Journal of Physiology (1989), 417, pp. 483-500 483 With 6 text-figures Printed in Great Britain BARBITURATE REGULATION OF KINETIC PROPERTIES OF THE GABAA RECEPTOR CHANNEL OF MOUSE SPINAL NEURONES IN CULTURE BY ROBERT L. MACDONALD, CARL J. ROGERS AND ROY E. TWYMAN From the Department ofNeurology, University ofMichigan Medical Center, Ann Arbor, Michigan 48104, USA (Received 23 May 1988) SUMMARY 1. Barbiturate regulation of the kinetic properties of y-aminobutyric acidA (GABA) receptor channel chloride currents from somata of mouse spinal cord neurones were investigated using whole-cell and excised outside-out patch-clamp recording techniques. 2. GABA (2 1uM), GABA (2 /iM) plus phenobarbitone (PhB) (500 gM) and GABA (2,lM) plus pentobarbitone (PB) (50/,M), applied by pressure ejection from blunt perfusion micropipettes, evoked inward chloride currents when neurones or patches were voltage clamped at -75 mV and the chloride equilibrium potential was 0 mV. GABA receptor channel currents were increased by PhB and PB. 3. Single GABA receptor channel currents were recorded with a main conductance state of 27 pS and a less frequent subconductance state of 16-5 pS. The conductances of the two states were unchanged by the barbiturates. 4. The main conductance state kinetics were analysed. GABA alone or with the barbiturates gated the channel open singly and in groups of openings. 5. The barbiturates increased GABA receptor channel mean open time and shifted frequency histograms of channel open times to longer times. 6. Three exponential functions were required to fit the frequency histograms of GABA receptor channel open times, suggesting that the channel has at least three open states (Or, 02, 03). The time constants for the exponential functions (0-9, 2-7 and 7X8 ms, respectively) were unchanged by the barbiturates. The increases in mean open times and the shifts of the open-time frequency histograms by the barbiturates were due to a reduction in relative frequency of occurrence of the two short open states (O1 and 02) and to an increase in the relative frequency of occurrence of the longest open state (03). 7. Frequency histograms of GABA receptor channel closed times were fitted with five exponential functions, suggesting that the channel has multiple closed states. None of the time constants nor areas of the exponential functions were significantly changed by the barbiturates. 8. For analysis, a burst was defined as openings surrounded by closures greater than a critical closed time, tc, of 5 ms. For GABA (2 ,UM), frequency histograms of GABA receptor channel bursts were fitted with three exponential functions, NIS 7(X)1 16-2> 484 R. L. MACDONALD, C. J. ROGERS AND R. E. TWYMAN suggesting that the channel has three burst states (B1, B2, B3). The B1 burst state was probably a single opening to the 01 open state while the B2 and B3 burst states were probably composed of multiple openings to the 02 and 03 open states. With both barbiturates, the relative frequency of occurrence of the two shortest burst states (B1 and B2) was reduced and that of the longest burst state (B3) was increased. The time constant of the B3 burst state was increased slightly by the barbiturates. 9. We suggest that barbiturates increase GABA receptor channel current by increasing the relative frequency of occurrence of the long (B3) bursts which contain multiple long openings and decreasing the relative proportion of shorter (B1 and B2) bursts which contain shorter openings. This results in an increase in GABA receptor channel apparent mean open time and mean burst duration. Barbiturates do not measurably alter the closing rates of the open states, and therefore, do not alter their mean open times. Rather, barbiturates alter the rate constants which regulate entry into the three open states, such that entry into the longest, stable open state (03) is favoured over the two shorter and less stable open states (01 and 02)- INTRODUCTION The postsynaptic y-aminobutyric acidA (GABA) receptor channel is composed of an oligomeric complex of polypeptide subunits which form a chloride channel associated with binding sites for GABA, benzodiazepines and barbiturates (Olsen, 1982; Schofield, Darlison, Fujita, Burt, Stephenson, Rodriguez, Rhee, Rama- chandran, Reale, Glencorse, Seeburg & Barnard, 1987). GABA binds to the receptor and gates open the chloride channel (Curtis, H6sli, Johnston & Johnston, 1968; Hamill, Bormann & Sakmann, 1983). Barbiturates potentiate the inhibitory effects of GABA in the vertebrate central nervous system (Schmidt, 1963; Nicoll, 1972; Ransom & Barker, 1976; Macdonald & Barker, 1979). Based on studies using the noise analysis technique, it was proposed that: (1) the GABA receptor channel opens into a single open state with exponentially distributed open durations and (2) barbiturates potentiate GABAergic inhibition by increasing the mean duration of the single open state without altering the frequency of channel opening or the channel conductance (Barker & McBurney, 1979; Study & Barker, 1981; Barker, McBurney & MacDonald, 1982). However, the resolution of the noise analysis technique is low. Using the single-channel recording technique which has a higher temporal resolution (Hamill, Marty, Neher, Sakmann & Sigworth, 1981), single GABA receptor channel currents have been recorded and shown to be more complex (Jackson, Lecar, Mathers & Barker, 1982; Hamill et al. 1983; Sakmann, Hamill & Bormann, 1983; Bormann & Clapham, 1985; Martin, 1985; Mathers, 1985a; Bormann, Hamill & Sakmann, 1987; Macdonald, Rogers & Twyman, 1989). The GABA receptor channel has been shown to have multiple open and closed states (Macdonald et al. 1989) and to open in bursts (Hamill et al. 1983). The effect of barbiturates on the kinetic properties of GABA receptor channels has not been determined. We have used whole-cell and outside-out patch-clamp recording techniques to study the regulation of GABA receptor channel currents by the BARBITURATE MODULATION OF GABA Cl- CHANNELS 485 barbiturates phenobarbitone (PhB) and pentobarbitone (PB), using mouse spinal cord neurones grown in cell culture. METHODS Cell culture A single-cell suspension of neurones from mechanically dissociated spinal cords, dissected from 10 to 12-day-old murine fetuses, was grown in culture medium as previously described (Ransom, Neale, Henkart, Bullock & Nelson, 1977; Macdonald et al. 1989). The mothers were killed by cervical dislocation under CO2 narcosis prior to removal of the fetuses. Cultures were maintained for 2-5 weeks prior to being used in these experiments. Bathing and recording solutions The same extracellular and intrapipette solutions consisting of a symmetric chloride salt solution were used for both whole-cell and excised outside-out recordings. The extracellular solution consisted of (in mM): 142 NaCl, 8-1 KCI, 1 CaCl2, 6 MgCl2, 10 glucose, 10 Na+-HEPES (pH 7 4). The intrapipette solution consisted of (in mM): 153 KCI, 1 MgCl2, 10 Na+-HEPES, 5 EGTA, 1 NaOH, 2 KOH (pH 74). The specific glycine receptor antagonist strychnine (200 nM) (Sigma Chemical Co., St Louis, MO, USA) was added to all solutions to ensure that no glycine receptor channel currents were included in the analyses. Recordings were performed at room temperature (20-23 °C). This combination of extracellular and intrapipette solutions resulted in a chloride equilibrium potential (Ec1) of0 mV and a potassium equilibrium potential (EK) of -75 mV. Micropipettes Patch recording micropipettes were fabricated up to 24 h prior to use. Drugs and GABA were applied from pressure ejection or diffusion micropipettes. To avoid dilution in the drug micropipette tips, the drug micropipettes were removed from the bathing solution whenever the drugs were not being applied. Prior to application, a pressure pulse (5-10 s) was applied distant from the recording site to eject diluted solution from the micropipette tip. Equipment Both whole-cell and patch-clamp recordings were performed using a Model L/M EPC-7 amplifier (LIST-Medical Instruments, Darmstadt, FRG). For whole-cell recordings, responses were low-pass filtered (3 dB at 600 Hz, 8-pole Butterworth) (A.P. Circuit Corporation. New York, NY, USA) and recorded on a Gould chart recorder (Gould Inc., Cleveland, OH, USA). Patch-clamp command potentials and single-channel currents were recorded on a video cassette recording system (VCR) (SONY SL-2700, modified to 0-20 kHz) via a digital audio processor (SONY PCM-501 ES, 14-bit, 44 kHz). Simultaneously, these data were recorded on a chart recorder (Gould Inc.) using a low- pass (3 dB at 1 kHz) 8-pole Bessel filter (Frequency Devices, Haverhill, MA, USA). For single- channel analysis the data were played back from the VCR system and digitized (8 kHz, 14 bit, Tecmar A/D converter, Cleveland, OH, USA) for computer (80386 based processors) analysis with a low-pass (3 dB at 1 kHz), 8-pole Bessel filter interposed. Digitized data segments ranged in length from 20-25 s. All recordings were performed at room temperature (20-23 °C). Drug application GABA, PhB and PB were obtained from Sigma Chemical Co. GABA (2 FM) was prepared as previously described and applied using pressure micropipettes (Nowak, Young & Macdonald, 1982). The barbiturates PhB and PB were dissolved in solution and serial dilutions provided a final concentration of 500 /M for PhB and 50,uM for PB. These concentrations have been shown previously to enhance GABA responses on mouse spinal cord neurones in cell culture without directly activating GABA receptor channel current (Macdonald & Barker, 1978; Schulz & Macdonald, 1981). A mixture of GABA (2 /M) and barbiturate was applied to the neurone or patch 486 R. L. MACDONALD, C. J. ROGERS AND R. E. TWYMAN membrane via pressure ejection micropipettes in the presence of a diffusion pipette containing the barbiturate. Micropipettes were moved to within 50 ,um of the neurone or membrane only during the time of each application. Whole-cell and outside-out patch recording Whole-cell and excised outside-out patch recording techniques were similar to those of Hamill et al.