Vol. 290, Issue 1, 129-135, July 1999

Characterization of Interaction of 3,4,5-Trimethoxybenzoic Acid 8-(Diethylamino)octyl Ester with Torpedo californica Nicotinic Acetylcholine Receptor and 5-Hydroxytryptamine₃ Receptor¹

Hongwei Sun, Elizabeth A. McCardy, Tina K. Machu and Michael P. Blanton

Department of Pharmacology, Texas Tech University Health Sciences Center, Lubbock, Texas

The widely used calcium channel antagonist 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8) has been identified as a noncompetitive antagonist (NCA) and open-channel blocker of both muscle- and neuronal-type nicotinic acetylcholine receptors (AChRs). To further examine the interaction of TMB-8 with the AChR, the compound was tested as a competitor for the binding of two NCAs of the Torpedo californica AChR, phencyclidine and 3-trifluoromethyl-3-(m[¹²⁵I]iodophenyl)diazirine, for which the binding to the AChR has been pharmacologically well characterized and a channel binding loci has been established. TMB-8 fully inhibited specific photoincorporation of 3-trifluoromethyl-3-(m[¹²⁵I]iodophenyl)diazirine into the resting AChR channel (IC₅₀ = 3.1 µM) and inhibited high-affinity [³H]phencyclidine binding to the desensitized AChR (IC₅₀ = 2.4 µM). We conclude that TMB-8 is a potent NCA of the nicotinic AChR, interacting with the resting, open-channel, and desensitized channel conformations. TMB-8 was next tested as an inhibitor of the structurally homologous 5-hydroxytryptamine (5-HT)₃ receptor (5-HT₃R). Using 5-HT₃R containing Sf21 cell membranes, TMB-8 completely inhibited specific binding of the radiolabeled 5-HT₃R antagonist [³H]GR65630 (K_i = 2.5 µM). Furthermore, TMB-8 antagonized 5-HT-evoked currents of both mouse and human 5-HT₃Rs expressed in Xenopus laevis oocytes, and additional analysis was consistent with a competitive antagonistic mechanism of action. These results, taken together, indicate that TMB-8 antagonizes the function of the AChR and 5-HT₃R by different mechanisms. Given the sequence similarity and emerging evidence of structural homology in the channels of these two receptors, these results underscore the existence of subtle yet important structural differences in each channel.