The pharmacology of recombinant GABAA receptors containing bovine alpha 1, beta 1, gamma 2L sub-units stably transfected into mouse fibroblast L-cells.

Abstract

1. Responses to gamma-aminobutyric acid (GABA) were evoked in mouse fibroblast L-cells stably transfected with bovine, alpha 1, beta 1, gamma 2L sub-units of the GABAA receptor. Expression was stimulated via a steroid-inducible promoter system. 2. In near symmetrical intracellular and extracellular chloride concentrations, GABA evoked inward currents at negative holding potentials that reversed at +5 mV and displayed slight outward rectification. Concentration-response curves were fitted well by the logistic equation. GABA had a pEC50 = 5.1 +/- 0.1 and the curves had a slope of 1.9 +/- 0.1. 3. Responses to GABA were antagonized by bicuculline, picrotoxin and penicillin. The action of bicuculline was competitive (pA2 = 6.4) whilst the block by picrotoxin was uncompetitive and strongly agonist-dependent. 4. Benzodiazepine receptor agonists potentiated responses to 3 microM GABA. The rank order of potency was FG 8205 > flunitrazepam > zolpidem > C1218872. FG 8205 and C1218872 produced markedly lower maximal potentiations with efficacies 0.4 and 0.6 x that of flunitrazepam, respectively. The potencies of zolpidem and C1218872 observed are in agreement with the BZ1 type pharmacology of this sub-unit combination. The potentiation of GABA by flunitrazepam was antagonized by flumazenil with a Ki of 3.8 nM. 5. GABA responses were potentiated in the presence of pentobarbitone and alphaxalone. The response was also noticeably broadened by these compounds due to a decrease in the response decay rate. Concentrations of pentobarbitone of 100 microM and above evoked an inward current in the absence of GABA. Alphaxalone up to 10 microM did not evoke a direct response. 6. This expression system produced functional receptors that behaved in a fashion analogous to those found endogenously in other preparations. Thus, this system appears to provide a useful and versatile preparation for the analysis of sub-unit regulation of GABAA receptor pharmacology.