Regulation of voltage-dependent calcium channels in rat sensory neurones involves a Ras-mitogen-activated protein kinase pathway

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.