Pentobarbital Modulates g-Aminobutyric Acid-Activated Single- Channel Conductance in Rat Cultured Hippocampal Neurons

We examined the effect of a range of pentobarbital concentrations on 0.5 mM g-aminobutyric acid (GABA)-activated channels (10 6 1 pS) in inside-out or outside-out patches from rat cultured hippocampal neurons. The conductance increased from 12 6 4 to 62 6 9 pS as the pentobarbital concentration was raised from 10 to 500 mM and the data could be fitted by a Hill-type equation. At 100 mM pentobarbital plus 0.5 mM GABA, the conductance seemed to reach a plateau. The pentobarbital EC50 0.5 mM GABA value was 22 6 4 mM and n was 1.9 6 0.5. In 1 mM pentobarbital plus 0.5 mM GABA, the single-channel conductance decreased to 34 6 8 pS. This apparent inhibition of channel conductance was relieved by 1 mM diazepam. The channel conductance was 64 6 6 pS in the presence of all three drugs. The channels were open more in the presence of both GABA and pentobarbital than in the presence of either drug alone. Pentobarbital alone (100 mM) activated channels with conductance (30 6 2 pS) and kinetic properties distinct from those activated by either GABA alone or GABA plus pentobarbital. Whether pentobarbital induces new conformations or promotes conformations observed in the presence of GABA alone cannot be determined from our study, but the results clearly show that it is the combination of drugs present that determines the single-channel conductance and the kinetic properties of the receptors. g-Aminobutyric acid (GABA) is the main inhibitory transmitter in the brain. When it binds to GABAA receptors, a chloride conductance is activated. These receptors are the targets of many therapeutic drugs and their pharmacological profile is determined by their subunit composition (MacDonald and Olsen, 1994; Barnard et al., 1998). To date, 20 different GABAA subunits have been cloned (Barnard et al., 1998). They are grouped into a1–6, b1–4, g1–3, r1–3, p, e, d, and u subunit families and are thought to assemble into heteropentameric receptors. The relative prominence of the different subunits varies between regions of the brain. Subunit heterogeneity has been shown to contribute to the variability in channel conductance among GABAA receptors (Sigel et al., 1990; Verdoorn et al., 1990; Angelotti and MacDonald, 1993). Barbiturates are a class of drugs that prolong postsynaptic inhibitory GABA-activated currents and exert a depressant action on the central nervous system (Nicoll et al., 1975; Gage and Robertson, 1985; Franks and Lieb, 1994). In wholecell studies, the major effect of the barbiturate pentobarbital has been to shift the GABA dose-response curve to lower concentrations (Rho et al., 1996). How the barbiturates modulate the function of the single receptor is not well understood. Whether they induce new conformations or merely promote conformations observed in the presence of GABA alone is not known. Studies using fluctuation analysis and single-channel recordings on cultured neurons (Mathers and Barker, 1980; Study and Barker, 1981; Mathers, 1985; MacDonald et al., 1989; Rho et al., 1996) indicate that barbiturates increase the GABA-activated currents by increasing the open probability of the GABA-activated channels. Recently, the conductance of several ligand-gated receptors has been shown to be modulated by the ligand concentrations (Ruiz and Karpen, 1997; Rosenmund et al., 1998) or by allosteric modulators of the receptors (Eghbali et al., 1997; Derkach et al., 1999; Guyon et al., 1999). We examined what effect pentobarbital had on GABAA channels in rat cultured hippocampal neurons in the presence of 0.5 mM GABA. Our results show that not only does pentobarbital increase the open probability of channels but that the single-channel conductance is also increased. ABBREVIATIONS: GABA, g-aminobutyric acid; TES, N-tris(hydroxymethyl) methyl-2-aminoethanesulfonic acid. 0026-895X/00/030463-00$3.00/0 MOLECULAR PHARMACOLOGY Vol. 58, No. 3 Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics 112/844635 Mol Pharmacol 58:463–469, 2000 Printed in U.S.A. 463 at A PE T Jornals on Sptem er 0, 2017 m oharm .aspeurnals.org D ow nladed from Materials and Methods Neurons used in the experiments were dissociated from hippocampal slices from newborn rats and maintained in culture for 8 to 24 days using techniques described previously (Curmi et al., 1993). Experiments were done at room temperature (20–24°C) on insideout patches except where stated. Channels were activated either by GABA in the pipette (inside-out patches) or by flowing a solution containing GABA through a narrow tube superfusing the patch (outside-out patches). The volume of the bath was 0.4 ml and the flow rate was 4 ml/min. This ensured a rapid change of solution in the bath within the first 30 s. Pentobarbital was applied by switching the solution flowing through the bath to a solution containing pentobarbital or by flowing a solution containing pentobarbital through a narrow tube superfusing the patch. The second method gave a rapid change in drug concentration (Birnir et al., 1995) but results were similar. The bath solution contained 135 mM NaCl, 3 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), pH 7.4. Pipette solution contained 141 mM NaCl or choline, 0.3 mM KCl, 0.5 mM CaCl2, 2 mM MgCl2, 10 mM TES, pH 7.4. In experiments on outside-out patches, the pipette also contained 5 mM EGTA. GABA (Sigma, St. Louis, MO) and pentobarbital (Sigma) were dissolved in the bath solution. Diazepam (Hoffman-La Roche, Nutley, NJ) was first dissolved in dimethyl sulfoxide as described by Eghbali et al. (1997). Conventional patch-clamp techniques were used when establishing a gigaseal and forming patches (Hamill et al., 1981). Pipettes were made from borosilicate glass (Clark Electromedical, Reading, England), coated with Sylgard (Dow Corning, Midland, MI) and fire-polished. Their resistance ranged from 10 to 20 MV. Currents were recorded using an Axopatch 1C current-to-voltage converter (Axon Instruments, Burlingame, CA), filtered at 5 kHz, digitized at 44 kHz using a pulse code modulator (PCM 501; Sony, Tokyo, Japan), and stored on videotape. The currents were played back from the videotape through the Sony PCM and digitized at frequency of 10 kHz using a Tecmar analog-to-digital converter interfaced with an IBM-compatible PC. The currents were then digitally filtered at 5 kHz and analyzed using a computer program called CHANNEL2 written by Michael Smith (John Curtin School of Medical Research, Australian National University, Canberra, Australia). The amplitude of currents was measured either from all-points current amplitude probability histograms or from direct measurements of the amplitude of individual currents filtered at 5 kHz. Opening and closing transitions were detected by setting thresholds levels just above the baseline noise. The mean current was measured as the average of the deviations of all data points from zero (the middle of the baseline current) during periods of 30 s. The average open probability of channels was measured from opening and closing transitions detected by setting thresholds levels just above the baseline noise. Channel burst durations were measured by constructing burst duration histograms from current recordings. A burst was defined as an opening or group of openings separated by closed periods of less than a critical time, which was defined as 1 ms. A suitable critical time was chosen after inspection of the distributions of closed events before burst analysis was started. The fastest closed time constant for all drug conditions was found to be less than 1 ms; therefore, all closings briefer than 1 ms were considered to occur within a burst. Burst durations were placed into frequency histograms using logarithmic binning. The square roots of the frequency histograms were fitted with the sums of three or four exponential components (Sigworth and Sine, 1987). Data are expressed as means 6 S.E. (n 5