Enhanced Neurosteroid Potentiation of Ternary GABAA Receptors Containing the ␦ Subunit
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The Journal of Neuroscience, March 1, 2002, 22(5):1541–1549 Enhanced Neurosteroid Potentiation of Ternary GABAA Receptors Containing the ␦ Subunit Kai M. Wohlfarth,1 Matt T. Bianchi,2 and Robert L. Macdonald3,4,5 Department of 1Neurology and 2Neuroscience Graduate Program, University of Michigan, Ann Arbor, Michigan 48104- 1687, and Departments of 3Neurology, 4Molecular Physiology and Biophysics, and 5Pharmacology, Vanderbilt University, Nashville, Tennessee 37212 Attenuated behavioral sensitivity to neurosteroids has been effect on the rate or extent of apparent desensitization. Thus the ␦ reported for mice deficient in the GABAA receptor subunit. We polarity of THDOC modulation depended on GABA concentra- ␣  ␥ therefore investigated potential subunit-specific neurosteroid tion for 1 3 2L GABAA receptors. However, the same proto- ␣  ␦ pharmacology of the following GABAA receptor isoforms in a col applied to 1 3 receptors resulted in peak current en- transient expression system: ␣13␥2L, ␣13␦, ␣63␥2L, and hancement by THDOC of Ͼ800% and prolonged deactivation. ␣  ␦ 6 3 . Potentiation of submaximal GABAA receptor currents by Interestingly, THDOC induced pronounced desensitization in the neurosteroid tetrahydrodeoxycorticosterone (THDOC) was the minimally desensitizing ␣13␦ receptors. Single channel greatest for the ␣13␦ isoform. Whole-cell GABA concentra- recordings obtained from ␣13␦ receptors indicated that tion–response curves performed with and without low concen- THDOC increased the channel opening duration, including the trations (30 nM) of THDOC revealed enhanced peak GABAA introduction of an additional longer duration open state. Our ␦ receptor currents for isoforms tested without affecting the results suggest that the GABAA receptor subunit confers ␣  ␦ Ͼ GABA EC50. 1 3 currents were enhanced the most ( 150%), increased sensitivity to neurosteroid modulation and that the ␣  ␦ whereas the other isoform currents were enhanced 15–50%. At intrinsic gating and desensitization kinetics of 1 3 GABAA a higher concentration (1 M), THDOC decreased peak receptors are altered by THDOC. ␣  ␥ ␦ 1 3 2L receptor current amplitude evoked by GABA (1 mM) Key words: GABAA receptor; subunit; neurosteroid; desen- concentration jumps and prolonged deactivation but had little sitization; single channel; gating Fast synaptic inhibition in the mammalian CNS is mediated steroids can directly activate GABAA receptor channels (Lam- mainly by activation of GABAA receptor channels (Macdonald bert et al., 1995). and Olsen, 1994; Whiting et al., 1995). GABAA receptor function The GABAA receptor is a pentameric structure formed by the is modulated by various clinically important drugs that act on coassembly of subunit polypeptides from a large multigene family allosteric modulatory sites (Macdonald and Olsen, 1994; Sieghart, (McKernan and Whiting, 1996; Barnard et al., 1998) that are 1995). For example, neurosteroids, which represent a class of differentially expressed both temporally and spatially throughout molecules that are synthesized in the nervous system, have been the brain (Zheng et al., 1993, 1995). This heterogeneous expres- demonstrated to have anxiolytic, hypnotic, anesthetic, and anti- sion confers specific physiological and pharmacological properties convulsant effects (Baulieu and Robel, 1990; Paul and Purdy, of GABAA receptors (Sigel et al., 1990; Mathews et al., 1994). For 1992; Macdonald and Olsen, 1994; Lambert et al., 1995) and may example, it has been demonstrated that the presence of ␣ and ␥ be involved in memory enhancement, behavioral actions, and subunits can affect neurosteroid modulation. The ␣ subunit sub- neuroprotection (Frye, 1995; Green et al., 2000; Yoo et al., 1996). type was found to influence efficacy, whereas the ␥ subunit Several studies have shown that neurosteroids bind to GABA A subtype influenced both efficacy and EC50 for neurosteroid inter- receptors at sites different from GABA, benzodiazepines, and action with GABAA receptors (Gee and Lan, 1991; Lan et al., barbiturates (Gee et al., 1988; Turner et al., 1989) and can act as 1991; Sapp et al., 1992). Also, Zhu et al. (1996) reported that the positive or negative modulators of receptor function (Majewska presence of ␦ subunits inhibited neurosteroid modulation but not et al., 1986; Gee et al., 1988; Puia et al., 1990; Gee and Lan, 1991; direct activation, of GABAA receptors. However, a recent study Park-Chung et al., 1999). Neurosteroid enhancement of submaxi- (Mihalek et al., 1999) demonstrated that mice lacking the mal GABAA receptor currents occurs through increases in both ␦ GABAA receptor subunit had attenuated behavioral responses channel open frequency and open duration (Puia et al., 1990; to systemic neurosteroid administration. This suggested an im- Twyman and Macdonald, 1992). At high concentrations, neuro- portant role for the ␦ subunit either in the neurosteroid modula- tion of GABAA receptor currents or in the neural circuits rele- vant to the behavioral effects of neurosteroids. Approximately Received Oct. 26, 2001; revised Nov. 29, 2001; accepted Dec. 6, 2001. ␦ 30% of cerebellar GABAA receptors are thought to contain the This work was supported by National Institutes of Health Grant R01-NS33300 ␦ (R.L.M.), the Deutsche Forschungsgemeinschaft WO 770/1-1 (K.M.W.), and a subunit. mRNA is also found in the hippocampus and thalamus National Institute on Drug Abuse training fellowship T32-DA07281-03 (M.T.B.). (Benke et al., 1991; Laurie et al., 1992a,b; McKernan and Correspondence should be addressed to Dr. Robert L. Macdonald, Department of Whiting, 1996). Neurology, Vanderbilt University, 2100 Pierce Avenue, Nashville, TN 37212. E- mail: Robert.Macdonald@mcmail.vanderbilt.edu. We used whole-cell and single-channel patch-clamp recordings Copyright © 2002 Society for Neuroscience 0270-6474/02/221541-09$15.00/0 and applied GABA using an ultra fast application system to • 1542 J. Neurosci., March 1, 2002, 22(5):1541–1549 Wohlfarth et al. Neurosteroid Modulation of GABAA Receptors investigate neurosteroid allosteric modulation of GABAA recep- tiple channels, were observed in most patches, they would not affect the tor currents in mammalian cells transiently transfected with re- open duration histograms. Open duration histograms were generated ␣ ␣  and fitted using Interval5 software (Dr. Barry S. Pallotta, University of combinant GABAA receptors containing 1or 6 with 3 and ␥ ␦ North Carolina, Chapel Hill, NC). The number of exponential functions 2L or subunits. required to describe the data was determined by a log-likelihood method (additional components were accepted if they significantly improved the MATERIALS AND METHODS fit). Events with durations Ͻ150 sec (1.5 times the system dead time) were shown in the plots but were not considered by the fitting routine. Expression of recombinant GABAA receptors. The cDNAs encoding rat ␣ ␣  ␦ ␥ Additional data reduction and filtering were implemented for figure 1, 6, 3, , and 2L GABAA receptor subunit subtypes were individ- display purposes only. Numerical data were expressed as mean Ϯ SEM. ually subcloned into the plasmid expression vector pCMVNeo. All Statistical significance, using Student’s t test (two-tailed, paired, or un- subunits have been sequenced and are identical to published sequences. paired as appropriate) was taken as p Ͻ 0.05. Human embryonic kidney cells (HEK293T; a gift from P. Connely, COR Therapeutics, San Francisco, CA) were maintained in DMEM, supple- mented with 10% fetal bovine serum, at 37°Cin5%CO2/95% air. Cells RESULTS were transfected with 4 g of each subunit plasmid along with 1–2 gof pHOOK (Invitrogen, Carlsbad, CA) for immunomagnetic bead separa- Direct activation by THDOC depended on tion (Greenfield et al., 1997), using a modified calcium phosphate copre- subunit composition cipitation technique as described previously (Angelotti et al., 1993). The next day, cells were replated, and recordings were made 18–30 hr later. All constructs produced THDOC-sensitive currents in Electrophysiology and drug application. Patch-clamp recordings were HEK293T cells. Cells were voltage clamped at Ϫ65 mV, and performed on transfected fibroblasts bathed in an external solution whole-cell currents were recorded in response to increasing consisting of (in mM): NaCl 142, KCl 8, MgCl2 6, CaCl2 1, HEPES 10, concentrations of THDOC (Fig. 1A, C). The GABA receptor glucose 10, pH 7.4, 325 mOsm). Electrodes were formed from soda lime A (whole cell), thin-walled borosilicate (whole cell), or thick-walled boro- isoforms exhibited different THDOC sensitivities (EC50 val- silicate (excised patch) glass (World Precision Instruments, Pittsburgh, ues) (Fig. 1C). Although there is little mechanistic information PA) with a Flaming Brown electrode puller (Sutter Instrument Co., San in this analysis, it is necessary to describe direct activation so Rafael, CA). Electrodes had resistances of 0.8–8.0 M⍀ when filled with that appropriate concentrations can be chosen for subsequent an internal solution consisting of (in mM): KCl 153, MgCl2 1, MgATP 2, modulation experiments (see below). Additionally, we ob- HEPES 10, EGTA 5, pH 7.3, 300 mOsm. Lower resistance electrodes were used for experiments in which cells were lifted from the recording served a subunit and subtype dependence of the direct effects ␣ dish (see Fig. 5). Higher resistance electrodes were used for single- of THDOC. 6 subtype-containing receptors had lower EC50 channel recordings and were coated with Q-dope. The combination of values for THDOC