Rapid, Opioid-sensitive Mechanisms Involved in Transient Receptor Potential Vanilloid 1 Sensitization

Sarah J Roberts-Thomson,Wei Cheng,Jie Zheng,Madusha Peiris,Bruce D Wyse,Irina Vetter,Gregory R Monteith,Peter J Cabot

doi:10.1074/jbc.m707865200

Abstract

TRPV1 is a nociceptive, Ca2+-selective ion channel involved in the development of several painful conditions. Sensitization of TRPV1 responses by cAMP-dependent PKA crucially contributes to the development of inflammatory hyperalgesia. However, the pathways involved in potentiation of TRPV1 responses by cAMP-dependent PKA remain largely unknown. Using HEK cells stably expressing TRPV1 and the mu opioid receptor, we demonstrated that treatment with the adenylate cyclase activator forskolin significantly increased the multimeric TRPV1 species. Pretreatment with the mu opioid receptor agonist morphine reversed this increased TRPV1 multimerization. FRET analysis revealed that treatment with forskolin did not cause multimerization of pre-existing TRPV1 monomers on the plasma membrane and that intracellular pools of TRPV1 exist mostly as monomers in this model. This suggests that increased TRPV1 multimerization occurred from an intracellular store of inactive TRPV1 monomers. Treatment with forskolin also caused an increase in TRPV1 expression on the plasma membrane not resulting from increased TRPV1 expression, and this rapid TRPV1 translocation was inhibited by treatment with morphine. Thus, potentiation of TRPV1 responses by cAMP-dependent PKA involves plasma membrane insertion of functional TRPV1 multimers formed from an intracellular store of inactive TRPV1 monomers. This potentiation occurs rapidly and can be dynamically modulated by activation of the mu opioid receptor under conditions where cAMP levels are raised, such as with inflammation. Increased translocation and multimerization of TRPV1 channels provide a cellular mechanism for fine-tuning of nociceptive responses that allow for rapid modulation of TRPV1 responses independent of transcriptional changes.

Highlights

While cAMP analogues acting directly at protein kinase (PKA) lead to a similar sensitization, opioids, by virtue of their antihyperalgesic activity resulting from adenylate cyclase inhibition, are unable to prevent transient receptor potential vanilloid 1 (TRPV1) sensitization by direct PKA activators [12]
Potentiation of TRPV1 responses by cAMP-dependent PKA is crucial to the development of inflammatory hyperalgesia
Our results propose a mechanism for potentiation of TRPV1 responses by cAMP-dependent PKA incorporating both increased TRPV1 translocation and multimerization (Fig. 8)

Summary

Introduction

Capsaicin Responses Potentiated by cAMP-dependent PKA Are Inhibited by Morphine—Potentiation of TRPV1-mediated Ca2ϩ responses to addition of varying concentrations of capsaicin was assessed after a short (15 min) incubation with 0.1% DMSO (control) or forskolin (50 ␮M, Fig. 1A). Treatment with the Transglutaminase Inhibitor Dansylcadaverine Inhibits Capsaicin Responses Potentiated by Forskolin— To determine if TRPV1 functionality is affected when TRPV1 multimers are disrupted, we preincubated TRPV1/MOP HEK cells with varying concentrations of the transglutaminase inhibitor dansylcadaverine and assessed calcium responses to the addition of 300 nM capsaicin in control and forskolin-stimulated cells (Fig. 3A).

Results

Conclusion