BENZODIAZEPINE AND BETA-CARBOLINE REGULATION OF SINGLE GABA(A) RECEPTOR CHANNELS OF MOUSE SPINAL NEURONS IN CULTURE

1. The effects of the benzodiazepine receptor agonist, diazepam (DZ), and the inverse agonist, ,7-dimethoxyl-4-ethyl-beta-carboline-3-carbox ylate (DMCM), on gamma-aminobutyric acid (GABA(A)) receptor single cha nnel currents were characterized. Outside-out patches were obtained fr om somata of cultured mouse spinal cord neurones and voltage clamped a t -75 mV (E(cl) = 0 mV). 2. GABA (2 mu M) alone or with DZ (20-1000 nM ) or DMCM (20-100 nM) was applied to patches by pressure ejection from blunt micropipettes. DZ enhanced GABA(A) receptor currents with an in verted U-shaped concentration-response curve. Mean steady-state curren ts were increased by low concentrations of DZ (20-50 nM). At higher co ncentrations of DZ, the enhancement was diminished. Mean steady-state currents were decreased by DMCM at all concentrations. 3. GABA(A) rece ptor channels opened most frequently to a 27 pS main conductance level and less frequently to a 19 pS subconductance level. Neither DZ nor D MCM altered the proportion of time spent at either of the conductance levels. The kinetic properties of the main conductance level were stud ied. 4. Neither DZ nor DMCM altered the mean GABA(A) receptor channel open or burst durations. Sums of three exponential functions were requ ired to fit best open and burst duration-frequency histograms for GABA alone or with DZ or DMCM. No significant changes in the three time co nstants or areas of the three exponential functions for open or burst duration histograms were produced by DZ or DMCM. 5. With increasing co ncentrations of DZ up to 50 nM, GABA evoked an increased frequency of channel openings and bursts. With higher DZ concentrations, the magnit udes of the increase in channel opening and burst frequencies were red uced. At all concentrations of DMCM, GABA evoked a decreased frequency of channel openings and bursts. 6. Closed duration-frequency histogra ms for GABA alone or with DZ or DMCM were best fitted by sums of at le ast six exponential functions. The three shortest closed duration time constants were unchanged by DZ or DMCM, The three longest closed dura tion time constants were altered by DZ and DMCM, consistent with alter ations in opening frequency. 7. DZ increased and DMCM decreased steady -state GABA(A) receptor current by increasing or decreasing channel op ening frequency without altering mean channel open duration. We propos e that DZ and DMCM alter GABA(A) receptor current by acting reciprocal ly to increase or decrease only, respectively, the apparent agonist as sociation rate at the first of two proposed GABA binding steps without altering channel gating. The basis for the altered apparent associati on rate is discussed.