cAMP-dependent phosphorylation of the tetrodotoxin-resistant voltage-dependent sodium channel SNS

1. Protein kinase A (PKA) modulation of tetrodotoxin-resistant (TTX-r) volt age-gated sodium channels may underly the hyperalgesic responses of mammali an sensory neurones. We have therefore examined PKA phosphorylation of the cloned a-subunit of the rat sensory neurone-specific TTX-r channel SNS. Pho sphorylation of SNS was compared with that of a mutant channel, SNS(SA), in which all five PKA consensus sites (RXXS) within the intra cellular I-II l oop had been eliminated by site-directed mutagenesis (serine to alanine). 2. In vitro PKA phosphorylation and tryptic peptide mapping of SNS and muta nt SNS(SA) I-II loops expressed as glutathione-X-transferase (GST) fusion p roteins confirmed that the five mutated serines were the major PKA substrat es within the SNS I-II loop. 3. SNS and SNS(XA) channels were transiently expressed in COS-7 cells and t heir electrophysiological properties compared. In wild-type SNS channels, f orskolin and 8-bromo cAMP produced effects consistent with PKA phosphorylat ion. Mutant SNS(SA) currents, however, were not significantly affected by e ither 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(XA ) current was shifted to depolarized potentials compared with SNS. This was associated with a slowing of SNS(XA) current inactivation at hyperpolarize d potentials and suggested a tonic PKA phosphorylation of wild-type channel s under basal conditions. 5. We conclude that the major substrates involved in functional PKA modulat ion of the SNS channel, are located within the intracellular I-II loop.