title: Putative TRP channel antagonists, SKF 96365, flufenamic acid and 2-APB, are non-competitive antagonists at recombinant human α1β2γ2 GABA(A) receptors.
abstract: Although transient receptor potential (TRP) channel biology research has expanded rapidly in recent years, the field is hampered by the widely held, but relatively poorly investigated, belief that most of the pharmacological tools used to investigate TRP channel function may not be particularly selective for their intended targets. The objective of this study was therefore to determine if this was indeed the case by systematically evaluating the effects of three routinely used putative TRP channel antagonists, SKF 96365, flufenamic acid (FF) and 2-aminoethoxydiphenyl borate (2-APB) against one of the most widely expressed CNS receptor subtypes CNS, the human α1β2γ2 GABA(A) receptor. Using whole cell patch-clamp recording to record responses to rapidly applied GABA in the absence and presence of the three putative antagonists in turn we found that SKF 96365 (1-100 μM) and FF (1-100 μM) significantly inhibited GABA responses of recombinant human α1β2γ2 GABA(A) receptor stably expressed in HEK293 cells with IC(50) values of 13.4 ± 5.1 and 1.9 ± 1.4 μM, respectively, suppressing the maximal response to GABA at all concentrations used in a manner consistent with a non-competitive mode of action. SKF 96365 and FF also both significantly reduced desensitisation and prolonged the deactivation kinetics of the receptors to GABA (1mM; P<0.05). 2-APB (10-1000 μM) also inhibited responses to GABA at all concentrations used with an IC(50) value of 16.7 ± 5.4 μM (n=3-5) but had no significant effect on the activation, desensitisation or deactivation kinetics of the GABA responses. Taken together this investigation revealed that these widely utilised TRP channel antagonists display significant 'off-target' effects at concentrations that are routinely used for the study of TRP channel function in numerous biological systems and as such, data which is obtained utilising these compounds should be interpreted with caution.
authors.affiliation: Department of Physiology, University College Cork, Ireland. m.rae@ucc.ie
authors.forename: M G
authors.initials: MG
authors.lastname: Rae
authors.forename: J
authors.initials: J
authors.lastname: Hilton
authors.forename: J
authors.initials: J
authors.lastname: Sharkey
mesh_terms: D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000378:metabolism*; D001667:Binding, Competitive; D001896:Boron Compounds / Q000378:metabolism* / Q000494:pharmacology; D005439:Flufenamic Acid / Q000378:metabolism / Q000494:pharmacology*; D058787:GABA-A Receptor Antagonists / Q000378:metabolism / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D050052:TRPC Cation Channels / Q000037:antagonists & inhibitors*
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D001896:Boron Compounds; D058787:GABA-A Receptor Antagonists; D007093:Imidazoles; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D050052:TRPC Cation Channels; D005439:Flufenamic Acid; C109986:2-aminoethoxydiphenyl borate; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole