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

Abstract

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.

Figure 1. The effect of V12Ras and V12S186Ras expression on calcium current density

The averaged current density-voltage relationships for control GFP-, V12Ras- and V12S186Ras-expressing cells were evoked using 200 ms depolarising steps from −50 to +55 mV in 5 mV increments, from a holding potential (V_h) of −80 mV. Individual curves were fitted with a Boltzmann function as outlined in the legend to Table 1. Dashed lines denote the Boltzmann fits to these averaged curves. Repeated measures ANOVA indicates that V12Ras and V12S186Ras have significant effects on the current-voltage relationships (F_2,43, 14.13; P < 0.001). The inset shows the average peak current traces at +15 mV for each of the three treatment groups. Individual traces within each treatment group were normalised to show current density and then averaged.

Figure 2. Lack of effect of V12Ras and V12S186Ras expression on the inactivation of calcium current

The histogram shows percentage inactivation of peak current amplitude measured at 1.5 s in GFP- (n = 5), V12Ras- (n = 8) and V12S186Ras-transfected cells (n = 6). The inset shows the averaged current traces normalised to the peak, for GFP- (n = 5), V12Ras- (n = 8) and V12S186Ras-expressing cells (n = 6). Individual currents were evoked by 1500 ms pulses to +15 mV from a V_h of −80 mV. The decaying phase of the current was best fitted by a double exponential, as indicated by the continuous line (V12Ras trace), to give time constants for fast (τ_fast, 107.7 ± 40.1 ms) and slow (τ_slow, 639.5 ± 48.7 ms) inactivation.

Figure 3. Effect of Ras effector region mutants V12S35Ras, V12C40Ras and V12G37Ras

Averaged current density-voltage relationships and associated average current traces for each Ras effector loop mutant are shown with the relevant control. A, effect of V12S35Ras expression; B, effect of V12C40Ras expression; C, effect of V12G37Ras expression. For current-voltage plots, individual currents were evoked using 200 ms depolarising test pulses in 5 mV steps, from a V_h of −80 mV. The averaged current traces shown were obtained at a test potential of +15 mV. Asterisks denote statistically significant differences between the mutant-expressing and control GFP-expressing cells: *P < 0.05 and **P < 0.01.

Figure 4. Effects of MEK inhibitor U0126 and lack of effect of PtdI 3-K inhibitor LY294002

A, current density-voltage relationships for control and U0126 pre-treated cells. Cells were pre-incubated for 30 min in culture medium containing 10 μm U0126 and the currents compared with those recorded from control cells which had been pre-incubated with normal culture medium for 30 min. The inset shows the averaged current traces for control and U0126-treated cells obtained using a depolarising pulse to +15 mV. Asterisks indicate significant differences between control and U0126 pre-treated cells: *P < 0.05. B, representative time courses for acute application of 20 μm U0126 and 20 μm U0124, plotted as percentage change in peak current. Drug application was by continuous perfusion. The inset shows the current traces recorded at time points 1 and 2, before and after application of U0126, as indicated on the plot. Currents were evoked using 100 ms test pulses to +15 mV. Vertical scale bars indicate the absolute current amplitude. C, average current density-voltage plots for control and LY294002 pre-treated cells. As described above, cells were pre-incubated for 30 min with 50 μm LY294002-containing culture medium. Control cells were pre-treated with normal culture medium only. The inset shows average currents evoked from a V_h−80 mV using 200 ms depolarising pulses to +15 mV.

Figure 5. Lack of effect of V12S35Ras on the relative proportions of VDCC subtypes in DRGs

A, histogram showing the percentage inhibition of peak current by nicardipine (5 μm) and ω-conotoxin GVIA (ω-CgTx GVIA, 0.5 μm) in GFP-expressing control cells compared with V12S35Ras-expressing cells. B, representative current traces showing the effect of nicardipine followed by ω-CgTx GVIA on GFP control and V12S35Ras-expressing cells. Individual traces were evoked using 100 ms depolarising pulses to +15 mV from a V_h of −80 mV. Vertical scale bars indicate the absolute current amplitude.