GABAA receptor subunit γ2 and δ subtypes confer unique kinetic properties on recombinant GABAA receptor currents in mouse fibroblasts

GABAA receptors (GABARs) mediate the majority of fast inhibitory neurotransmission in the mammalian brain. Functional GABARs are ligand-gated chloride ion channels composed of five individual subunits. These subunits derive from six identified families, many with multiple subtypes (α1-6, β1-3, γ1-3, δ, e and π). When expressed in Xenopus oocytes or mammalian cells, different GABAR subunit combinations form receptors with unique pharmacological and biophysical properties (Macdonald & Olsen, 1994). These subunits do not assemble randomly, however, for while αβ subunit combinations readily express in mammalian cells, addition of a γ subunit drives expression of αβγ isoforms (Angelotti & Macdonald, 1993). The majority of native receptors are believed to be formed by combinations of αβγ and αβδ subunits (McKernan & Whiting, 1996), although the recently characterized e and π subunits may substitute for γ or δ subunits in some instances (Hedblom & Kirkness, 1997; Davies et al. 1997). In the rat, the γ2 subtype becomes the dominant γ subunit expressed at later developmental stages, and mRNA and membrane protein for this subtype are expressed in most brain regions. In contrast, the δ subunit is restricted only to a few cell populations in the postnatal rat that include thalamic relay neurons, cerebellar granule neurons and dentate granule neurons of the hippocampus (Laurie et al. 1992a,b; Wisden et al. 1992; Sperk et al. 1997). While the δ subunit has been shown to combine preferentially with the α6 subtype in cerebellar granule neurons (Jones et al. 1997), the GABAR subtypes that it combines with in dentate granule neurons remain unknown. The potential importance of hippocampal δ subunit-containing GABARs is underscored, however, by δ subunit knockout mice that exhibit spontaneous seizures (Olsen et al. 1997). For this investigation, we chose the α1β3γ2L and α1β3δ GABAR isoforms to determine the roles of γ and δ subunits in shaping GABAR currents. α1β3γ2L and α1β3δ GABAR whole-cell currents have been characterized previously in L929 fibroblasts, where incorporation of the δ subunit resulted in higher apparent GABA affinity, slower and less complete whole-cell current desensitization, and smaller whole-cell currents compared with receptors containing the γ2 subtype (Saxena & Macdonald, 1994). α1β3 currents also desensitized more rapidly than α1β3δ currents, although both faster (Fisher & Macdonald, 1997) and slower desensitization (Dominguez-Perrot et al. 1996) relative to α1β3γ2L currents have been reported. In addition, α1β3 single channels had a smaller main channel conductance level (13 pS), while α1β3γ2L and α1β3δ channels had a similar larger main conductance level (27 pS). The γ2L subtype, however, conferred a change on the open and closed properties of the receptor, leading to a tendency for longer duration openings and longer bursts of openings (Fisher & Macdonald, 1997). While suggesting major differences in channel gating and desensitization, these analyses did not resolve the rapid phases of activation, desensitization and deactivation of GABAR currents. These rapid kinetic properties are critical to understanding the potential synaptic roles of the α1β3γ2L and α1β3δ GABAR isoforms. In previous studies of native receptors, rapid application of GABA to outside-out membrane patches containing many GABARs reproduced the rapid activation and deactivation of IPSCs (Maconochie et al. 1994; Jones & Westbrook, 1995; Tia et al. 1996; Galaretta & Hestrin, 1997; Mellor & Randall, 1997, 1998). Also, with this rapid application protocol, it was demonstrated that GABAR desensitization was an important factor in shaping the deactivation time course of macropatch responses (Jones & Westbrook, 1995, 1996). In addition, macropatch deactivation kinetics were altered by allosteric modulators of GABARs such as benzodiazepines (Lavoie & Twyman, 1996; Mellor & Randall, 1997) and the anaesthetic propofol (Zhu & Vicini, 1997), as well as by intracellular phosphatase activity (Jones & Westbrook, 1997). Moreover, different recombinant GABAR isoforms displayed unique rapid kinetic properties (Verdoorn, 1994; Tia et al. 1996; Lavoie et al. 1997) that probably contribute to the diversity in GABAergic synaptic responses. In addition to predicting the synaptic behaviour of recombinant GABAR isoforms, rapid kinetic analysis of macroscopic currents may serve as a bridge between single-channel and whole-cell analysis, allowing for the development of more comprehensive kinetic models of GABAR behaviour that incorporate desensitization (Macdonald & Twyman, 1992). For this study, we implemented a GABA application system that allowed very rapid solution exchange (10–90 % rise time < 400 μs) during electrophysiological recordings from outside-out membrane patches containing multiple receptor channels. Using this technique, we determined the rapid activation, desensitization and deactivation kinetics of α1β3γ2L and α1β3δ GABAR currents and used these kinetic data in combination with steady-state single-channel analysis to develop comprehensive models of GABAR kinetic behaviour for these isoforms. In some instances, the α1β3 isoform was examined so that contributions of the γ and δ subunits could be more thoroughly assessed.