Probing the molecular dimensions of general anaesthetic target sites in tadpoles (Xenopus laevis) and model systems using cycloalcohols

Abstract

1 The series of cycloalcohols C6, C7, C8 and C10 have been used to probe the molecular dimensions of a variety of general anaesthetic target sites. 2 The general anaesthetic EC50 concentrations of the cycloalcohols were determined for tadpoles (Xenopus laevis). All of the cycloalcohols tested were found to be potent general anaesthetics (on average EC50/C(sat) = 0.03). 3 The effects of the cycloalcohols on highly purified luciferase enzymes from fireflies (Photinus pyralis) and bacteria (Vibrio harveyi) were also investigated. Both enzymes were inhibited competitively, with the cycloalcohols competing with firefly luciferin for binding to the firefly enzyme and with n-decanal for binding to the bacterial enzyme. 4 The binding site on the firefly enzyme could accommodate two molecules of cycloalcohols C6 and C7 but only a single molecule of the larger cycloalcohols (C8 and C10), implying a volume of the binding site of about 250 cm3 mol-1. In contrast, the binding site on the bacterial luciferase could bind only a single cycloalcohol molecule between C6 and C10. While all of the cycloalcohols were potent inhibitors of the firefly luciferase enzyme (on average EC50/C(sat) = 0.015), they were very weak inhibitors of the bacterial luciferase enzyme (on average EC50/C(sat) = 0.12). Since both enzymes bind long-chain aliphatic n-alcohols tightly, the differing affinities of the cycloalcohols for the two enzymes is probably a consequence of geometrical factors. 6 The cycloalcohols produced very small effects on lipid bilayers. At EC50 concentrations which produce general anaesthesia, lipid bilayer phase transitions were shifted, on average, by only 0.43°C. 7 We conclude that the general anaesthetic effects of the cycloalcohols can most economically be explained by assuming that the cycloalcohols act at protein binding sites in the central nervous system. These target sites would have binding properties similar to those of the anaesthetic-binding site on firefly luciferase, but their average volume would be somewhat smaller than 250 cm3 mol-1.