The TRPV1 (VR1) receptor is a nociceptor-specific ligand-gated cation channel capable of integrating a cocktail of noxious stimuli including capsaicin, protons and heat. Mutagenesis studies have revealed seemingly distinct regions involved in mediating ligand binding and channel activation at the capsaicin binding site. Residue 512 mediates capsaicin gating of the channel (Jordt et al, 2002) and residue 547 mediates the cross species differences seen with RTX (Chou et al, 2004 ; Phillips et al, 2004; Gavva et al, 2004). The aim of this present study was to use whole-cell patch clamp electrophysiology to investigate cross-talk between these two regions, by utilising specific ligands thought to interact at both these sites. Recordings were made from HEK cells stably expressing rat TRPV1 and CHO cells expressing human TRPV1. HEK cells were also transiently transfected with mutant TRPV1 cDNA constructs together with GFP to identify expressing cells. Initial experiments using the S512Y mutant channel confirmed the importance of residue 512 for functional interaction of capsaicin, and species-specific exchange of residue 547 mediated a species difference in capsaicin-dependent gating (Table 1). Likewise, the potency of I-RTX, as well as RTX was also altered when the residue at position 547 was switched between the human and rat homologues of the receptor. Interestingly, the mutation of S512Y converted the activity of the antagonist, I-RTX, into an intrinsic agonist (EC50= 259 nM), albeit with a lower potency than its parent compound, RTX.

Recent studies have proposed a novel model for the receptor, based on the X-ray crystal structure of the Kvap channel (Jiang et al, 2003). This model places these two seemingly distinct residues, 512 and 547, within close proximity of one another. These data further support the model whereby intracellular ligand interaction near the S3 domain mediates movement of this region during channel gating.