Effects of γ2S subunit incorporation on GABAA receptor macroscopic kinetics
Introduction
GABAA receptors are the major type of inhibitory neurotransmitter receptor in the mammalian central nervous system. They are composed of five subunits, arranged around a central pore that conducts chloride ions upon agonist binding (Nayeem et al., 1994). Several distinct GABAA subunits have been identified in recent years grouped according to sequence homology into several different subunit subtypes, including α1–6, β1–3, γ1–3, δ, ϵ, π and θ (Barnard et al., 1998, Bonnert et al., 1999). The composition of GABAA receptors varies with development (Montpied et al., 1989), and localization in the CNS (Sieghart and Sperk, 2002), but the most common receptors in vivo are likely composed of α1, β2, and γ2 subunits (Benke et al., 1994, Benke et al., 1991, Laurie et al., 1992, Stephenson, 1995).
Because subunit composition is a major factor that determines the pharmacological and physiological characteristics of GABAA receptors (Angelotti and Macdonald, 1993, Fisher and Macdonald, 1997a, Fisher and Macdonald, 1997b, Gingrich et al., 1995, Haas and Macdonald, 1999, Sigel and Baur, 2000), and thus of inhibitory synapses, understanding the influence of particular subunits on receptor properties is of considerable interest. Usually this is approached by studying recombinant receptors that have been expressed in heterologous expression systems such as Xenopus oocytes or HEK 293 cells, with combinations of different subunits. However, even when comparing the same subunit combinations, substantial kinetic and pharmacological heterogeneity can be observed in such experiments (Ebert et al., 1996, Li and Pearce, 2000, Boileau et al., 2002a). Comparisons between these studies are sometimes complicated by possible difference in the source of the cDNA, the cell type or strain used for expression, the speed of solution exchange, and even the use of different flow apparatus techniques and materials (Flood and Coates, 2000, Papke et al., 1994).
Another possible explanation for the variability is that mixtures of receptors may exist in individual cells. For example, when expressing combinations of α, β and γ subunits, some receptors may contain α, β and γ subunits, and others only α and β subunits. Previous work in our laboratory and others demonstrated that in Xenopus oocytes, over-expression of the γ2S subunit is required to elicit maximal potentiation of GABA-mediated currents by BDZs (Boileau et al., 2002a, Boileau and Czajkowski, 1999, Boileau et al., 1998). Presumably this is due to the presence of mixtures of αβ and αβγ receptors when α, β and γ subunits are expressed in a 1:1:1 ratio (Boileau et al., 2002a, Ebert et al., 1994, Tretter et al., 1997). Here, we characterize the properties of αβ and αβγ receptors with rapid agonist application applied to HEK 293 cells, using variable over-expression of γ subunit cDNA in transient transfection with α1, β2, and γ2s subunits.
Section snippets
Cell culture and DNA transfection
HEK293 cells were transiently transfected with cDNAs of GABAA receptor subunits. Cells (American Type Culture Collection CRL 1573) were maintained in standard culture conditions (37 °C, 5% CO2), in culture medium consisting of minimal essential medium with Earle’s salts (Invitrogen, Carlsbad, CA) and 10% fetal bovine serum (Harlan Bioproducts for Sciences, Indianapolis, IN). Cells were plated in 60 mm culture dishes 24–72 h before transient transfection. cDNAs for rat GABAA receptor subunits α1
Results
To test whether γ subunit over-expression influences kinetic properties of receptors expressed in a mammalian expression system, we used rapid drug application techniques to examine cells that differed in the ratio of the γ2S subunit to α1 and β2 subunits expressed (Fig. 1). When 20 s pulses of a high concentration (10 mM) of GABA were applied to whole cells transfected with αβ (1:1), αβγ (1:1:1), αβγ (1:1:7) or αβγ (1:1:10) ratios, cells transfected with a 1:1:1 ratio exhibited variable
Discussion
Previous studies of recombinant expressed receptors have shown that subunit composition influences a number of pharmacologic and physiologic characteristics of GABAA receptors (Angelotti and Macdonald, 1993, Fisher and Macdonald, 1997a, Fisher and Macdonald, 1997b, Gingrich et al., 1995, Haas and Macdonald, 1999, Sigel and Baur, 2000). For heteromultimeric receptors, combinations of subunits are generally co-expressed using equal amounts of cDNA or RNA that codes for each subunit. However, in
Acknowledgements
We thank Dr. Mathew Jones for useful discussions and suggestions, and Abdalla Saad and José Luis Mercado for experimental assistance. Funded in part by NIH grants NS34727 to C.C., MH66406 to A.B. and C.C., GM55719 to R.P. and German Research Society grant BE2380/1-1 to C.B.
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