Berecki Géza, McArthur Jeffrey R, Cuny Hartmut, Clark Richard J, Adams David J
Health Innovations Research Institute, RMIT University, Melbourne, Victoria 3083, Australia.
J Gen Physiol. 2014 Apr;143(4):465-79. doi: 10.1085/jgp.201311104.
Neuronal Cav2.1 (P/Q-type), Cav2.2 (N-type), and Cav2.3 (R-type) calcium channels contribute to synaptic transmission and are modulated through G protein-coupled receptor pathways. The analgesic α-conotoxin Vc1.1 acts through γ-aminobutyric acid type B (GABAB) receptors (GABABRs) to inhibit Cav2.2 channels. We investigated GABABR-mediated modulation by Vc1.1, a cyclized form of Vc1.1 (c-Vc1.1), and the GABABR agonist baclofen of human Cav2.1 or Cav2.3 channels heterologously expressed in human embryonic kidney cells. 50 µM baclofen inhibited Cav2.1 and Cav2.3 channel Ba(2+) currents by ∼40%, whereas c-Vc1.1 did not affect Cav2.1 but potently inhibited Cav2.3, with a half-maximal inhibitory concentration of ∼300 pM. Depolarizing paired pulses revealed that ∼75% of the baclofen inhibition of Cav2.1 was voltage dependent and could be relieved by strong depolarization. In contrast, baclofen or Vc1.1 inhibition of Cav2.3 channels was solely mediated through voltage-independent pathways that could be disrupted by pertussis toxin, guanosine 5'-[β-thio]diphosphate trilithium salt, or the GABABR antagonist CGP55845. Overexpression of the kinase c-Src significantly increased inhibition of Cav2.3 by c-Vc1.1. Conversely, coexpression of a catalytically inactive double mutant form of c-Src or pretreatment with a phosphorylated pp60c-Src peptide abolished the effect of c-Vc1.1. Site-directed mutational analyses of Cav2.3 demonstrated that tyrosines 1761 and 1765 within exon 37 are critical for inhibition of Cav2.3 by c-Vc1.1 and are involved in baclofen inhibition of these channels. Remarkably, point mutations introducing specific c-Src phosphorylation sites into human Cav2.1 channels conferred c-Vc1.1 sensitivity. Our findings show that Vc1.1 inhibition of Cav2.3, which defines Cav2.3 channels as potential targets for analgesic α-conotoxins, is caused by specific c-Src phosphorylation sites in the C terminus.
神经元Cav2.1（P/Q型）、Cav2.2（N型）和Cav2.3（R型）钙通道参与突触传递，并通过G蛋白偶联受体途径进行调节。镇痛性α-芋螺毒素Vc1.1通过B型γ-氨基丁酸（GABAB）受体（GABABRs）发挥作用，抑制Cav2.2通道。我们研究了Vc1.1、Vc1.1的环化形式（c-Vc1.1）以及GABABR激动剂巴氯芬对在人胚肾细胞中异源表达的人Cav2.1或Cav2.3通道的GABABR介导的调节作用。50 μM巴氯芬使Cav2.1和Cav2.3通道的Ba(2+)电流抑制约40%，而c-Vc1.1不影响Cav2.1，但能有效抑制Cav2.3，半数最大抑制浓度约为300 pM。去极化配对脉冲显示，巴氯芬对Cav2.1的抑制作用约75%是电压依赖性的，可通过强去极化解除。相比之下，巴氯芬或Vc1.1对Cav2.3通道的抑制作用仅通过百日咳毒素、鸟苷5'-[β-硫代]二磷酸三锂盐或GABABR拮抗剂CGP55845可破坏的电压非依赖性途径介导。激酶c-Src的过表达显著增强了c-Vc1.1对Cav2.3的抑制作用。相反，共表达催化无活性的c-Src双突变体形式或用磷酸化的pp60c-Src肽预处理可消除c-Vc1.1的作用。对Cav2.3的定点突变分析表明，第37外显子中的酪氨酸1761和1765对于c-Vc1.1抑制Cav2.3至关重要，并参与巴氯芬对这些通道的抑制作用。值得注意的是，在人Cav2.1通道中引入特定c-Src磷酸化位点的点突变赋予了c-Vc1.1敏感性。我们的研究结果表明，Vc1.1对Cav2.3的抑制作用是由C末端特定的c-Src磷酸化位点引起的，这将Cav2.3通道定义为镇痛性α-芋螺毒素的潜在靶点。
