Abstract
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 (Ki = 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.
Footnotes
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Send reprint requests to: Dr. Michael P. Blanton at the Department of Pharmacology, Texas Tech University Health Sciences Center, Lubbock, TX 79430. E-mail: phrmpb{at}ttuhsc.edu
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↵1 This research was supported in part by National Institutes of Health Grants R29-NS35786 (M.P.B.) and AA10561 (T.K.M).
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↵2 The concentration dependence of the reduction in [125I]TID labeling by TMB-8 into the AChR α subunit (in the resting state conformation) was also determined by gamma-counting of a ∼20-kDa S. aureus V8 protease fragment of the α subunit that contains the channel-linning M2 segment (αSer-173-Glu-338; Blanton et al., 1998b). TMB-8 reduced the amount of specific [125I]TID incorporation by >95% (total [125I]TID incorporation reduced by ∼80%) with a calculated IC50 value of 3.1 μM (nH = 0.96).
- Abbreviations:
- ACh
- acetylcholine
- AChR
- nicotinic acetylcholine receptor
- TMB-8
- 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester
- [125I]TID
- 3-trifluoromethyl-3-(m[125I]iodophenyl)diazirine
- PCP
- phencyclidine
- 5-HT
- 5-hydroxytryptamine
- 5-HT3R
- 5-hydroxytryptamine3 receptor
- Received November 23, 1998.
- Accepted March 4, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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