Division of Biomedical Sciences, Cranmer St. George's, University of London, London, UK.
FASEB J. 2012 Jan;26(1):409-19. doi: 10.1096/fj.11-185611. Epub 2011 Oct 3.
Ca(2+)-permeable cation channels consisting of canonical transient receptor potential 1 (TRPC1) proteins mediate Ca(2+) influx pathways in vascular smooth muscle cells (VSMCs), which regulate physiological and pathological functions. We investigated properties conferred by TRPC1 proteins to native single TRPC channels in acutely isolated mesenteric artery VSMCs from wild-type (WT) and TRPC1-deficient (TRPC1(-/-)) mice using patch-clamp techniques. In WT VSMCs, the intracellular Ca(2+) store-depleting agents cyclopiazonic acid (CPA) and 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) both evoked channel currents, which had unitary conductances of ∼2 pS. In TRPC1(-/-) VSMCs, CPA-induced channel currents had 3 subconductance states of 14, 32, and 53 pS. Passive depletion of intracellular Ca(2+) stores activated whole-cell cation currents in WT but not TRPC1(-/-) VSMCs. Differential blocking actions of anti-TRPC antibodies and coimmunoprecipitation studies revealed that CPA induced heteromeric TRPC1/C5 channels in WT VSMCs and TRPC5 channels in TRPC1(-/-) VSMCs. CPA-evoked TRPC1/C5 channel activity was prevented by the protein kinase C (PKC) inhibitor chelerythrine. In addition, the PKC activator phorbol 12,13-dibutyrate (PDBu), a PKC catalytic subunit, and phosphatidylinositol-4,5-bisphosphate (PIP(2)) and phosphatidylinositol-3,4,5-trisphosphate (PIP(3)) activated TRPC1/C5 channel activity, which was prevented by chelerythrine. In contrast, CPA-evoked TRPC5 channel activity was potentiated by chelerythrine, and inhibited by PDBu, PIP(2), and PIP(3). TRPC5 channels in TRPC1(-/-) VSMCs were activated by increasing intracellular Ca(2+) concentrations (Ca(2+)), whereas increasing Ca(2+) had no effect in WT VSMCs. We conclude that agents that deplete intracellular Ca(2+) stores activate native heteromeric TRPC1/C5 channels in VSMCs, and that TRPC1 subunits are important in determining unitary conductance and conferring channel activation by PKC, PIP(2), and PIP(3).
钙（Ca（2+））通透性阳离子通道由经典瞬时受体电位 1（TRPC1）蛋白组成，在血管平滑肌细胞（VSMCs）中调节生理和病理功能的 Ca（2+）内流途径。我们使用膜片钳技术研究了 TRPC1 蛋白赋予急性分离的野生型（WT）和 TRPC1 缺陷型（TRPC1（-/-））小鼠肠系膜动脉 VSMCs 中天然单 TRPC 通道的特性。在 WT VSMCs 中，细胞内 Ca（2+）储存耗竭剂环孢菌素 A（CPA）和 1,2-双（2-氨基苯氧基）乙烷-N，N，N'，N'-四乙酸（BAPTA-AM）均诱发通道电流，其单元电导约为 2 pS。在 TRPC1（-/-）VSMCs 中，CPA 诱导的通道电流具有 14、32 和 53 pS 的 3 种亚电导状态。WT VSMCs 中细胞内 Ca（2+）储存的被动耗竭激活了全细胞阳离子电流，但 TRPC1（-/-）VSMCs 中没有激活。抗 TRPC 抗体的差异阻断作用和共免疫沉淀研究表明，CPA 在 WT VSMCs 中诱导异源 TRPC1/C5 通道，在 TRPC1（-/-）VSMCs 中诱导 TRPC5 通道。PKC 抑制剂Chelerythrine 可阻止 CPA 诱导的 TRPC1/C5 通道活性。此外，PKC 激活剂佛波醇 12,13-二丁酸酯（PDBu）、PKC 催化亚基和磷脂酰肌醇-4,5-二磷酸（PIP（2））和磷脂酰肌醇-3,4,5-三磷酸（PIP（3））激活 TRPC1/C5 通道活性，Chelerythrine 可阻止其活性。相反，Chelerythrine 增强 CPA 诱导的 TRPC5 通道活性，PDBu、PIP（2）和 PIP（3）抑制其活性。TRPC1（-/-）VSMCs 中的 TRPC5 通道被增加的细胞内 Ca（2+）浓度[Ca（2+）]（i）激活，而 WT VSMCs 中增加[Ca（2+）]（i）没有影响。我们得出结论，耗竭细胞内 Ca（2+）储存的试剂激活 VSMCs 中的天然异源 TRPC1/C5 通道，并且 TRPC1 亚基对于确定单元电导和赋予 PKC、PIP（2）和 PIP（3）的通道激活很重要。
