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. 1999 Apr 15;516 ( Pt 2)(Pt 2):433-46.
doi: 10.1111/j.1469-7793.1999.0433v.x.

cAMP-dependent Phosphorylation of the Tetrodotoxin-Resistant Voltage-Dependent Sodium Channel SNS

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Free PMC article

cAMP-dependent Phosphorylation of the Tetrodotoxin-Resistant Voltage-Dependent Sodium Channel SNS

E M Fitzgerald et al. J Physiol. .
Free PMC article

Abstract

1. Protein kinase A (PKA) modulation of tetrodotoxin-resistant (TTX-r) voltage-gated sodium channels may underly the hyperalgesic responses of mammalian sensory neurones. We have therefore examined PKA phosphorylation of the cloned alpha-subunit of the rat sensory neurone-specific TTX-r channel SNS. Phosphorylation of SNS was compared with that of a mutant channel, SNS(SA), in which all five PKA consensus sites (RXXS) within the intracellular I-II loop had been eliminated by site-directed mutagenesis (serine to alanine). 2. In vitro PKA phosphorylation and tryptic peptide mapping of SNS and mutant SNS(SA) I-II loops expressed as glutathione-S-transferase (GST) fusion proteins confirmed that the five mutated serines were the major PKA substrates within the SNS I-II loop. 3. SNS and SNS(SA) channels were transiently expressed in COS-7 cells and their electrophysiological properties compared. In wild-type SNS channels, forskolin and 8-bromo cAMP produced effects consistent with PKA phosphorylation. Mutant SNS(SA) currents, however, were not significantly affected by either agent. Thus, elimination of the I-II loop PKA consensus sites caused a marked reduction in PKA modulation of wild-type channels. 4. Under control conditions, the voltage dependence of activation of SNS(SA) current was shifted to depolarized potentials compared with SNS. This was associated with a slowing of SNS(SA) current inactivation at hyperpolarized potentials and suggested a tonic PKA phosphorylation of wild-type channels under basal conditions.5. We conclude that the major substrates involved in functional PKA modulation of the SNS channel are located within the intracellular I-II loop.

Figures

Figure 1
Figure 1. Schematic diagram of the α-subunit of SNS
The α-subunit of voltage-dependent sodium channels comprises four homologous domains, I-IV, each of which is composed of six membrane spanning segments, S1-S6. Within the intracellular linker region IS6 and IIS1 (I-II loop) of the SNS α-subunit there are five sites matching PKA consensus sequences at serine residues 463, 487, 499, 510 and 536. Site-directed mutagenesis was used to remove these PKA consensus sequences by substituting alanine residues for serines at these positions to create a mutant channel, SNS(SA). In vitro PKA phosphorylation was studied using GST fusion proteins of the I-II loop (residues Leu-424 to Gln-592; shown as thick line) of either wild-type GST-SNS or mutant GST-SNS(SA). An additional three putative PKA phosphorylation sites, two serines and a threonine, originally identified in the sequence of PN3 (Sangameswaran et al. 1996), are also present in the SNS sequence.
Figure 2
Figure 2. Phosphorylation of the wild-type GST-SNS and mutant GST-SNS(SA) fusion proteins by PKA
A, analysis of wild-type GST-SNS fusion protein phosphorylation by PKA. GST-SNS (5 μg) was phosphorylated in vitro by PKA (20 ng (100 μl buffer)−1) for varying time periods (min) as shown. The extent of phosphorylation was then assessed by SDS-PAGE followed by autoradiography. GST-SNS was rapidly phosphorylated to a final stoichiometry of 0·95 moles of phosphate per mole of protein. B, phosphoamino acid analysis of wild-type GST-SNS and mutant GST-SNS(SA) fusion proteins. PKA-phosphorylated proteins were digested with trypsin followed by acid hydrolysis. The resulting phosphopeptides were then separated by TLC and visualized by autoradiography. The migration of standard phosphoamino acids is indicated. Wild-type GST-SNS is phosphorylated primarily on serine residues with trace levels of phosphothreonine. Mutant GST-SNS(SA) is phosphorylated to a lesser extent on serine residues. C, PKA phosphorylation of GST-SNS and mutant GST-SNS(SA) was compared. The extent of phophorylation was examined as in A. Mutation of the five serine residues markedly reduced the stoichiometry of phosphorylation to 0·18 moles of phosphate per mole of protein.
Figure 3
Figure 3. Tryptic peptide mapping of wild-type GST-SNS and mutant GST-SNS(SA)
Wild-type and mutant fusion proteins were phosphorylated as described in Fig. 2. Excised gel slices containing the respective phosphoproteins were digested with trypsin and subjected to 2-dimensional peptide mapping followed by autoradiography. PKA phosphorylation of GST-SNS generates 9 reproducible phosphopeptides, suggesting phosphorylation at multiple serine residues. Mutation of these five serines to alanine residues, however, resulted in 8 of the 9 phosphopeptides being eliminated.
Figure 4
Figure 4. Characterization of SNS expressed in COS-7 cells
A, typical inward currents recorded in a COS-7 cell transiently transfected with SNS cDNA. These currents were evoked from a holding potential, Vh, of -90 mV by means of 40 ms depolarizing steps to -40, -15, 0, +20 and +40 mV. No inward currents were observed in either mock-transfected (expression vector only) or non-transfected cells using the same voltage protocol. B, representative traces showing the lack of effect of 50 μM Cd2+ on currents elicited at +15 mV from a Vh of -90 mV. C, typical traces illustrating the effects of 100 μM veratridine. During depolarizing steps to +15 mV, veratridine reduced the amplitude of inward current. Repolarization to -90 mV revealed a slowing decaying tail current of markedly increased amplitude compared with controls.
Figure 5
Figure 5. Comparison of voltage dependence of current activation in wild-type SNS and mutant SNS(SA) which lacks five PKA consensus sites within the intracellular I-II loop
A, conductance-voltage relationships were derived from individual current-voltage plots as described in the legend to Table 1. The derived values for voltage dependence of activation in SNS (•, n= 15) were V½= -2·1 ± 1·4 mV and k= 8·5 ± 0·6 mV; and for SNS(SA) (^, n= 15), V½= 8·8 ± 1·5 mV, k= 9·8 ± 0·6 mV. Currents were activated by depolarizing steps from -50 to +60 mV in 5 mV increments from a holding potential of -90 mV. B, representative currents and associated current-voltage plots from a wild-type SNS-transfected cell (•) and a cell transfected with mutant SNS(SA) (^). Currents were evoked using 200 ms depolarizing pulses to -40, -15, -10 and +15 mV from Vh of -90 mV. The current-voltage plots were fitted with a Boltzmann function to give the following values for SNS: V½= -11·5 mV, k= 5·0 mV, Vrev= 70·0 mV; and for SNS(SA): V½= -5·1 mV, k= 6·9 mV, Vrev= 67·1 mV.
Figure 6
Figure 6. Current kinetics compared in SNS and SNS(SA)
A, the time constant of activation (τact) for SNS (▪) and SNS(SA) (□). The rising phase of current at each voltage step was fitted with a single exponential and τact derived as a measure of activation rate. B, the time constant of inactivation (τinact) derived from single exponential fits to the decaying phase of current. Asterisks indicate statistically significant differences between SNS and SNS(SA): *P < 0·05 and **P < 0·01 (Student's unpaired t test). The inset shows averaged currents for SNS- (n= 14) and SNS(SA)-transfected cells (n= 12). Individual currents were evoked by short 40 ms pulses to +15 mV and normalized to the peak amplitude prior to averaging. Continuous lines indicate single exponential fits of τact and τinact for SNS (τact= 0·86 ms, τinact= 10·79 ms) and SNS(SA) (τact= 0·69 ms, τinact= 15·27 ms).
Figure 7
Figure 7. Effects of 10 μM dideoxyforskolin, 10 μM forskolin and 100 μM 8-bromo cAMP on SNS and SNS(SA)
Each panel (A-F) shows a representative time course, plotted as the percentage change in current amplitude relative to peak current amplitude at time t= 0 s (0 %). The inset for each panel shows the associated current traces recorded at time points 1 and 2 as indicated on the plots. Scale bars refer to the absolute values of current amplitude in pA. A, lack of significant effect of dideoxyforskolin on SNS. B, example of SNS current steadily increasing in amplitude following application of forskolin. C, increase in SNS current amplitude observed in the presence of 8-bromo cAMP. D, dideoxyforskolin had no significant effect on SNS(SA) current. E, forskolin was ineffective on SNS(SA) currents as was 8-bromo cAMP, although in the example shown here (F) there was a slight increase in current amplitude. No effects on current kinetics were observed during any of the above treatments in either SNS or SNS(SA). In all cases, currents were activated from a Vh of -90 mV by stepping to depolarized potentials, typically +15 mV for SNS currents or +20 mV for SNS(SA) currents.
Figure 8
Figure 8. Summary bar-chart showing the effects of dideoxyforskolin (10 μM), forskolin (10 μM) and 8-bromo cAMP (100 μM) on SNS and SNS(SA)
Each bar represents the mean percentage change in current amplitude for SNS (▪) and SNS(SA) (formula image). Asterisks indicate statistically significant differences between SNS and SNS(SA): *P < 0·05 and **P < 0·01 (Student's unpaired t test).

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