Pharmacological profiling of the TRPV3 channel in recombinant and native assays.

BACKGROUND AND PURPOSE: Transient receptor potential vanilloid subtype 3 (TRPV3) is implicated in nociception and certain skin conditions. As such, it is an attractive target for pharmaceutical research. Understanding of endogenous TRPV3 function and pharmacology remains elusive as selective compounds and native preparations utilizing higher throughput methodologies are lacking. In this study, we developed medium-throughput recombinant and native cellular assays to assess the detailed pharmacological profile of human, rat and mouse TRPV3 channels. EXPERIMENTAL APPROACH: Medium-throughput cellular assays were developed using a Ca(2+) -sensitive dye and a fluorescent imaging plate reader. Human and rat TRPV3 pharmacology was examined in recombinant cell lines, while the mouse 308 keratinocyte cell line was used to assess endogenous TRPV3 activity. KEY RESULTS: A recombinant rat TRPV3 cellular assay was successfully developed after solving a discrepancy in the published rat TRPV3 protein sequence. A medium-throughput, native, mouse TRPV3 keratinocyte assay was also developed and confirmed using genetic approaches. Whereas the recombinant human and rat TRPV3 assays exhibited similar agonist and antagonist profiles, the native mouse assay showed important differences, namely, TRPV3 activity was detected only in the presence of potentiator or during agonist synergy. Furthermore, the native assay was more sensitive to block by some antagonists. CONCLUSIONS AND IMPLICATIONS: Our findings demonstrate similarities but also notable differences in TRPV3 pharmacology between recombinant and native systems. These findings offer insights into TRPV3 function and these assays should aid further research towards developing TRPV3 therapies.

Sazetidine-A is a potent and selective agonist at native and recombinant alpha 4 beta 2 nicotinic acetylcholine receptors.

Sazetidine-A has been recently proposed to be a "silent desensitizer" of alpha4beta2 nicotinic acetylcholine receptors (nAChRs), implying that it desensitizes alpha4beta2 nAChRs without first activating them. This unusual pharmacological property of sazetidine-A makes it, potentially, an excellent research tool to distinguish between the role of activation and desensitization of alpha4beta2 nAChRs in mediating the central nervous system effects of nicotine itself, as well as those of new nicotinic drugs. We were surprised to find that sazetidine-A potently and efficaciously stimulated nAChR-mediated dopamine release from rat striatal slices, which is mediated by alpha4beta2(*) and alpha6beta2(*) subtypes of nAChR. The agonist effects on native striatal nAChRs prompted us to re-examine the effects of sazetidine-A on recombinant alpha4beta2 nAChRs in more detail. We expressed the two alternative stoichiometries of alpha4beta2 nAChR in Xenopus laevis oocytes and investigated the agonist properties of sazetidine-A on both alpha4(2)beta2(3) and alpha4(3)beta2(2) nAChRs. We found that sazetidine-A potently activated both stoichiometries of alpha4beta2 nAChR: it was a full agonist on alpha4(2)beta2(3) nAChRs, whereas it had an efficacy of only 6% on alpha4(3)beta2(2) nAChRs. In contrast to what has been published before, we therefore conclude that sazetidine-A is an agonist of native and recombinant alpha4beta2 nAChRs but shows differential efficacy on alpha4beta2 nAChRs subtypes.

Stable expression and characterisation of a human alpha 7 nicotinic subunit chimera: a tool for functional high-throughput screening.

A chimera comprising the N-terminal region of the human alpha7 nicotinic acetylcholine receptor, fused to the transmembrane/C-terminal domains of the mouse serotonin 5-HT3 receptor, was constructed. Injection of the chimera cDNA into Xenopus oocytes, or transient transfection in human embryonic kidney (HEK-293) cells, resulted in the expression of functional channels that were sensitive to nicotinic acetylcholine, but not serotonin receptor ligands. In both systems, the responses obtained from chimeric receptors inactivated more slowly than those recorded following activation of wild-type alpha7 receptors. A stable HEK-293 cell line expressing the human alpha7/mouse 5-HT3 chimera was established, which showed that the chimera displayed a similar pharmacological profile to wild-type alpha7 receptors. Use of this chimera in high-throughput screening may enable the identification of novel pharmacological agents that will help to define further the role of alpha7 nicotinic receptors in physiology and disease.