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

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


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 8-Bromo Cyclic Adenosine Monophosphate / pharmacology
  • Animals
  • COS Cells
  • Colforsin / pharmacology
  • Cyclic AMP / physiology*
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Drug Resistance
  • Electrophysiology
  • Glutathione Transferase / metabolism
  • Ion Channel Gating / drug effects
  • Ion Channel Gating / physiology
  • Molecular Conformation
  • Mutagenesis, Site-Directed / physiology
  • NAV1.8 Voltage-Gated Sodium Channel
  • Peptide Mapping
  • Phosphopeptides / metabolism
  • Phosphorylation
  • Rats
  • Sodium Channels / drug effects
  • Sodium Channels / genetics
  • Sodium Channels / physiology*
  • Tetrodotoxin / pharmacology*
  • Transfection / genetics
  • Transfection / physiology


  • NAV1.8 Voltage-Gated Sodium Channel
  • Phosphopeptides
  • Scn10a protein, rat
  • Sodium Channels
  • Colforsin
  • 8-Bromo Cyclic Adenosine Monophosphate
  • Tetrodotoxin
  • Cyclic AMP
  • Glutathione Transferase
  • Cyclic AMP-Dependent Protein Kinases