Skip to main content
NCBI home page
The Journal of Physiology logoLink to The Journal of Physiology
. 2000 Sep 15;527(Pt 3):433–444. doi: 10.1111/j.1469-7793.2000.00433.x

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

E M Fitzgerald 1
PMCID: PMC2270090  PMID: 10990531

Abstract

  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 α-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 ω-conotoxin GVIA- and nicardipine-sensitive N- (∼40 %) and L- (∼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.

Intracellular calcium plays a pivotal role in cell growth, and calcium influx through voltage-dependent calcium channels (VDCCs) has been implicated in a number of related processes including differentiation (Hagag et al. 1986), survival (Larmet et al. 1992), neurite outgrowth (Cohan et al. 1987; Gomez & Spitzer, 1999), and gene expression (Murphy et al. 1991; Ghosh et al. 1994). Many of the signalling proteins which control cell growth, in particular the small G-protein Ras, receptor tyrosine kinases (RTKs), phosphatidylinositol 3-kinase (PtdI 3-K) and mitogen-activated protein kinases (MAPKs), are increasingly being shown to play a role in ion channel regulation. Ras enhancement of N-, L- and T-type VDCC current has been documented in several neuronal cell types (e.g. Hescheler et al. 1991; Hahnel et al. 1992; Pollack & Rane, 1996; Fitzgerald & Dolphin, 1997; Lei et al. 1998), and nerve growth factor (NGF), an upstream activator of Ras, is widely reported to have similar effects (Levine et al. 1995; Wildering et al. 1995; Fitzgerald & Dolphin, 1997; Lei et al. 1998). A role for L-type VDCCs in NGF-independent activation of the Ras-MAPK pathway has also been proposed (Finkbeiner & Greenberg, 1996). Downstream from Ras, MAPKs and PtdI 3-K, have been implicated in the upregulation of neuronal N- and L-type VDCCs (Lei et al. 1998; Blair et al. 1999), whilst in a recent report, activation of the Ras-MAPK pathway was also shown to suppress endogenous T-type channels in Swiss 3T3 cells (Strobeck et al. 1999). In sensory neurones, endogenous Ras is involved in the tonic upregulation of VDCCs (Fitzgerald & Dolphin, 1997). This process largely involves NGF-RTK activation of Ras but the downstream effectors involved have not yet been identified (Fitzgerald & Dolphin, 1997).

Activated Ras has three main downstream effector pathways: the MAPK pathway, essential for cell differentiation and proliferation (Marshall, 1996); the PtdI 3-K pathway, involved in cell proliferation and survival (Toker & Cantley, 1997), and the Ral guanine nucleotide dissociation stimulator (RalGDS) pathway, which plays a role in cell morphology and cytoskeletal development (Khosravi-Far et al. 1998; Vojtek & Der, 1998). Relatively subtle mutations within the effector region of Ras (residues 32–40) can produce partial loss of function mutants in which the interaction with one effector is maintained but with the others is lost (White et al. 1995; Joneson et al. 1996). Here, a variety of Harvey-Ras (Ha-Ras) mutants, in a constitutively active V12 background (Rodriguez-Viciana et al. 1997), were expressed in primary cultured rat dorsal root ganglion neurones. Mutations which prevent activation of MAPK failed to enhance the basal calcium current density whereas mutations which activate only the MAPK pathway were sufficient to increase current density, implying a role for MAPK signalling in Ras modulation of VDCCs. All components of calcium current were upregulated via the Ras-MAPK pathway, suggesting a common mechanism for interaction at the channel level.

METHODS

Cell culture

Rat dorsal root ganglion neurones were cultured as previously described by Fitzgerald & Dolphin (1997). Briefly, dorsal root ganglia were dissected from 1- to 2-day-old rats that had been killed by decapitation. Ganglia were then incubated with collagenase (1.25 μg ml−1; Sigma, Poole, UK) for 13 min and then with trypsin (2.5 μg ml−1, Sigma) for 6 min in Ham's F14 nutrient medium (Gibco-Life Technologies, Paisley, UK) containing 10 % heat-inactivated horse serum (Gibco-Life Technologies), glutamine (2 mm, Sigma), penicillin (50 IU ml−1) and streptomycin (50 μg ml−1). Following this, ganglia were mechanically dissociated using a fire-polished pipette, in the presence of DNase (1600 kunitz ml−1; Sigma). Dorsal root ganglion neurones were plated onto poly-L-lysine/laminin (Sigma) at a density of approximately 1–3 × 104 cells per coverslip (22 mm × 22 mm) in the above medium containing nerve growth factor (1 ng ml−1; Sigma). After 24 h in culture at 37°C in air containing 5 % CO2, the cells were transfected as described below. Recordings were made between 1 and 2 days after transfection.

Plasmids and transfection

The following human Ha-Ras (Kraus et al. 1984) mutant plasmids were a gift from Dr J. Downward (ICRF, Lincoln's Inn Fields, London): V12, V12S186, V12S35, V12C40, and V12G37. Within a constitutively active V12 background (glycine 12 to valine), each construct has been point-mutated within the effector region (residues 32–40) to create a partial loss of function mutant which preferentially binds to one effector whilst failing to bind the other two (White et al. 1995; Joneson et al. 1996; Rodriguez-Viciana et al. 1997). V12S35Ras (threonine 35 to serine) preferentially activates Raf-1. V12C40Ras (tyrosine 40 to cysteine) preferentially binds the p110 catalytic α-subunit of PtdI 3-K, and V12G37Ras (glutamic acid 37 to glycine) activates RalGDS. V12S186Ras (cysteine 186 to serine) is a farnesylation-defective mutant which does not activate any downstream effectors. The mut-3 mutant of green fluorescent protein (GFP) was a gift from Dr T. Hughes (Yale University, New Haven, CT, USA). All cDNAs were inserted into pcDNA3 expression vector under the control of a cytomegalovirus (CMV)-based promoter.

Dorsal root ganglion neurones were transfected by means of the Biolistic PDS-1000/He system (Bio-Rad, Hemel Hempstead, Herts, UK) which utilises a combination of helium pressure and vacuum circuits to bombard DNA-coated subcellular sized gold particles (1.6 μm) into the nucleus of target cells. After washing sequentially with ethanol (70 % and 100 %) and water, the gold particles were suspended in 50 % glycerol to give a final concentration of 60 mg ml−1. cDNA (either 6 μl GFP + 6 μl Ras mutant DNA, or 6 μl GFP alone as a control), 50 μl CaCl2 (2.5 M) and 20 μl spermidine (0.1 M) were added sequentially to 50 μl of gold particle suspension. After vortexing the suspension for 15 min the supernatant was discarded and the gold particles washed again in 70 %, followed by 100 % ethanol. Just prior to transfection, the DNA-coated gold particles were re-suspended in 100 % ethanol (48 μl per 6 bombardments). Following bombardment at 500 p.s.i., the cells were returned to the incubator at 37°C until used in experiments. Cells were viewed under fluorescence at approximately 20 h after transfection. Successfully transfected cells were identified for electrophysiological recording by expression of GFP. On average, between 10–20 % of cells were fluorescent.

Electrophysiology and solutions

Electrophysiological recordings were performed between 1 and 2 days after transfection. Mean cell capacitance was approximately 15 pF. Cells were viewed briefly with a fluorescein filter block, and only small fluorescent cells expressing GFP, which were spatially isolated and had a compact morphology with short fine neurites, were used in experiments. The whole-cell configuration of the patch-clamp technique (Hamill et al. 1981) was used to record barium currents in the following solutions. The internal solution contained (mm): caesium aspartate 140.0, MgCl2 2.0, CaCl2 0.1, Hepes 20, EGTA 5, K2ATP 1.0; adjusted to pH 7.2 with CsOH, and to 310 mosmol l−1 with sucrose. The external solution contained (mm): TEABr 160.0, MgCl2 1.0, KCl 5, NaHCO3 1.0, Hepes 10.0, glucose 4.0, BaCl2 1.0, tetrodotoxin 0.0005; adjusted to pH 7.4 with Tris-base, and to 320 mosmol l−1 with sucrose. All experiments were performed at room temperature (20–22°C). In some cells, a time-dependent increase in peak barium current (IBa) associated with a hyperpolarising shift in the current-voltage relationship was observed during the initial period after establishing whole-cell mode. All the data presented here were therefore taken from currents which had been allowed to stabilise for up to 10 min before any measurements were made.

Patch pipettes of 2–4 MΩ resistance were pulled from thin-walled borosilicate glass tubing, fire-polished and coated with Sigmacote (Sigma). An Axopatch-1D amplifier (Axon Instruments, Foster City, CA, USA) was used for recordings which were filtered at 2 kHz (4-pole Bessel filter) and digitised at 5–44 kHz using a Digidata 1200 A/D converter (Axon Instruments). The cells were held at a potential of −80 mV where the holding current was less than −0.05 nA and series resistance normally less than 10 MΩ. Currents were recorded with the cell capacitance compensated and leak currents subtracted using either a P/4 or P/8 subtraction protocol. Series resistance was compensated up to 80 % and only cells which were adequately space clamped, as judged by a graded current activation, were used. Voltage errors from residual uncompensated series resistance were less than 5 mV. The liquid junction potential was 5 mV and all data have been corrected accordingly. Data acquisition and analysis was performed using pCLAMP software (version 6.0.4, Axon Instruments) and Origin version 3.5/5.0 (Microcal Software, Northampton, MA, USA).

Drug or control solutions were applied under continuous perfusion. U0126 (Promega, Southampton, UK) and U0124 (Calbiochem, Nottingham, UK) were first dissolved in DMSO and then added to external solution to give a final concentration of 10 or 20 μm. Similarly, LY294002 (Biomol-Affiniti Research, Mamhead, Devon, UK) was also dissolved in DMSO and subsequently added to external solution to be used at a final concentration of 50 μm. There were no adverse effects of DMSO (0.01 %) on calcium currents in DRGs (data not shown). Nicardipine (Sigma) was first dissolved in 100 % ethanol and used at 5 μm whilst ω-conotoxin GVIA (Alomone Laboratories, Jerusalem, Israel) was dissolved in dH2O and used at a final concentration of 0.5 μm. All the reagents used were stored as aliquots of stock solution at −20°C, diluted, and used freshly each day.

All data are presented as the mean ±s.e.m. Unless stated otherwise, statistical analysis was carried out using Student's t test, using 95 % confidence limits (Origin version 5.0). Where more than two factors were tested, one-way ANOVA and/or repeated measures ANOVA were used as appropriate (Statistica version 4.0, StatSoft Inc., Tulsa, OK, USA). Due to the inherent variability within and between different DRG cultures, treated cells were compared with the relevant control for each separate group of experiments (see Tables 1 and 2).

Table 1.

Biophysical properties of wholecell calcium current in V12Ras-, V12S186Ras-, V12S35Ras-, V12C40Ras-and V12G37Ras-expressing DRG neurones

Treatment Imax (pA pF−1) V50 (mV) k (mV) Vrev (mV) τact at −5 mV (ms) Capacitance (pF)
GFP −53.9 ± 5.3 (17) −1.4 ± 0.9 (17) 5.3 ± 0.2 (17) 51.4 ± 1.5 (17) 11.2 ± 5.9 (17) 12.2 ± 1.6 (17)
V12Ras −76.3 ± 8.4 (16) −2.7 ± 0.8 (16) 5.4 ± 0.3 (16) 55.5 ± 1.0 (16) 11.5 ± 5.9 (16) 12.0 ± 1.4 (16)
V12S186Ras −28.9 ± 2.2 (13) −1.6 ± 1.2 (13) 5.6 ± 0.2 (13) 46.3 ± 2.2 (13) 15.8 ± 5.9 (11) 13.1 ± 2.7 (13)
F2,43    14.13***      1.13 0.39     8.22      0.30      0.10
GFP −44.7 ± 3.9 (15) −0.7 ± 0.7 (15) 5.5 ± 0.2 (15) 48.4 ± 1.3 (15) 7.7 ± 1.7 (15) 14.3 ± 2.0 (15)
V12S35Ras −74.8 ± 6.4 (15)** −3.5 ± 1.2 (15) 5.4 ± 0.3 (15) 52.1 ± 1.2 (15) 11.7 ± 8.9 (15) 16.4 ± 2.6 (15)
GFP −47.4 ± 8.9 (11) −0.7 ± 1.0 (11) 5.4 ± 0.3 (11) 47.4 ± 2.1 (11) 11.2 ± 3.5 (11) 14.2 ± 2.1 (11)
V12C40Ras −60.8 ± 8.6 (18) −3.4 ± 1.0 (18) 5.3 ± 0.4 (18) 52.8 ± 1.1 (18) 15.1 ± 3.1 (18) 12.0 ± 1.1 (18)
GFP −57.3 ± 7.1 (10) −1.7 ± 1.5 (10) 5.2 ± 0.2 (10) 53.8 ± 1.7 (10) 9.5 ± 3.5 (10) 14.8 ± 2.1 (10)
V12G37Ras −39.9 ± 4.8 (10) −1.7 ± 0.8 (10) 5.7 ± 0.2 (10) 52.0 ± 1.6 (10) 10.4 ± 2.7 (10) 16.2 ± 1.9 (10)
Open in a new tab
Imax is the maximum peak current density. Individual current densityvoltage plots were fitted with a Boltzmann function:
graphic file with name tjp0527-0433-mu1.jpg
where Vrev is the reversal potential, V50 is the voltage for halfmaximal activation of current, g is the conductance and k is the slope factor. The time constants of current activation (τact) were derived by fitting single exponentials to the rising phases of individual currents, typically evoked using 200 ms pulses. Capacitance refers to the wholecell capacitance obtained directly from the amplifier. Significant differences, determined using either Student's t test or oneway ANOVA, are denoted
**

P < 0.01

***

P < 0.001. The number of cells per treatment is shown in parentheses.

Table 2.

Effects of U0126 and LY294002 on the biophysical properties of wholecell calcium current in DRGs

Treatment Imax (pA pF−1) V50 (mV) k (mV) Vrev (mV) Capacitance (pF)
Control −65.8 ± 6.6 (8) −1.2 ± 1.0 (8) 5.1 ± 0.2 (8) 60.2 ± 1.5 (8) 15.5 ± 1.8 (8)
U0126 (10 μm) −40.4 ± 7.6 (8) −0.7 ± 1.2 (8) 5.0 ± 0.2 (8) 52.9 ± 2.4 (8) 12.7 ± 1.7 (8)
U0124 (10 μm) −66.4 ± 13.9 (5) −3.8 ± 2.9 (5) 4.8 ± 0.4 (5) 52.9 ± 3.1 (5) 18.8 ± 3.4 (5)
F2,18    4.36*     0.94     0.15     0.76     1.77
Control −81.9 ± 7.8 (17) −3.1 ± 1.0 (17) 5.4 ± 0.3 (17) 53.3 ± 1.4 (17) 15.4 ± 1.9 (17)
LY294002 (50 μm) −64.9 ± 8.6 (17) −3.1 ± 1.0 (17) 5.5 ± 0.3 (17) 50.8 ± 1.5 (17) 13.9 ± 0. 9 (17)
Open in a new tab

Imax, V50, k and Vrev are as previously defined in the legend to Table 1. Data were analysed using either Student's t test or one-way ANOVA

*

(P < 0.05). U0124/U0126 data were log transformed to meet assumptions of normality. The number of cells per treatment is shown in parentheses.

RESULTS

Effect of V12Ras and V12S186Ras expression

In control GFP-expressing cells, the average peak current density (Imax) was −53.9 ± 5.3 pA pF−1, with the peak generally occurring at +15 mV (Table 1 and Fig. 1). This inward current was composed almost exclusively of high voltage-activated (HVA) current. Only a small proportion of cells (< 10 %), exhibited evidence of low voltage-activated (LVA) current (data not shown). Hence, all the effects on calcium current reported here can essentially be considered to affect HVA calcium current. Expression of constitutively active V12Ras resulted in an increase in Imax of 41 % to −76.3 ± 8.4 pA pF−1, whereas expression of the farnesylation-defective mutant V12S186Ras, resulted in a 47 % decrease of Imax to −26.9 ± 2.2 pA pF−1 (one-way ANOVA; F2,43, 14.13; P < 0.001). This effect was consistent throughout the voltage range tested (repeated measures ANOVA; F2,43, 14.24; P < 0.001; Fig. 1). The voltage for half-maximal activation of current (V50) was derived from current-voltage plots as outlined in the legend to Table 1. In spite of the changes in current density, the voltage dependence of current activation, as indicated by the V50, was not significantly affected by expression of either V12Ras or V12S186Ras (see Fig. 1 and Table 1). The averaged current traces shown in Fig. 1 suggested that the current kinetics were also largely unaffected by expression of either V12Ras or V12S186Ras. By fitting a single exponential to the rising phase of current at −5 mV, average values for the time constant of activation (τact) were derived for each treatment group. As shown in Table 1, τact at −5 mV was indeed unaffected by expression of either Ras mutant. All cells exhibited some degree of current inactivation over relatively short pulses of 200 ms (Fig. 1). To investigate inactivation more closely, peak currents elicited using 1500 ms test pulses to +15 mV were compared (Fig. 2). The decaying phase of current was best fitted by a double exponential to give time constants for slow (τslow) and fast (τfast) inactivation of current. Peak control currents recorded at +15 mV inactivated by 63 ± 6 % over 1500 ms, and at this time current inactivation was still not complete. For these control currents, τfast was 86 ± 28 ms and τslow was 910 ± 192 ms (n = 5). In V12Ras-transfected cells, the percentage current inactivation at 1500 ms was 59 ± 3 %, with a τfast of 108 ± 40 ms and τslow of 640 ± 49 ms (n = 8). In cells over-expressing V12S186Ras, τfast was 59 ± 9 ms and τslow 768 ± 63 ms, which was associated with 73 ± 4 % inactivation (n = 6). Thus, although the average currents shown in Fig. 2 may suggest a slight increase in the rate of inactivation for V12S186Ras- compared with some slowing for V12Ras-expressing cells, neither the percentage inactivation measured at 1500 ms, nor the rates of inactivation of these currents were significantly different from controls. Consequently, the principal effect of upregulating the activity of Ras by expressing constitutively active V12Ras was to increase the basal current density. Expression of the V12S186Ras mutant, which is unable to activate any downstream effectors, reduced the basal current density. Importantly, the whole-cell capacitance, an approximate measure of cell size, was the same in all three groups of cells, implying that the changes in current density did not simply reflect an increase or decrease in overall cell size resulting for example from extension or retraction of neurites (Table 1).

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

Figure 1

Open in a new tab

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 (Vh) 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 (F2,43, 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.

Figure 2

Open in a new tab

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 Vh 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 (τfast, 107.7 ± 40.1 ms) and slow (τslow, 639.5 ± 48.7 ms) inactivation.

Effects of Ras effector region mutants V12S35-, V12C40- and V12G37Ras

In order to determine which downstream effector(s) of Ras is involved in VDCC regulation, three Ras effector region mutants (V12S35, V12C40 and V12G37) were expressed in DRGs.

The V12S35Ras mutant preferentially interacts with Raf-1 to activate the MAPK pathway (Rodriguez-Viciana et al. 1997). Expression of V12S35Ras increased the peak current density, Imax, by 67 % compared with control cells transfected with GFP alone (P < 0.01). No significant shift in the current-voltage relationship was observed as a result of V12S35Ras expression (Table 1 and Fig. 3A). Similarly, there were no obvious effects of V12S35Ras on either current activation (see Table 1) or inactivation. The time constants for inactivation fitted to Imax at +15 mV in V12S35Ras-expressing cells (τfast, 71 ± 7 ms; τslow, 673 ± 37 ms; n = 7) were not significantly different from those obtained previously for control cells (see earlier). Furthermore, the percentage inactivation of current over 1500 ms, 68 ± 7 % (n = 7) was also unaffected. Thus, as with expression of constitutively active V12Ras, the principal effect of V12S35Ras expression was an increase in basal current density. Again, the whole-cell capacitance was unaffected, implying that the increase in current density was not due simply to increased neurite outgrowth following expression of V12S35Ras (Table 1).

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

Figure 3

Open in a new tab

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 Vh 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.

The V12C40Ras mutant preferentially interacts with the p110 catalytic α-subunit of PtdI 3-K, another well established Ras effector with important roles in cell growth and survival (e.g. Toker & Cantley, 1997; Klesse & Parada, 1998). Expression of V12C40Ras caused a slight though not significant increase in the peak current density (28 %), and no significant effects on the voltage dependence of activation were observed (Table 1 and Fig. 3B). Current activation and inactivation kinetics were also unaffected by V12C40Ras expression (Table 1). In control cells the time constant for current activation (τact) at −5 mV was 11 ± 4 ms (n = 11) compared with a τact of 15 ± 3 ms (n = 18) in V12C40Ras-expressing cells. Peak current at +15 mV in V12C40Ras cells inactivated by 72 ± 1 % and could be fitted with a double exponential to give the following time constants, τfast = 64 ± 11 ms (n = 6) and τslow = 639 ± 53 ms (n = 6), which were not significantly different from the previously obtained control values.

Expression of V12G37Ras preferentially activates the RalGDS pathway (Khosravi-Far et al. 1998; Vojtek & Der, 1998). In DRGs, the peak current density recorded in V12G37Ras-expressing cells was not significantly different from controls although Imax was reduced by 30 % (Table 1 and Fig. 3C). This result was supported by repeated measures ANOVA (F1,19, 3.60; not significant). Again, no significant effects on either the current kinetics or voltage dependence of current activation were observed (see Table 1). When measured at −5 mV τact in V12G37-expressing cells was 10 ± 3 ms (n = 10) compared with 10 ± 4 ms (n = 10) in GFP-expressing control cells. Similarly, the rate of inactivation was unaffected by expression of V12G37Ras. For Imax at +15 mV, the percentage inactivation was 70 ± 4 with a τfast of 54 ± 7 ms (n = 3) and τslow of 727 ± 93 ms (n = 3).

Together the data obtained using the Ras effector mutants suggest that activation of Raf-1 is sufficient to account for Ras-mediated enhancement of calcium current in DRG neurones.

Effects of U0126 and LY294002

Ras activation of Raf-1 initiates the MAPK signalling cascade where Raf-1 phosphorylates MEK (MAPK kinase) which in turn phosphorylates MAPKs (extracellular signal-regulated kinases, ERK1/ERK2) (Marshall, 1996). The enhancement of calcium current by V12S35Ras therefore suggests a role for MAPK signalling in the upregulation of calcium channels in DRGs. In addition to the molecular approach described above, the MEK-specific inhibitor U0126 was also tested. U0126 is a highly specific inhibitor which binds to the active forms of MEK1 and MEK2, thereby inhibiting the activation of MAPKs (ERK1 or ERK2) (DeSilva et al. 1998; Favata et al. 1998). Pre-incubation with 10–50 μm U0126 for 15 min has previously been shown to block MAPK activation in a variety of cell types including COS-7 cells (Favata et al. 1998), REF52 cells (Slack et al. 1999) and T cells (DeSilva et al. 1998). In the present study, DRGs were pre-incubated with 10 μm U0126 for 20–30 min before recording of calcium currents. As shown in Table 2 and Fig. 4A, the average peak current density in U0126-treated cells was 39 % less than that in control cells which had been pre-incubated with fresh culture medium for the same length of time (one-way ANOVA; F2,18, 4.36; P < 0.05). In contrast, the average peak current density (Imax) recorded from cells pre-treated with the inactive analogue U0124, was not significantly different from the control cells (Table 2). No obvious effects on the voltage dependence of activation of current or on the activation and inactivation kinetics were observed in the presence of either U0126 or U0124 (data not shown). In additional experiments, U0126 (20 μm) applied directly to cells under continuous perfusion induced a steady decrease in current amplitude of 45 ± 7 % (P < 0.05,n = 5) over a period of 10–15 min from the start of application (Fig. 4B). Further experiments are required to determine whether or not this effect is reversible. In contrast, the inactive analogue U0124 had no significant effect, reducing Imax by only 12 ± 3 % (n = 3) over the same period (Fig. 4B). The percentage change in peak current density was significantly greater in U0126- versus U0124-perfused cells (P < 0.05). Together these data confirm the involvement of the MAPK pathway in tonic upregulation of VDCCs. Furthermore, the short time course for the effect of U0126 implies a role for Ras-MAPK signalling which is consistent with modulation of existing channels, rather than an effect mediated by gene expression.

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

Figure 4

Open in a new tab

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 Vh−80 mV using 200 ms depolarising pulses to +15 mV.

Expression of V12C40Ras caused a slight, though not statistically significant increase in basal current compared with control cells. Whilst this suggests that Ras-activated PtdI 3-K may not be involved in Ras-mediated regulation of VDCCs in DRGs, it is possible that Ras-independent activation of PtdI 3-K could be important. Consequently, the PtdI 3-K-specific inhibitor, LY294002, was also tested (Sanchez-Margalet et al. 1994; Vlahos et al. 1994). Although the IC50 for LY294002 blocking PtdI 3-K activity in vitro is 1.4 μm, concentrations ranging from 10–100 μm are often required for inhibition in intact cells (Vlahos et al. 1994; Yao & Cooper, 1996). In the present study, DRGs were pre-incubated for 30 min with 50 μm LY294002 and the calcium currents compared with those from control cells that had been pre-incubated with fresh culture medium alone. In LY294002-treated cells, Imax was 21 % smaller than control Imax, although again this was not statistically significant (Fig. 4C). No effects on either the voltage dependence of activation or the current kinetics were observed (Table 2). This result combined with the lack of a significant effect of V12C40Ras expression would suggest that PtdI 3-K is unlikely to play a major role in Ras-mediated regulation of calcium channels in rat sensory neurones.

Determination of the calcium channel subtypes affected

All calcium channel subtypes, L-, N-, P-, Q-, R- and T-type, are functionally expressed in rat sensory neurones (e.g. Scroggs & Fox, 1992; Rusin & Moises, 1995). In the present study, approximately 70–80 % of current in control untransfected cells could be accounted for by L- and N-type HVA calcium channels, as determined by 1,4-dihydropyridine (nicardipine) and ω-conotoxin GVIA (ω-CgTx GVIA) sensitivity, respectively (data not shown). In order to determine which calcium channel subtypes might be upregulated by the Ras-MAPK pathway, the proportion of nicardipine- and ω-CgTx GVIA-sensitive current in V12S35Ras-expressing cells was compared with that in control cells expressing GFP alone. Sequential application of nicardipine (5 μm) followed by ω-CgTx GVIA (0.5 μm) + nicardipine (5 μm) reduced peak current amplitude at +15 mV by 81 % in control cells. On average, 41 ± 7 % of control current was nicardipine sensitive, i.e. L-type (n = 6), and 40 ± 9 % was ω-CgTx GVIA-sensitive, i.e. N-type (n = 5) (Fig. 5A). In the V12S35Ras-expressing cells, similar proportions of current were found to be nicardipine- (44 ± 4 %, n = 8) and ω-CgTx GVIA-sensitive (39 ± 4 %, n = 8) (Fig. 5B). The average peak current density in cells expressing V12S35Ras was −77.2 ± 9.1 pA pF−1 (n = 8) compared with an Imax of −50.6 ± 5.9 pA pF−1 in controls (P < 0.05,n = 6). Thus, in spite of a 53 % increase in current density resulting from V12S35Ras expression, the relative proportions of N- and L-type currents were the same for both control and V12S35-expressing cells. This implies that all components of calcium current in these cells, including nicardipine/ω-CgTx GVIA-insensitive current, are upregulated by the Ras-MAPK pathway. This result was confirmed by comparing the nicardipine and ω-CgTx GVIA-sensitivity of U0126 pre-treated and control cells. After 30 min pre-incubation with 10 μm U0126, Imax was reduced by 39 % (P < 0.05) from −72.7 ± 9.1 pA pF−1 (n = 8) in control cells, to −47.1 ± 9.4 pA pF−1 (n = 8) in U0126 pre-treated cells, although the proportions of nicardipine- and ω-CgTx GVIA-sensitive current were no different from controls. Thus in control cells, 49 ± 4 % (n = 8) of current was nicardipine-sensitive whilst the ω-CgTx GVIA-sensitive component constituted 23 ± 2 % (n = 7). Similarly, in U0126-treated cells some 47 ± 5 % (n = 8) was nicardipine-sensitive with 31 ± 6 % being ω-CgTx GVIA-sensitive (n = 8).

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

Figure 5

Open in a new tab

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 Vh of −80 mV. Vertical scale bars indicate the absolute current amplitude.

DISCUSSION

In rat sensory neurones, Fitzgerald & Dolphin (1997) showed that endogenous Ras was involved in the tonic upregulation of VDCCs, a process which largely involved NGF-RTK activation of Ras. Using a variety of Ha-Ras mutants expressed in rat DRGs, the present study has shown that mutants which activate the MAPK pathway enhance VDCC current, whereas mutants lacking the ability to activate MAPK fail to increase current significantly. Pharmacological inhibition of the MAPK pathway using the MEK inhibitor, U0126, reduced VDCC current with a time course which is consistent with modulation of VDCCs by Ras-MAPK signalling rather than gene-expression-mediated effects. Interestingly, all components of HVA calcium current appeared to be upregulated via Ras-MAPK signalling, suggesting a common mechanism for interaction at the channel level.

Ras activation of the MAPK pathway is required for VDCC current upregulation

Previous studies have shown that manipulating the activity of endogenous Ras can have a profound influence on VDCC function. Increasing Ras activity by acute microinjection of constitutively active V12Ras protein has been reported to enhance N-, L- and T-type VDCC currents in rat DRGs (Fitzgerald & Dolphin, 1997), embryonic chick DRGs (Hahnel et al. 1992) and NG108-15 cells (Hescheler et al. 1991). It has also recently been reported that suppression of endogenous T-type current via a Ras-MAPK pathway is required for morphogenesis of Swiss 3T3 cells (Strobeck et al. 1999). More usually, however, suppression of Ras activity causes a reduction in VDCC current. For example, expression of the dominant negative Ras mutant (N17Ras) or microinjection of Ras neutralising antibodies have both been found to reduce HVA calcium currents in PC12 cells (Pollack & Rane, 1996) and rat DRGs (Fitzgerald & Dolphin, 1997). In the present study, transient transfection of DRG neurones with constitutively active V12Ras mutant cDNA caused a marked increase in basal current density. In contrast, over-expression of a farnesylation-defective mutant of Ras, V12S186, reduced basal current density compared with controls. Ras proteins are synthesised as cytosolic precursors and must undergo post-translational modifications at the C-terminal before becoming biologically active. These modifications include farnesylation of the cysteine residue (C186), followed by proteolytic cleavage and methyl esterification (Hancock et al. 1990; Qui et al. 1991). Consequently, V12S186Ras in which cysteine 186 has been replaced with a serine residue, cannot activate any downstream effectors of Ras, and over-expression of this mutant essentially suppresses Ras function (Rodriguez-Viciana et al. 1997). Together these results agree with the previously reported effects of Ras on calcium currents in DRG neurones, in that upregulation of Ras activity increases current whilst suppressing Ras activity reduces basal current (Fitzgerald & Dolphin, 1997).

Three Ras effector region mutants, also within a constitutively active V12 background, were used to determine which downstream effector(s) is involved in regulation of VDCCs. Each of these mutants acted as a partial loss of function mutant, preferentially activating one effector whilst failing to activate the other two (Rodriguez-Viciana et al. 1997). A similar approach was recently used to investigate the role of T-type calcium channels in Ras-induced transformation of Swiss 3T3 cells (Strobeck et al. 1999).

In the RalGDS pathway, Ras binds and activates RalGDS which in turn activates effectors such as CDC42 and Rac1, members of the Rho subfamily of small G-proteins which are important in regulation of cell morphology and development of the cytoskeleton (Khosravi-Far et al. 1998; Vojtek & Der, 1998). Expression of V12G37Ras in DRGs caused a slight though not statistically significant reduction of calcium current density. Since the V12G37 mutant of Ras preferentially activates RalGDS whilst failing to activate either Raf-1 or PtdI 3-K (Rodriguez-Viciana et al. 1997), it would seem that activation of RalGDS alone cannot account for Ras enhancement of calcium current in this system. This is perhaps not so surprising given that there is little evidence in the literature to suggest a role for RalGDS in ion channel regulation. Nonetheless, the possibility that RalGDS could act in combination with other effector(s) to upregulate VDCCs cannot be ruled out on the basis of the evidence presented here.

PtdI 3-K is known to be important in cell proliferation and survival (reviewed in Toker & Cantley, 1997). In embryonic DRGs, NGF-activated Ras-PtdI 3-K was found by Klesse & Parada (1998) to be required for survival, and insulin-like growth factor receptor-1 induced activation of PtdI 3-K is suggested to play a role in VDCC regulation and survival of cerebellar granule neurones (Blair et al. 1999). Here though, expression of V12C40Ras, which preferentially activates PtdI 3-K, did not cause significant enhancement of VDCC current in DRG neurones. Whilst this suggests that Ras-activated PtdI 3-K on its own is insufficient to cause significant upregulation of VDCCs, it remains a possibility that PtdI 3-K acts in combination with other effector(s) to achieve significant current enhancement. Furthermore, PtdI 3-K can also be activated independently of Ras via TrkA binding with the p85 regulatory subunit of PtdI 3-K (Soltoff et al. 1992; Obermeier et al. 1993). Since previous work has suggested that tonic upregulation of VDCC current in DRGs can also be induced by NGF binding to TrkA (Fitzgerald & Dolphin, 1997), it is possible that NGF activation rather than Ras activation of PtdI 3-K could be involved in regulation of VDCC current. However, pre-incubation of DRGs with a relatively high concentration of LY294002, a specific inhibitor of PtdI 3-K, failed to induce a significant effect on the basal calcium current density. Although concentrations up to 100 μm LY294002 are occasionally required to block PtdI 3-K in intact cells, concentrations of 10–20 μm are usually sufficient to suppress its activity (e.g. Rodriguez-Viciana et al. 1997; Klesse & Parada, 1998; Blair et al. 1999). Consequently, the lack of effect of LY294002 in the present study would suggest that PtdI 3-K does not have a major role in regulation of VDCCs in DRG neurones, at least with respect to short term modulation of channels. Whether longer term manipulations of PtdI 3-K, over several days, would induce significant effects on calcium current density remains unclear. Numerous reports in the literature suggest a role for gene expression and increased synthesis of channels to explain NGF enhancement of VDCC current (e.g. Levine et al. 1995; Lei et al. 1998).

The best characterised pathway downstream of Ras is the MAPK serine-threonine protein kinase cascade, essential for cell differentiation and proliferation (Marshall, 1996). In this pathway, activated Ras promotes movement of the protein serine/threonine kinase Raf-1, to the plasma membrane where it becomes a functional kinase (Vojtek et al. 1993; Warne et al. 1993). Activated Raf-1 then phosphorylates another protein kinase MEK, a dual specificity kinase which in turn activates MAPKs (ERKs) by phosphorylation of both threonine and tyrosine residues. Once activated, MAPKs phosphorylate serine residues in a variety of target proteins, including transcription factors and protein kinases. Several studies have suggested a role for the MAPK pathway in ion channel regulation (Zhong, 1995; Lei et al. 1998; Strobeck et al. 1999). Expression of V12S35Ras in DRGs also caused a significant increase in basal calcium current density. This increase in basal current was of a similar magnitude to that observed in response to V12Ras expression. Since the V12S35Ras mutant preferentially activates Raf-1, it would seem that activation of Raf-1 and the MAPK pathway is both necessary and sufficient for Ras enhancement of VDCC current in DRGs.

The earliest that recordings were made from transfected DRGs was approximately 20 h after transfection. With such a time course it was possible that the increase in basal current density observed with expression of V12- and V12S35Ras could have been due to synthesis of new channels. Such a mechanism would certainly be consistent with the importance of the MAPK pathway in gene expression. However, the previous work of Fitzgerald & Dolphin (1997) reported that intracellular application of blocking phosphopeptides, which prevented NGF activation of Ras via Shc adaptor protein, reduced VDCC current over a short time course of 10–15 min, suggesting that Ras modulated existing channels. In the present study, a pharmacological blocker of the MAPK pathway U0126, which potently inhibits activated MEK downstream of Ras, was also tested. Pre-incubation of DRGs in 10 μm U0126 for 30 min induced a significant reduction of basal calcium current density compared with controls. Furthermore, acute application of U0126 by continuous perfusion induced a similar decrease in current amplitude which occurred over a period of 10–15 min. Together these data confirm a role for the MAPK pathway in VDCC regulation in these cells, and also support the hypothesis that Ras-MAPK enhancement of calcium current involves modulation of existing channels rather than gene expression-mediated synthesis of new channels.

Taken together, the evidence presented so far suggests that neither RalGDS nor PtdI 3-K have a major role in the Ras-mediated regulation of VDCCs in DRG neurones. In contrast, activation of the MAPK pathway does appear to be sufficient to explain the marked increase in VDCC current observed with expression of constitutively active V12Ras. Thus, although involvement of PtdI 3-K and/ or RalGDS cannot be completely ruled out, it seems most likely that activation of the Ras-MAPK pathway is responsible for the tonic modulation of VDCCs by Ras, previously reported in DRGs by Fitzgerald & Dolphin (1997).

Multiple VDCC subtypes are modulated via Ras-MAPK signalling

As stated earlier, all VDCC channel subtypes are functionally expressed in sensory neurones (Scroggs & Fox, 1992; Rusin & Moises, 1995). Throughout neuronal development the relative proportions of individual VDCC subtypes undergo a progressive change, with LVA T-type channels generally decreasing during development as the proportion of HVA channels increases (McCobb et al. 1989; Thompson & Wong, 1991). In the present study, DRGs were isolated from 1- to 2-day-old rat neonates and cells maintained in culture for a maximum of 3 days. Whole-cell calcium currents recorded from DRGs at this stage exhibited almost exclusively HVA current, of which L- and N-type current constituted approximately 80 %, as determined by nicardipine and ω-CgTx GVIA sensitivity. In view of this, L- and/ or N-type VDCCs were considered most likely to be modulated by Ras-MAPK signalling. Comparison of nicardipine- and ω-CgTx GVIA-sensitivity in control GFP-expressing cells versus V12S35Ras-expressing cells showed that in spite of a 50 % increase in basal current density in the V12S35Ras cells, the relative proportions of L- and N-type current were the same. This implied that all components of calcium current were upregulated via the Ras-MAPK pathway. Similarly, although peak current density was reduced by approximately 40 % after 30 min pre-incubation with the MEK inhibitor U0126, the proportions of N- and L-type current were not significantly different from control cells. Importantly, although there is a possibility of overlap in the selectivity of nicardipine for L-type over N-, P/Q- and R-type calcium channels (Furukawa et al. 1999), this does not affect the conclusion that all components of VDCC current are modulated via Ras-MAPK signalling in DRGs. Thus, Ras enhancement of VDCC current in these cells is likely to involve a common mechanism for interaction at the channel level.

Manipulation of the Ras-MAPK pathway caused marked changes in basal calcium current density. By comparison with modulation of VDCCs by other protein kinases, changes in the voltage dependence of current activation and/or current kinetics might have been expected. However, no significant effects on either the current-voltage relationships or the current kinetics were observed. In Aplysia neurones, the effects of PKC on VDCCs have been suggested to involve recruitment of previously covert calcium channels (Strong et al. 1987). Whilst a similar explanation for Ras-MAPK enhancement of VDCC current in DRGs cannot be competely ruled out, the rapid time course for U0126-induced inhibition of calcium current in these cells is more suggestive of modulation of channels which are already activated. Changes in current kinetics, particularly inactivation, may well be obscured in this system where L-type (sustained) and N-type (inactivating) currents together constitute up to 80 % of whole-cell current. Whilst it is therefore difficult to speculate about possible mechanisms for Ras-MAPK modulation, direct MAPK phosphorylation of the channels is one possibility since MAPK consensus sites can be identified in the sequences of some VDCC α-subunit clones, including α1C (L-type) and α1B (N-type). VDCC accessory subunits, β and α2-δ, could also be phosphorylated. Alternatively, the MAPK pathway may activate intermediate signalling molecules which themselves interact directly with the channels. These and other possible mechanisms are currently under investigation.

Specific VDCC subtypes tend to be associated with specific cellular processes, e.g. N-type channels are linked with neurotransmitter release (Yu et al. 1992; Gruner & Silva, 1994), and L-type channels are implicated in neuronal growth and survival (Murphy et al. 1991; Larmet et al. 1992). Thus, in terms of the cellular function of VDCC modulation by Ras, it seems most likely that specific upstream activators, rather than downstream effectors of Ras would be required to initiate regulation of specific VDCC subtypes. It was recently reported that isoforms of both Shc A and Shc C, intermediate signalling proteins involved in RTK activation of Ras, are expressed in adult rat DRG neurones (Ganju et al. 1998). Furthermore, NGF was shown to phosphorylate Shc A whereas epidermal growth factor (EGF) phosphorylated Shc C (Ganju et al. 1998). Although it was reported that tonic upregulation of VDCCs in neonatal DRGs largely involved NGF-TrkA activation of Ras (Fitzgerald & Dolphin, 1997) it is possible that EGF and/or other growth factors may also regulate VDCCs in these cells.

Conclusions

The evidence presented here, combined with that previously reported by Fitzgerald & Dolphin (1997) has shown that activation of the MAPK pathway is necessary and sufficient to account for Ras upregulation of VDCC current in rat DRG neurones. Enhancement of VDCC current appears to involve Ras-MAPK modulation of existing channels rather than increased synthesis of new channels via Ras-mediated gene expression. All components of VDCC current in these cells seem to be affected, suggesting a common mechanism for interaction at the channel level.

Acknowledgments

My thanks to N. Mahmoud and S. Martin for technical assistance, to K. Page for advice on subcloning, to A. C. Dolphin for the use of molecular biology facilities and to J. Fernandez for use of the Biolistic PDS-1000/He apparatus. Thanks also to A.C.D. and K.P. for critical reading of the manuscript, and to G. Masters for statistical advice. This work was supported by the Wellcome Trust (Wellcome Research Career Development Fellowship to E.M.F.).

References

  1. Blair LAC, Bence-Hanulec KK, Mehta S, Franke T, Kaplan D, Marshall J. Akt-dependent potentiation of L channels by insulin-like growth factor-1 is required for neuronal survival. Journal of Neuroscience. 1999;19:1940–1951. doi: 10.1523/JNEUROSCI.19-06-01940.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cohan CS, Conner JA, Kater SB. Electrically and chemically mediated increases in intracellular calcium in neuronal growth cones. Journal of Neuroscience. 1987;7:3588–3599. doi: 10.1523/JNEUROSCI.07-11-03588.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. DeSilva DR, Jones EA, Favata MF, Jaffee BD, Magolda RL, Trzaskos JM, Scherle PA. Inhibition of mitogen-activated protein kinase kinase blocks T-cell proliferation but does not induce or prevent anergy. Journal of Immunology. 1998;160:4175–4181. [PubMed] [Google Scholar]
  4. Favata MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, Van Dyk DE, Pitts WJ, Earl RA, Hobbs F, Copeland RA, Magolda RL, Scherle PA, Trzaskos PA. Identification of a novel inhibitor of mitogen activated protein kinase kinase. Journal of Biological Chemistry. 1998;273:18623–18632. doi: 10.1074/jbc.273.29.18623. [DOI] [PubMed] [Google Scholar]
  5. Finkbeiner S, Greenberg ME. Ca2+-dependent routes to Ras: mechanisms for neuronal survival, differentiation, and plasticity? Neuron. 1996;16:233–236. doi: 10.1016/s0896-6273(00)80040-9. [DOI] [PubMed] [Google Scholar]
  6. Fitzgerald EM, Dolphin AC. Regulation of rat neuronal voltage-dependent calcium channels by endogenous p21-ras. European Journal of Neuroscience. 1997;9:1252–1261. doi: 10.1111/j.1460-9568.1997.tb01480.x. [DOI] [PubMed] [Google Scholar]
  7. Furukawa T, Yamakawa T, Midera T, Sagawa T, Mori Y, Nukada T. Selectivities of dihydropyridine derivatives in blocking Ca2+ channel subtypes expressed in Xenopus oocytes. Journal of Pharmacology and Experimental Therapeutics. 1999;291:464–473. [PubMed] [Google Scholar]
  8. Ganju P, O'Bryan JP, Der C, Winter J, James IF. Differential regulation of SHC proteins by nerve growth factor in sensory neurons and PC12 cells. European Journal of Neuroscience. 1998;10:1995–2008. doi: 10.1046/j.1460-9568.1998.00209.x. [DOI] [PubMed] [Google Scholar]
  9. Ghosh A, Ginty DD, Bading H, Greenberg ME. Calcium regulation of gene expression in neuronal cells. Journal of Neurobiology. 1994;25:294–303. doi: 10.1002/neu.480250309. [DOI] [PubMed] [Google Scholar]
  10. Gomez TM, Spitzer NC. In vivo regulation of axon extension and pathfinding by growth-cone calcium transients. Nature. 1999;397:350–355. doi: 10.1038/16927. [DOI] [PubMed] [Google Scholar]
  11. Gruner W, Silva LR. ω-Conotoxin sensitivity and presynaptic inhibition of glutamatergic sensory neurotransmission in vitro. Journal of Neuroscience. 1994;14:2800–2808. doi: 10.1523/JNEUROSCI.14-05-02800.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hagag N, Halegoua S, Viola M. Inhibition of growth factor-induced differentiation of PC12 cells by microinjection of antibody to ras. Nature. 1986;319:680–682. doi: 10.1038/319680a0. [DOI] [PubMed] [Google Scholar]
  13. Hahnel C, Gottmann K, Wittinghofer A, Lux H-D. p21ras oncogene protein selectively increases low-voltage-activated Ca2+ current density in embryonic chick dorsal root ganglion neurons. European Journal of Neuroscience. 1992;4:361–368. doi: 10.1111/j.1460-9568.1992.tb00883.x. [DOI] [PubMed] [Google Scholar]
  14. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Archiv. 1981;391:85–100. doi: 10.1007/BF00656997. [DOI] [PubMed] [Google Scholar]
  15. Hancock JF, Paterson H, Marshall CJ. A polybasic domain or palmitoylation is required in addition to the CAAX motif to localise p21Ras to the plasma membrane. Cell. 1990;63:133–139. doi: 10.1016/0092-8674(90)90294-o. [DOI] [PubMed] [Google Scholar]
  16. Hescheler J, Klinz F-J, Schultz G, Wittinghofer A. Ras proteins activate calcium channels in neuronal cells. Cellular Signalling. 1991;3:127–133. doi: 10.1016/0898-6568(91)90019-q. [DOI] [PubMed] [Google Scholar]
  17. Joneson T, White MA, Wigler MH, Bar-Sagi D. Stimulation of membrane ruffling and MAP kinase activation by distinct effectors of Ras. Science. 1996;271:810–812. doi: 10.1126/science.271.5250.810. [DOI] [PubMed] [Google Scholar]
  18. Khosravi-Far R, Campbell S, Rossman KL, Der CJ. Increasing complexity of Ras signal transduction: involvement of Rho family proteins. Advances in Cancer Research. 1998;72:57–107. doi: 10.1016/s0065-230x(08)60700-9. [DOI] [PubMed] [Google Scholar]
  19. Klesse LJ, Parada LF. p21Ras and phosphatidylinositol-3 kinase are requird for survival of wild-type and NF1 mutant sensory neurons. Journal of Neuroscience. 1998;18:10420–10428. doi: 10.1523/JNEUROSCI.18-24-10420.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kraus MH, Yuasa Y, Aaronson SA. A position 12-activated H-ras oncogene in all HS578T mammary carcinosarcoma cells but not normal mammary cells of the same patient. Proceedings of the National Academy of Sciences of the USA. 1984;17:5384–5388. doi: 10.1073/pnas.81.17.5384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Larmet Y, Dolphin AC, Davies AM. Intracellular calcium regulates the survival of early sensory neurons before they become dependent on neurotrophic factors. Neuron. 1992;9:563–574. doi: 10.1016/0896-6273(92)90193-h. [DOI] [PubMed] [Google Scholar]
  22. Lei S, Dryden WF, Smith PA. Involvement of Ras/MAP kinase in the regulation of Ca2+ channels in adult bullfrog sympathetic neurons by nerve growth factor. Journal of Neurophysiology. 1998;80:1352–1361. doi: 10.1152/jn.1998.80.3.1352. [DOI] [PubMed] [Google Scholar]
  23. Levine ES, Dreyfus CF, Black IB, Plummer MR. Differential effects of NGF and BDNF on voltage-gated calcium currents in embryonic basal forebrain neurons. Journal of Neuroscience. 1995;15:3084–3091. doi: 10.1523/JNEUROSCI.15-04-03084.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McCobb DP, Best PM, Beam KG. Development alters expression of calcium currents in chick limb motoneurones. Neuron. 1989;2:1633–1643. doi: 10.1016/0896-6273(89)90052-4. [DOI] [PubMed] [Google Scholar]
  25. Marshall CJ. Ras effectors. Current Opinion in Cell Biology. 1996;8:197–204. doi: 10.1016/s0955-0674(96)80066-4. [DOI] [PubMed] [Google Scholar]
  26. Murphy TH, Worley PF, Baraban JM. L-type voltage-sensitive calcium channels mediate synaptic activation of immediate early genes. Neuron. 1991;7:625–635. doi: 10.1016/0896-6273(91)90375-a. [DOI] [PubMed] [Google Scholar]
  27. Obermeier A, Lammers R, Weismuller K-H, Jung G, Schlessinger J, Ullrich A. Identification of Trk binding sites for SHC and phosphatidylinositol 3-kinase and formation of a multimeric signaling complex. Journal of Biological Chemistry. 1993;268:22963–22966. [PubMed] [Google Scholar]
  28. Pollack JD, Rane SG. p21-ras signaling is necessary but not sufficient to mediate neurotrophin-induction of calcium channels in PC12 cells. Journal of Biological Chemistry. 1996;271:8008–8014. doi: 10.1074/jbc.271.14.8008. [DOI] [PubMed] [Google Scholar]
  29. Qui M-S, Pitts AF, Winters TR, Green SH. Ras isoprenylation is required for Ras-induced but not for NGF-induced neuronal differentiation of PC12 cells. Journal of Cell Biology. 1991;115:795–808. doi: 10.1083/jcb.115.3.795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rodriguez-Viciana P, Warne PH, Khwaja A, Marte BM, Pappin D, Das P, Waterfield MD, Ridley A, Downward J. Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras. Cell. 1997;89:457–467. doi: 10.1016/s0092-8674(00)80226-3. [DOI] [PubMed] [Google Scholar]
  31. Rusin KI, Moises HC. μ-Opioid receptor activation reduces multiple components of high-threshold calcium current in rat sensory neurons. Journal of Neuroscience. 1995;15:4315–4327. doi: 10.1523/JNEUROSCI.15-06-04315.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sanchez-Margalet V, Goldfine ID, Vlahos CJ, Sung CK. Role of phosphatidylinositol-3-kinase in insulin receptor signalling: studies with inhibitor, LY294002. Biochemical and Biophysical Research Communications. 1994;204:446–452. doi: 10.1006/bbrc.1994.2480. [DOI] [PubMed] [Google Scholar]
  33. Scroggs RS, Fox AP. Calcium current variation between acutely isolated adult rat dorsal root ganglion neurons of different size. The Journal of Physiology. 1992;445:639–658. doi: 10.1113/jphysiol.1992.sp018944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Slack JK, Catling AD, Eblen ST, Weber MJ, Parsons JT. c-Raf-mediated inhibition of epidermal growth factor-stimulated cell migration. Journal of Biological Chemistry. 1999;274:27177–27184. doi: 10.1074/jbc.274.38.27177. [DOI] [PubMed] [Google Scholar]
  35. Soltoff SP, Rabin SL, Cantley LC, Kaplan DR. Nerve growth factor promotes the activation of phosphatidylinositol 3-kinase and its association with the trk tyrosine kinase. Journal of Biological Chemistry. 1992;267:17472–17477. [PubMed] [Google Scholar]
  36. Strobeck MW, Okuda M, Yamaguchi H, Schwartz A, Fukasawa K. Morphological transformation induced by activation of the mitogen-activated protein kinase pathway requires suppression of the T-type Ca2+ channel. Journal of Biological Chemistry. 1999;274:15694–15700. doi: 10.1074/jbc.274.22.15694. [DOI] [PubMed] [Google Scholar]
  37. Strong JA, Fox AP, Tsien RW, Kaczmarek LK. Stimulation of protein kinase C recruits covert calcium channels in Aplysia bag cell neurons. Nature. 1987;325:714–717. doi: 10.1038/325714a0. [DOI] [PubMed] [Google Scholar]
  38. Thompson SM, Wong RKS. Development of calcium current subtypes in isolated rat hippocampal pyramidal cells. The Journal of Physiology. 1991;439:671–689. doi: 10.1113/jphysiol.1991.sp018687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Toker A, Cantley LC. Signalling through the lipid products of phosphoinositide-3-OH kinase. Nature. 1997;387:673–676. doi: 10.1038/42648. [DOI] [PubMed] [Google Scholar]
  40. Vlahos CJ, Matter WF, Hui KY, Brown RF. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholynyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) Journal of Biological Chemistry. 1994;269:5241–5248. [PubMed] [Google Scholar]
  41. Vojtek AB, Der CJ. Increasing complexity of the Ras signalling pathway. Journal of Biological Chemistry. 1998;273:19925–19928. doi: 10.1074/jbc.273.32.19925. [DOI] [PubMed] [Google Scholar]
  42. Vojtek AB, Hollenberg SM, Cooper JA. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell. 1993;74:205–214. doi: 10.1016/0092-8674(93)90307-c. [DOI] [PubMed] [Google Scholar]
  43. Warne PH, Rodriguez-Viciana PR, Downward J. Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro. Nature. 1993;364:352–355. doi: 10.1038/364352a0. [DOI] [PubMed] [Google Scholar]
  44. Wildering WC, Lodder JC, Kits KS, Bulloch AG. Nerve growth factor (NGF) acutely enhances high-voltage-activated calcium currents in molluscan neurons. Journal of Neurophysiology. 1995;74:2778–2781. doi: 10.1152/jn.1995.74.6.2778. [DOI] [PubMed] [Google Scholar]
  45. White MA, Nicolette C, Minden A, Polverino A, Vanaelst L, Karin M, Wigler MH. Multiple Ras functions can contribute to mammalian cell transformation. Cell. 1995;80:533–541. doi: 10.1016/0092-8674(95)90507-3. [DOI] [PubMed] [Google Scholar]
  46. Yao R, Cooper GM. Growth factor-dependent survival of rodent fibroblasts requires phosphatidylinositol 3-kinase but is independent of pp70S6K activity. Oncogene. 1996;13:343–351. [PubMed] [Google Scholar]
  47. Yu C, Lin PX, Fitzgerald S, Nelson P. Heterogeneous calcium currents and neurotransmitter release in cultured mouse spinal cord and dorsal root ganglion neurons. Journal of Neurophysiology. 1992;67:561–575. doi: 10.1152/jn.1992.67.3.561. [DOI] [PubMed] [Google Scholar]
  48. Zhong Y. Mediation of PACAP-like neuropeptide transmission by activation of Ras/Raf and cAMP signal transduction pathways in Drosophila. Nature. 1995;375:588–592. doi: 10.1038/375588a0. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Physiology are provided here courtesy of The Physiological Society

RESOURCES