The mode of action of antagonists of the excitatory response to acetylcholine in Aplysia neurones.

Abstract

The mode of action of various antagonists of acetylcholine (ACh) excitatory effects on Aplysia californica neurons was studied under voltage clamp. ACh was applied by iontophoresis whereas antagonists were applied in the bath. Tubocurarine and hexamethonium were the most thoroughly studied compounds. The elementary current, calculated as the ratio of the variance of ACh noise to the mean ACh induced current, was not modified by any of the antagonists tested. The evolution of the ACh induced current after a voltage jump, which is normally described by a single exponential, was modified by all the antagonists tested. A common feature of the modified relaxations was the appearance, over a certain concentration range of the antagonist, of 2 successive and opposite exponential components. The characteristics of the composite relaxations depended on the antagonist. For a given antagonist they varied with membrane potential, ACh concentration, and antagonist concentration. The noise power spectra of the ACh induced current showed changes consistent with those of the relaxations. In the absence of antagonists, the current induced by a steady application of ACh increased linearly with hyperpolarization. In the presence of antagonists, the I-V curve showed a marked curvature, indicating a proportionally larger reduction of the ACh response at more negative membrane potentials. The voltage sensitivity of the blocking action of hexamethonium and decamethonium was noticeably stronger than that of monovalent antagonists. A model was proposed to account for the observed effects. It assumed that the antagonists studied bound preferentially to the activated ACh-receptor complex, and converted it to a non-conducting state. Kinetic constants could be calculated for this reaction; e.g., for curare, at 12.degree. C and -80 mV, the dissociation and association constants were estimated at 0.1 s-1 and 4 .times. 105 M s-1. Partial replacement of the extracellular Na by Tris modified the relaxations observed in the presence of hexamethonium. Hexamethonium appeared less effective in the presence of Tris, which supported the hypothesis that the binding site of the antagonists was linked to the ionic channel.