ET(A) receptor antagonist BQ123 and the phospholipase C (PLC) inhibitor U73122. The diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) also stimulated I(cat), but the protein kinase C (PKC) inhibitor chelerythrine did not inhibit either the OAG- or ET-1-induced I(cat). Inositol 1,4,5-trisphosphate (IP(3)) did not activate I(cat), but greatly potentiated the response to OAG and this effect was blocked by heparin. Bath application of anti-TRPC3 and anti-TRPC7 antibodies to inside-out patches markedly inhibited ET-1-evoked I(cat), but antibodies to TRPC1, C4, C5 and C6 had no effect. Immunocytochemical studies demonstrated preferential TRPC7 expression in the plasmalemma, whereas TRPC3 was distributed throughout the myocyte, and moreover co-localization of TRPC3 and TRPC7 signals was observed at, or close to, the plasma membrane. Flufenamic acid, Gd(3+), La(3+) and extracellular Ca(2+) inhibited I(cat) with IC(50) values of 2.45 microm, 3.8 microm, 7.36 microm and 22 microm, respectively. These results suggest that in rabbit coronary artery myocytes ET-1 evokes a Ca(2+)-permeable non-selective cation channel with properties similar to TRPC3 and TRPC7, and indicates that these proteins may be important components of this conductance.
在本研究中，我们运用膜片吸管技术，研究了一种新型的钙通透阳离子通道的特性，该通道由强效冠状动脉血管收缩剂内皮素-1（ET-1）激活，存在于新鲜分离的兔冠状动脉心肌细胞中。在细胞贴附式记录中，向浴槽施加10 nM的ET-1可诱发阳离子通道电流（I(cat)），其亚电导状态约为18、34、51和68 pS，反转电位为0 mV。当细胞外钙作为电荷载体时，ET-1可诱发通道活性，表明其具有显著的钙通透性。ET-1诱导的反应可被ET(A)受体拮抗剂BQ123和磷脂酶C（PLC）抑制剂U73122抑制。二酰基甘油类似物1-油酰基-2-乙酰基-sn-甘油（OAG）也可刺激I(cat)，但蛋白激酶C（PKC）抑制剂白屈菜红碱既不抑制OAG诱导的I(cat)，也不抑制ET-1诱导的I(cat)。肌醇1,4,5-三磷酸（IP(3)）不能激活I(cat)，但可显著增强对OAG的反应，且这种作用可被肝素阻断。将抗TRPC3和抗TRPC7抗体施加于内面向外的膜片上，可显著抑制ET-1诱发的I(cat)，但抗TRPC1、C4、C5和C6抗体则无此作用。免疫细胞化学研究表明，TRPC7在质膜上有优先表达，而TRPC3分布于整个心肌细胞，此外，在质膜处或靠近质膜处观察到TRPC3和TRPC7信号的共定位。氟芬那酸、钆（Gd(3+)）、镧（La(3+)）和细胞外钙可抑制I(cat)，其IC(50)值分别为2.45 μM、3.8 μM、7.36 μM和22 μM。这些结果表明，在兔冠状动脉心肌细胞中，ET-1可诱发一种钙通透的非选择性阳离子通道，其特性与TRPC3和TRPC7相似，提示这些蛋白可能是该电导的重要组成部分。
