Desensitization of Capsaicin-activated Currents in the Vanilloid Receptor TRPV1 Is Decreased by the Cyclic AMP-dependent Protein Kinase Pathway

Durga P Mohapatra,Carla Nau

doi:10.1074/jbc.m306619200

Abstract

Proinflammatory prostaglandin E2 is known to sensitize sensory neurons to noxious stimuli. This sensitization is mediated by the cAMP-dependent protein kinase (PKA) signal pathway. The capsaicin receptor TRPV1, a non-selective cation channel of sensory neurons involved in the sensation of inflammatory pain, is a target of PKA-mediated phosphorylation. Our goal was to investigate the influence of PKA on Ca(2+)-dependent desensitization of capsaicin-activated currents. By using site-directed mutagenesis, we created point mutations at PKA consensus sites and studied wild-type and mutant channels transiently expressed in HEK293t cells under whole-cell voltage clamp. We found that forskolin, a stimulator of adenylate cyclase, decreased desensitization of TRPV1. The selective PKA inhibitor H89 inhibited this effect. Mimicking phosphorylation at PKA consensus sites by replacing Ser-6, Ser-116, Thr-144, Thr-370, Ser-502, Ser-774, or Ser-820 with aspartate resulted in five mutations (S116D, T144D, T370D, S774D, and S820D) that exhibited decreased desensitization as well. However, disrupting phosphorylation by replacing respective sites with alanine resulted in four mutations (S6A, T144A, T370A, and S820A) with desensitization properties resembling those of the aspartate mutations. Significant changes in relative permeabilities for Ca2+ over Na+ or in capsaicin sensitivity could not explain changes in desensitization properties of mutant channels. In mutations S116A, S116D, T370A, and T370D, pretreatment of cells with forskolin did not reduce desensitization as compared with wild-type and other mutant channels. We conclude that Ser-116 and possibly Thr-370 are the most important residues involved in the mechanism of PKA-dependent reduction of desensitization of capsaicin-activated currents.

Highlights

The sensation of pain is initiated when pain producing thermal, mechanical, or chemical stimuli excite nociceptive primary afferent neurons
We conclude that Ser-116 and possibly Thr-370 are the most important residues involved in the mechanism of protein kinase (PKA)-dependent reduction of desensitization of capsaicin-activated currents
Phosphorylation by Protein Kinase A but Not Protein Kinase C Decreases Ca2ϩ-dependent Desensitization of Capsaicin-activated Currents—TRPV1-WT and mutant channels were transiently expressed in HEK 293t cells and studied in whole-cell voltage clamp mode

Summary

Introduction

The sensation of pain is initiated when pain producing thermal, mechanical, or chemical stimuli excite nociceptive primary afferent neurons. One target of the cAMP/PKA signal pathway to sensitize primary sensory neurons is the capsaicin receptor TRPV1. TRPV1 is a nonselective cation channel that is predominantly expressed by nociceptive primary sensory neurons and that is involved in the detection of noxious stimuli. Treatment with PGE2 or activation of PKA transiently sensitizes capsaicin-activated currents in rat sensory neurons [13]. Potentiation was reduced in TRPV1 channels mutated at the predicted PKA phosphorylation sites Thr-144, Thr-370, and Ser-502 [15]. Another study demonstrated that PKA reduces desensitization of capsaicin- and proton-activated currents of TRPV1 heterologously expressed in Chinese hamster ovary-K1 cells and directly phosphorylates TRPV1 [16]. It was concluded that Ser-116 was one of several sites dephosphorylated by capsaicin but the major site through which phosphorylation by PKA reverses capsaicininduced dephosphorylation [16]

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