Cross-inhibition between native and recombinant TRPV1 and P2X₃ receptors

Abstract 

Small- to medium-sized neurons in the dorsal root ganglion (DRG) convey nociceptive information to the spinal cord. The co-expression of TRPV1 receptors (sensitive to vanilloids, heat and acidic pH) with P2X₃ receptors (sensitive to extracellular ATP) has been found in many DRG neurons. We investigated whether the co-activation of these two receptor classes in small-diameter cells leads to a modulation of the resulting current responses shaping the intensity of pain sensation. The whole-cell patch clamp method was used to record agonist-induced currents in cultured rat DRG neurons and in HEK293 cells transfected with the respective wild-type recombinant receptors or their mutants. Co-immunoprecipitation studies were used to demonstrate the physical association of TRPV1 and P2X₃ receptors. At a negative holding potential, the P2X₃ receptor agonist α,β-meATP induced less current in the presence of the TRPV1 agonist capsaicin than that in its absence. This inhibitory interaction was not changed at a positive holding potential, in a Ba²⁺-containing superfusion medium, or when the buffering of intrapipette Ca²⁺ was altered. The C-terminal truncation at Glu362 of P2X₃ receptors abolished the TRPV1/P2X₃ cross-talk in the HEK293 expression system. Co-immunoprecipitation studies with polyclonal antibodies generated against TRPV1 and P2X₃ showed a visible signal in HEK293 cells transfected with both receptors. It is concluded that the two pain-relevant receptors TRPV1 and P2X₃ interact with each other in an inhibitory manner probably by a physical association established by a motif located at the C-terminal end of the P2X₃ receptor distal to Glu362.

Abbreviations: DRG, dorsal root ganglion, HEK cells, human embryonic kidney cells, TRPV1, transient receptor potential channel of the vanilloid receptor subtype 1, α,β-meATP, α,β-methylene ATP, h, human, r, rat, HBSS, Hanks’ balanced salt solution, DMEM, Dulbecco’s minimal essential medium, WT, wild-type, BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, TNP-ATP, 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate, TrkA, receptor tyrosine kinase, NGF, nerve growth factor, GDNF, glial cell-derived neurotrophic factor, nACh receptor, nicotinic acetylcholine receptor

Keywords: P2X₃ receptor, TRPV1 receptor, Receptor interaction, Dorsal root ganglion, HEK293 cell, ATP, Capsaicin