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 P2X(3) 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 P2X(3) receptors. At a negative holding potential, the P2X(3) receptor agonist alpha,beta-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 Ba(2+)-containing superfusion medium, or when the buffering of intrapipette Ca(2+) was altered. The C-terminal truncation at Glu362 of P2X(3) receptors abolished the TRPV1/P2X(3) cross-talk in the HEK293 expression system. Co-immunoprecipitation studies with polyclonal antibodies generated against TRPV1 and P2X(3) showed a visible signal in HEK293 cells transfected with both receptors. It is concluded that the two pain-relevant receptors TRPV1 and P2X(3) 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 P2X(3) receptor distal to Glu362.

Combined genetic and pharmacological inhibition of TRPV1 and P2X3 attenuates colorectal hypersensitivity and afferent sensitization

“…In agreement with previous studies in rat lumbar DRG neurons (16,41,48), the present work demonstrates that a substantial proportion of mouse PN colorectal afferent somata coexpress TRPV1 with P2X3. Previously, we reported deficits in colorectal mechanosensation and sensitization with single deletion of either TRPV1 or P2X3 (19,20,39).…”

“…The results suggest an additive, if not greater than additive, attenuation of afferent response to stretch by simultaneous antagonism of both channels. This may be secondary to a combined effect of two independent processes, or it may indicate an interaction that could be mediated through shared Ca 2ϩ -dependent signaling cascades (10,21,33,36,47,55) or modulatory conformational spread (4) via direct physical association of TRPV1 and P2X3 as demonstrated previously (41). Importantly, this combined antagonist/agonist experiment additionally confers pharmacological validity to the selectivity of the drugs used (e.g., TNP-ATP has effects only on REs that respond to ␣,␤-meATP).…”

Neuromodulation by Extracellular ATP and P2X Receptors in the CNS

“…Similar experiments have now been repeated with several ion channel combinations showing that cross inhibition between P2X receptors and members of the nicotinic receptor-like family are common (Barajas-Lopez et al, 1998; Barajas-Lopez et al, 2002; Boue-Grabot et al, 2003; Boue-Grabot et al, 2004a; Boue-Grabot et al, 2004b). Most recently, functional interactions have been reported for P2X receptors and acid sensing ion channels (ASIC) (Birdsong et al, 2010) as well as between P2X3 receptors and TRPV1 channels (Stanchev et al, 2009). We comment here on general themes that emerge.…”

Phosphoinositide signaling in somatosensory neurons

“…Accordingly, capsaicin has been shown to inhibit several different ion channels in DRG neurons, and for inhibition of KCNQ channels (Zhang et al, 2011) and for inhibition of TRPA1 (Akopian et al, 2007) inclusion of PI(4,5)P 2 in the whole cell patch pipette alleviated capsaicin-induced inhibition. For inhibition of Voltage-gated Ca 2+ channels (Wu et al, 2006), P2X channels (Stanchev et al, 2009), HCN channels (Kwak, 2012) and for voltage gated Na + channels (Liu et al, 2001), the role of phosphoinositides has not been tested, see details at the discussion of the individual channels. Local capsaicin evokes intense burning pain, but paradoxically, it is also used as a local analgesic, because of the desensitization of the nociceptor neurons, which includes many processes, including desensitization of the channel itself, and potentially inhibition of some of the other ion channels described here (Szallasi and Blumberg, 1999).…”

“…It was shown that in rat DRG neurons activation of P2Y2 receptors inhibit P2X3 via PI(4,5)P 2 depletion (Mo et al, 2013). Capsaicin also inhibits P2X3 in TRPV1 expressing cells (Stanchev et al, 2009), and in rat DRG neurons (Piper and Docherty, 2000) but the involvement of phosphoinositides were not examined.…”