cAMP‐dependent phosphorylation of the tetrodotoxin‐resistant voltage‐dependent sodium channel SNS
EM Fitzgerald, K Okuse, JN Wood… - The Journal of …, 1999 - Wiley Online Library
The Journal of physiology, 1999•Wiley Online Library
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 α‐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 …
channels may underly the hyperalgesic responses of mammalian sensory neurones. We
have therefore examined PKA phosphorylation of the cloned α‐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 …
- 1Protein 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 α‐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).
- 2In 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.
- 3SNS 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.
- 4Under 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.
- 5We conclude that the major substrates involved in functional PKA modulation of the SNS channel are located within the intracellular I‐II loop.
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