Enhancement of g-Aminobutyric AcidA Receptor Activity by a-Chloralose

a-Chloralose is widely used as an anesthetic in the laboratory due to its minimal effects on autonomic and cardiovascular systems, yet little is known about its mechanism of action. We examined the effects of a-chloralose on g-aminobutyric acid type A (GABAA) receptor activity because recent studies have shown that several classes of general anesthetics modulate the function of this receptor. GABAA receptor activity was assayed by measuring the GABA-induced current in Xenopus oocytes expressed with human GABAA receptor alpha-1, beta-1 and gamma-2L subunits. a-Chloralose produced a concentrationdependent potentiation of the GABA-induced current with an EC50 value of 49 mM and a maximal effect of 239% of control. Membrane current was not affected by a-chloralose in the absence of GABA. a-Chloralose (100 mM) increased the affinity for GABA 5-fold and produced a small (17%) increase in the efficacy of GABA. Measurement of the reversal potentials for the a-chloralose response suggested that the effect is mediated through increased Cl conductance. Studies of a-chloralose interactions with other allosteric modulators determined that a-chloralose binds to a site on the GABAA receptor complex distinct from the benzodiazepine, neurosteroid and barbiturate sites. Chloral hydrate, trichloroethanol and urethane also augmented GABA-induced currents. a-Chloralose had no effect on the hydroxytryptamine-induced currents in oocytes expressed with the 5-hydroxytryptamine3 receptor. These data extend the number of classes of anesthetics that allosterically modulate GABAA receptor activity and indicate that GABAA receptors may be a common site of action for diverse classes of general anesthetics. General anesthetics have been in use for .150 years, yet only within the past 10 to 15 years have we begun to understand the molecular mechanism of action of these compounds. Recent research has focused on examining effects of anesthetics on ion channels in neuronal membranes. To date, each of the anesthetics studied modulate GABAA receptor activity by enhancing the GABA-induced chloride conductance (Harris et al., 1995). Previous work has primarily focused on the anesthetics used in the clinic; however, little work has been done with nonvolatile anesthetics used in the laboratory and by veterinarians. a-Chloralose is frequently used as an anesthetic due to its ease of administration, long duration and maintenance of autonomic reflexes (Balis and Monroe, 1964; Silverman and Muir, 1993), yet despite the wide application of this agent, there is little understanding of the mechanisms of action of this anesthetic. Limited studies suggest that a-chloralose affects GABAA receptor activity (Nicoll and Wojtowicz, 1980; Moody et al., 1988; Ishizuka et al., 1989; Kumamoto and Murata, 1996); however, the potency of a-chloralose for modulation of GABAA receptor activity, mechanism of action and binding site on the receptor complex is not known. Using GABAA receptors expressed in Xenopus laevis oocytes as a model system, the effects of a-chloralose on GABA-induced currents were examined to determine whether modulation of GABAA receptor activity may play a role in the anesthetic action of this compound.