Comparison of the Effects of Ruthenium Red on Different TRPA1 Mammalian Orthologs

We have cloned and characterized the guinea pig (Cavia porcellus) TRPA1 ion channel and compared the effects of the tool TRP channel blocker molecule, Ruthenium Red (RuR), on the guinea pig with several other mammalian TRPA1 constructs, all heterologously expressed in CHO cells. Using a plate-based Ca+2 detection assay, human, mouse, rat and guinea pig TRPA1 responded to the TRPA1 agonists thymol and cinnamaldehyde with nearly indistinguishable potency and efficacy. We then tested the ability of RuR to block the agonist-induced changes in [Ca+2]internal and found that RuR efficaciously blocked agonist responses of human, mouse and rat clones over predicted ranges comparable to published studies, while the guinea pig ortholog was comparatively refractory to block by RuR, a phenomenon we confirmed using plate-based electrophysiology. An amino acid sequence comparison of S5-S6 regions, containing the putative site(s) of action of RuR, revealed that the guinea pig channel contained three non-conservative residue substitutions that distinguished it from the other TRPA1 clones. We singly mutated these residues to their human counterparts and also generated double and triple guinea pig mutants. Electrophysiological characterization of the mutant TRPA1 channels revealed that block by RuR was left-shifted toward its human TRPA1 potency without shifting responses to agonists or the structurally-unrelated small molecule blocker A-967079. In summary, our results provide the first molecular evidence for distinct mechanisms of action of multiple TRPA1 blockers and add to the growing body of literature highlighting marked species differences between TRPA1 orthologs.