Chemical kinetic measurements of the effect of trans- and cis-3,3'-bis[(trimethylammonio)methyl]azobenzene bromide on acetylcholine receptor mediated ion translocation in Electrophorus electricus and Torpedo californica

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Abstract
A quench-flow technique was used to study the effect of trans- and cis-3,3''-bis[(trimethylammonio)methyl]azobenzene bromide (trans- and cis-Bis-Q), photoisomerizable ligands, on the acetylcholine receptor in vesicles prepared from the electric organ of Electrophorus electricus and of Torpedo californica. In E. electricus, two rate coefficients of the receptor-mediated translocation of 86Rb+ induced with trans-Bis-Q were measured: JA, the rate coefficient for ion flux, and .alpha., the rate coefficient for receptor inactivation (desensitization). Both rate coefficients increase with increasing concentrations of Bis-Q up to 50 .mu.M. At higher concentrations JA decreases in a concentration-dependent manner while .alpha. remains unchanged. This effect was previously observed with suberyldicholine [Pasquale, E. B., Takeyasu, K., Udgaonkar, J., Cash, D. J., Severski, M. C., and Hess, G. P. (1983) Biochemistry 22, 5967-5973] and with acetylcholine [Takeyasu, K., Udgaonkar, J., and Hess, G. P. (1983) Biochemistry 22, 5973-5978] and was analyzed in terms of a minimum mechanism that accounts for the properties of activation, desensitization, and inhibition of the receptors. Two molecules of trans-Bis-Q must be bound for the channel to open, but at concentrations greater than 50 .mu.M the population of open channels decreases because of the additional binding of one molecule of trans-Bis-Q to a regulatory inhibitory site, independent of the activating sites. cis-Bis-Q does not induce transmembrane ion flux, but it does inhibit the response of the receptor to acetylcholine and induces inactivation (desensitization) in the micromolar range. In T. californica no activity of the receptor could be observed in the presence of trans-Bis-Q, but trans-Bis-Q did inhibit acetylcholine-induced flux. The implication of the results to the use of Bis-Q as a photoactivatable ligand in kinetic studies is discussed.

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