Regulation of voltage-dependent calcium channels in rat sensory neurones involves a Ras-mitogen-activated protein kinase pathway
Regulation of voltage-dependent calcium channels in rat sensory neurones involves a Ras-mitogen-activated protein kinase pathway
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DOI：
10.1111/j.1469-7793.2000.00433.x
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发表时间：
2000-09-15
影响因子：
5.5
通讯作者：
Fitzgerald, EM
中科院分区：
文献类型：
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作者：
Fitzgerald, EM
1. 小g蛋白Ras是调节细胞生长信号通路的关键成分，参与了大鼠感觉神经元电压依赖性钙通道（VDCCs）的补性上调。为了研究Ras的哪些下游效应物参与了这一过程，在原代培养的大鼠背根神经节神经元（DRGs）中，一系列Ras突变体cdna与绿色荧光蛋白（GFP）共表达。与对照细胞（单独使用GFP）相比，组成活性V12Ras（甘氨酸12到缬氨酸）显着增加了41%的基础钙电流密度。相比之下，法酰化缺陷突变体V12S186Ras（半胱氨酸186到丝氨酸）没有激活下游效应物，显著降低了47%的钙电流密度。Ras效应区突变体V12C40（酪氨酸40转化为半胱氨酸，激活磷脂酰肌醇3-激酶p110 α亚基）和V12G37（谷氨酸37转化为甘氨酸，激活拉尔鸟嘌呤核苷酸解离刺激剂）对VDCC电流无显著影响。然而，V12S35Ras（苏氨酸35 -丝氨酸；激活Raf-1和丝裂原活化蛋白激酶（MAPK）途径）显著增加基础钙电流密度67%，表明Raf-1激活足以使Ras增强这些细胞中的钙电流。Raf-1激活MAPK通路中的MEK （MAPK激酶），MEK抑制剂U0126在10-15分钟后降低了45%的钙电流，而无活性类似物U0124则没有作用。MEK抑制的快速时间过程表明，通过Ras-MAPK途径直接调节vdcc，而不是基因表达介导的作用。V12S35Ras表达或U0126预处理均不影响omega- concontoxin GVIA-和nicardipine敏感的N-（接近40%）和L（接近40%）型电流的相对比例，这表明DRGs中钙电流的所有成分都通过这一途径增强。
1. The small G-protein Ras, a critical component in the signalling pathways regulating cell growth, is involved in the tonic upregulation of voltage-dependent calcium channels (VDCCs) in rat sensory neurones. To investigate which downstream effector(s) of Ras is involved in this process, a series of Ras mutant cDNAs were co-expressed with green fluorescent protein (GFP) in primary cultured rat dorsal root ganglion neurones (DRGs).2. Constitutively active V12Ras (glycine 12 to valine) markedly increased basal calcium current density by 41% compared with control cells (GFP alone). In contrast, a farnesylation-defective mutant, V12S186Ras (cysteine 186 to serine; activates no downstream effectors,, significantly reduced calcium current density by 47%.3. Ras effector region mutants V12C40 (tyrosine 40 to cysteine; activates the p110 alpha-subunit of phosphatidylinositol 3-kinase) and V12G37 (glutamic acid 37 to glycine; activates Ral guanine nucleotide dissociation stimulator) had no significant effect on VDCC current. However, V12S35Ras (threonine 35 to serine; activates Raf-1 and the mitogen-activated protein kinase (MAPK) pathway) markedly increased basal calcium current density by 67%, suggesting that Raf-1 activation is sufficient for Ras enhancement of calcium current in these cells.4. Raf-1 activates MEK (MAPK kinase) in the MAPK pathway, and the MEK inhibitor U0126 reduced calcium current by 45% after 10-15 min, whereas the inactive analogue U0124 had no effect. This rapid time course for MEK inhibition suggests direct modulation of VDCCs via the Ras-MAPK pathway rather than gene expression-mediated effects.5. The relative proportions of omega-conotoxin GVIA- and nicardipine-sensitive N- (similar to 40%) and L(similar to 40%) type currents were unaffected by either V12S35Ras expression or U0126 pretreatment, suggesting that all components of calcium current in DRGs, are enhanced via this pathway.