Regulation of voltage-dependent calcium channels in rat sensory neurones involves a Ras-mitogen-activated protein kinase pathway.
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). 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 %. 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. 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. The relative proportions of omega-conotoxin GVIA- and nicardipine-sensitive N- ( approximately 40 %) and L- ( approximately 40 %) type currents were unaffected by either V12S35Ras expression or U0126 pre-treatment, suggesting that all components of calcium current in DRGs, are enhanced via this pathway.
Fitzgerald
E M
EM
Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK. e.fitzgerald@ucl.ac.uk
D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D020864:Calcium Channels, N-Type / Q000187:drug effects* / Q000235:genetics; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004594:Electrophysiology; D004791:Enzyme Inhibitors; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D009475:Neurons, Afferent / Q000187:drug effects* / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010957:Plasmids / Q000235:genetics; D051381:Rats; D015854:Up-Regulation; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D002070:Butadienes; D020864:Calcium Channels, N-Type; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D009570:Nitriles; D000081082:Phosphoinositide-3 Kinase Inhibitors; C113580:U 0126; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins