Multiple Actions of Propofol on and GABAA Receptors

GABAA receptors are predominantly composed of and isoforms in the brain. It has been proposed that receptors mediate phasic inhibition, whereas receptors mediate tonic inhibition. Propofol (2,6-di-isopropylphenol), a widely used anesthetic drug, exerts its effect primarily by modulating GABAA receptors; however, the effects of propofol on the kinetic properties of and receptors are uncertain. We transfected human embryonic kidney (HEK293T) cells with cDNAs encoding rat 1, 6, 3, 2L, or subunits and performed whole-cell patch-clamp recordings to explore this issue. Propofol (3 M) increased GABA concentration-response curve maximal currents similarly for both 1 3 2L and 6 3 2L receptors, but propofol increased those for 1 3 and 6 3 receptors differently, the increase being greater for 1 3 than for 6 3 receptors. Propofol (10 M) produced similar alterations in 1 3 2L and 6 3 2L receptor currents when using a preapplication protocol; peak currents were not altered, desensitization was reduced, and deactivation was prolonged. Propofol enhanced peak currents for both 1 3 and 6 3 receptors, but the enhancement was greater for 1 3 receptors. Desensitization of these two isoforms was not modified by propofol. Propofol did not alter the deactivation rate of 1 3 receptor currents but did slow deactivation of 6 3 receptor currents. The findings that propofol reduced desensitization and prolonged deactivation of 2L subunit-containing receptors and enhanced peak currents or prolonged deactivation of subunitcontaining receptors suggest that propofol enhancement of both phasic and tonic inhibition may contribute to its anesthetic effect in the brain. GABAA receptors are ligand-gated pentameric chloride ion channels and mediate the majority of inhibition in the central nervous system. More than 16 different GABAA receptor subunit subtypes have been identified, including 1 through 6, 1 through 3, 1 through 3, , , , and (Olsen and Macdonald, 2002). McKernan and Whiting (1996) suggested that GABAA receptors may exist in vivo predominantly as and isoforms. The isoforms are mainly localized in GABAergic synapses, but isoforms were found on extraor perisynaptic membranes (Nusser et al., 1998; Wei et al., 2003), suggesting that receptors may mediate phasic inhibition and receptors may be involved in tonic inhibition (Bai et al., 2001; Stell et al., 2003). Recombinant receptors expressed in mammalian cells exhibited rapid desensitization (Haas and Macdonald, 1999; Bianchi and Macdonald, 2001; Scheller and Forman, 2002). However, 1 or 4 subunit-containing GABAA receptors had relatively less desensitization (Brown et al., 2002; Wohlfarth et al., 2002; Feng et al., 2004), although 6 subunit-containing receptors were more desensitizing (Bianchi et al., 2002). Several widely used general anesthetic drugs, including propofol (2,6-di-isopropylphenol), exert their effects in the central nervous system mainly by enhancing GABAA receptor currents (Olsen and Macdonald, 2002). Modulation of receptor current amplitudes by propofol has been substantially explored (Hill-Venning et al., 1997; Uchida et al., 1997; Lam and Reynolds, 1998; Pistis et al., 1999; Carlson et al., 2000; Davies et al., 2001; Krasowski et al., 2001; Williams and Akabas, 2002), but propofol effects on the kinetic properties of recombinant receptors are unclear. Although one study suggested that propofol slightly enhanced the function of 4 3 receptors (Brown et al., 2002), its effects on current kinetics of other isoforms are unknown. Therefore, modulation of propofol on recombinant and receptors was examined to explore the potential effects of propofol on phasic and tonic GABAergic inhibition. GABAA receptor 1 subunit mRNA is ubiquitously expressed in the brain, whereas 6 subunit mRNA is restrictively found in the cerebellum (Wisden et al., 1992). In addition, 6 subunits This work was supported by National Institutes of Health grant R01NS33300 (to R.L.M.). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.104.003426. ABBREVIATION: HEK, human embryonic kidney. 0026-895X/04/6606-1517–1524$20.00 MOLECULAR PHARMACOLOGY Vol. 66, No. 6 Copyright © 2004 The American Society for Pharmacology and Experimental Therapeutics 3426/108196 Mol Pharmacol 66:1517–1524, 2004 Printed in U.S.A. 1517 at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from preferably coassemble with subunits, and the 6 receptor is one of the predominant subunit-containing GABAA receptor isoforms in the brain (Poltl et al., 2003). It is of interest to determine whether propofol has different effects on 1 and 6 subunit-containing GABAA receptors. Therefore, modulation of recombinant 1 , 6 , 1 , and 6 receptors by propofol was examined to explore the potential subunitdependent effects of propofol. In the present study, we demonstrated subunit-specific propofol activation of 2L and subunit-containing GABAA receptors. Propofol evoked a greater maximal conductance change ( G) from 2L than from subunit-containing receptors. Propofol similarly decreased the desensitization and prolonged the deactivation of 1 3 2L and 6 3 2L receptors without affecting the peak current amplitudes. Although propofol modulation of GABAA receptor currents was relatively insensitive to the subunit subtype, subtypespecific effects of propofol were observed for receptors. Propofol produced a greater enhancement of peak current amplitudes for 1 3 than for 6 3 receptors and prolonged the deactivation of 6 3 receptor currents without altering deactivation of 1 3 receptor currents. Materials and Methods Expression of Recombinant GABAA Receptors in Human Embryonic Kidney Cells. HEK293T cells were grown in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) in an incubator at 37°C with 5% CO2 and 95% air. The cells were seeded at a density of 400,000/dish in 60-mm culture dishes (Corning Incorporated, Corning, NY) and transfected the following day with the combinations of cDNAs encoding rat 1, 6, 3, 2L, and GABAA receptor subunits (2 g of each subunit in different ternary combinations) along with 2 g of pHOOK (Invitrogen) using a modified calcium phosphate precipitation method (Fisher and Macdonald, 1997). The cells were incubated at 37°C for 4 h with 3% CO2 and then shocked for 30 s with 15% glycerol (Sigma-Aldrich, St. Louis, MO). The selection marker pHOOK encoded a cell surface antibody (sFv) that bound to the antigen (phOx) coated on the ferromagnetic beads (Invitrogen). The bead-bound transfected cells were separated from nontransfected cells using a magnetic stand (Greenfield et al., 1997). Electrophysiological recordings were obtained 24 h later. Eighty two percent of cells that bound beads also expressed GABAA receptors ( 80% for 1 3 2L receptors, 79% for 1 3 receptors, 86% for 6 3 2L receptors, and 87% for 6 3 receptors). Whole-Cell Recordings. Whole-cell macroscopic currents were recorded using the patch-clamp technique at room temperature. The recording electrodes were pulled from the thin-wall borosilicate glass tubing (i.d., 1.12 mm; o.d., 1.5 mm) (World Precision Instruments Inc., Sarasota, FL) on a P-2000 Quartz Micropipette Puller (Sutter Instrument Company, Novato, CA). The electrodes were fire-polished on an MF-830 Microforge (Narishige, Tokyo, Japan), and the resistances of the electrodes were 0.9 to 1.6 M when filled with an internal solution (see Chemicals, Solutions, and Drug Application for