Experimental studies on transient receptor potential (TRP) channels continue to be hampered by the lack of specific antagonists for many subtypes of this receptor superfamily. Due to this absence, a host of non-specific compounds have been utilised to aid identification of particular subtypes of TRP channels. For example, structurally diverse substances such as 1-[beta-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF96365), flufenamic acid (FF) and 2-aminoethoxydiphenyl borate (2-APB) are routinely used as antagonists to pharmacologically characterise members of the canonical subtype of TRP channels (TRPC)1,2, yet their selectivity for these and other ion channels remains largely uninvestigated.
The present study demonstrates that SKF96365, FF and 2-APB also act as non-competitive antagonists of GABA–induced currents mediated by this chloride-permeable, inhibitory ligand-gated ionotropic receptor.
Using whole cell patch-clamp recording and a rapid drug application system (<100ms exchange time) extracellularly applied GABA (0.1-10,000M) evoked concentration-dependent inward currents in Human Embryonic Kidney (HEK) cells stably transfected with GABAA receptors with an average peak amplitude of 4189.9±234.2pA (n=37) to 1mM GABA and an EC50 value of 47±2M and a Hill coefficient of 1.4±0.1 (n=3-41) corresponding to two GABA binding sites.
SKF 96365 (1–100M) inhibited GABA responses with an IC50 value of 13.4±5.1M (n=4) and suppressed the maximal response to GABA at all concentrations used (P<0.05, n=4–9) without any significant rightward shift in the concentration response curve consistent with a non-competitive mode of action. SKF96365 (10M) also significantly prolonged the inactivation time of the receptors to GABA at all concentrations examined (e.g. control inactivation time () for 100M GABA = 194.8±7.5ms-1, n=36; + 10M SKF96365, =308.8±12.7 ms-1, n=4, P<0.05).
Similarly, FF (1-100M) inhibited GABA responses with an IC50 value of 1.9±1.4M (n=3-6) in a manner again consistent with a non-competitive mode of action (P<0.05, n=4). FF (1, 10 & 100M) also significantly prolonged the inactivation kinetics of responses to GABA at all concentrations (e.g. 1mM GABA, =212.15.8ms-1, n=101; + 100M FF, =1003.433.6ms-1, n=6, P<0.01).
2-APB (10–1000M) inhibited responses to GABA at all concentrations (P<0.05, n=3-6), with an IC50 value of 16.7±5.4M (n=3-5) by both suppressing the maximal response to GABA and inducing a rightward shift of the concentration response curve (at >100M 2-APB) consistent with both competitive and non-competitive mechanisms. 2-APB had no significant effect on either the activation or inactivation kinetics of the GABA responses.
The current studies therefore caution against the sole use of these compounds to identify TRPC channels.
1. Chinopoulos, C., Gerencser, A. A., Doczi, J., Fiskum, G., Adam-Vizi, V. Inhibition of glutamate-induced delayed calcium deregulation by 2-APB and La3+ in cultured cortical neurones. J. Neurochem. 2004; 91; 471-83.
2. Inoue, R., Okada, T., Onoue, H., Hara, Y., Shimizu, S., Naitoh, S., Ito, Y., Mori, Y. The transient receptor potential protein homologue TRP6 is the essential component of vascular a1-adrenoceptor-activated Ca2+-permeable cation channel. Circ. Res., 2001; 88; 325 – 332.