Elsevier

Neuropharmacology

Volume 44, Issue 8, June 2003, Pages 1003-1012
Neuropharmacology

Effects of γ2S subunit incorporation on GABAA receptor macroscopic kinetics

https://doi.org/10.1016/S0028-3908(03)00114-XGet rights and content

Abstract

GABAA receptors, the major inhibitory neurotransmitter receptors in the mammalian central nervous system, are heteropentameric proteins. We are interested in understanding the contribution of the γ subunit to the kinetic properties of GABAA receptors. Studies in Xenopus oocytes have suggested that co-expression of α1, β2, and γ2S subunits results in the formation of both αβ and αβγ receptors (Boileau et al., 2002a, Boileau et al., 1998). Here, we have used an excess of the γ2S subunit in transfections of HEK293 cells to bias expression toward αβγ-containing receptors. Using rapid application and whole cell patch clamp techniques, we found that incorporation of the γ subunit eliminated the rapid phases of desensitization and accelerated deactivation, consistent with a proposed role of desensitization in slowing deactivation. In addition, αβγ receptors had an increased GABA EC50, reduced sensitivity to block by Zn2+, and did not display outward rectification as compared to αβ receptors.

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.

References (55)

  • A. Keramidas et al.

    M2 pore mutations convert the glycine receptor channel from being anion- to cation-selective

    Biophysical Journal

    (2000)
  • K. Krampfl et al.

    Desensitization characteristics of rat recombinant GABA(A) receptors consisting of alpha1beta2gamma2S and alpha1beta2 subunits expressed in HEK293 cells

    Neurosci. Letters

    (2000)
  • P. Montpied et al.

    Multiple GABAA receptor alpha subunit mRNAs revealed by developmental and regional expression in rat, chicken and human brain

    FEBS Letters

    (1989)
  • G.B. Smith et al.

    Functional domains of GABAA receptors

    Trends in Pharmacological Sciences

    (1995)
  • S. Tia et al.

    Distinct deactivation and desensitization kinetics of recombinant GABAA receptors

    Neuropharmacology

    (1996)
  • T.A. Verdoorn et al.

    Functional properties of recombinant rat GABAA receptors depend upon subunit composition

    Neuron

    (1990)
  • T.P. Angelotti et al.

    Assembly of GABAA receptor subunits: α1β1 and α1β1 γ2S subunits produce unique ion channels with dissimilar single-channel properties

    Journal of Neuroscience

    (1993)
  • M.I. Banks et al.

    Kinetic differences between synaptic and extrasynaptic GABA(A) receptors in CA1 pyramidal cells

    Journal of Neuroscience

    (2000)
  • E.A. Barnard et al.

    International Union of Pharmacology. XV. Subtypes of gamma-aminobutyric acidA receptors: classification on the basis of subunit structure and receptor function

    Pharmacological Reviews

    (1998)
  • Baumann, S.W., Baur, R., Sigel, E., 2002. Forced subunit assembly in alpha 1beta 2gamma 2 GABAA receptors: insight into...
  • M.T. Bianchi et al.

    Structural determinants of fast desensitization and desensitization- deactivation coupling in GABAA receptors

    Journal of Neuroscience

    (2001)
  • M.T. Bianchi et al.

    Slow phases of GABA(A) receptor desensitization: structural determinants and possible relevance for synaptic function

    Journal of Physiology

    (2002)
  • A.J. Boileau et al.

    Identification of transduction elements for benzodiazepine modulation of the GABA(A) receptor: three residues are required for allosteric coupling

    Journal of Neuroscience

    (1999)
  • A.J. Boileau et al.

    Molecular dissection of benzodiazepine binding and allosteric coupling using chimeric gamma-aminobutyric acidA receptor subunits

    Molecular Pharmacology

    (1998)
  • Boileau, A.J., Pearce, R.A., Czajkowski, C., 2002b. Heterologous expression of GABA-A receptor subunits: What are we...
  • T.P. Bonnert et al.

    theta, a novel gamma-aminobutyric acid type A receptor subunit

    Proceedings of the National Academy of Science USA

    (1999)
  • Y. Chang et al.

    Stoichiometry of a recombinant GABAA receptor

    Journal of Neuroscience

    (1996)
  • Cited by (52)

    • Methylmercury induces an initial increase in GABA-evoked currents in Xenopus oocytes expressing α<inf>1</inf> and α<inf>6</inf> subunit-containing GABA<inf>A</inf> receptors

      2017, NeuroToxicology
      Citation Excerpt :

      The GABAA receptors expressed in oocytes in the present study were either α1β2γ2S or α6β2γ2S subtype, because the former is the most common subtype of GABAA receptor in the brain including the cerebellum, whereas the latter is found specifically in cerebellar granule cells (Benke et al., 1991, 1994; Laurie et al., 1992). Each oocyte received a 50 nl injection of a mixture of α1 or α6, β2 and γ2S in the proportion of 1:1:10 (Boileau et al., 2003). After cRNA injection, occytes were incubated in ND96 at 19 °C for at least 2 days before electrophysiological experiments.

    • Comparison of γ-aminobutyric acid, type A (GABA<inf>A</inf>), receptor αβγ and αβδ expression using flow cytometry and electrophysiology: Evidence for alternative subunit stoichiometries and arrangements

      2016, Journal of Biological Chemistry
      Citation Excerpt :

      To determine the subunit requirements for receptor surface trafficking, we transfected HEK293T cells with all possible combinations of α1, β2, γ2L, and δ subunit cDNAs (excluding conditions with γ2L and δ subunit co-transfection), labeled subunits with fluorescently conjugated antibodies, and evaluated cell surface fluorescence levels using flow cytometry. Although there has been ongoing debate in the GABAA receptor literature regarding what subunit cDNA ratios should be transfected in recombinant receptor studies (27–29), we chose to begin with equimolar ratios because this should approximate the relative gene dosage in vivo (α1, β2, γ2, and δ GABAA receptor subunit genes are autosomal and none has been shown to be imprinted). Because no commercially available antibodies against γ2 or δ subunits were found to be suitable for flow cytometry (secondary to excessive nonspecific binding), the HA epitope (YPYDVPDYA) was inserted near the N termini of γ2L and δ subunits (see under “Experimental Procedures”), and levels of these subunits were detected using a fluorescently conjugated anti-HA antibody.

    • Comparison of kinetic and pharmacological profiles of recombinant α <inf>1</inf> γ <inf>2L</inf> and α <inf>1</inf> β <inf>2</inf> γ <inf>2L</inf> GABA <inf>A</inf> receptors - A clue to the role of intersubunit interactions

      2016, European Journal of Pharmacology
      Citation Excerpt :

      There are, however, exceptions from this stoichiometry. A high agonist affinity GABAA receptor can be assembled from α and β subunits only (Fisher and Macdonald, 1997; Boileau et al., 2003) and these receptors were implicated in the tonic conductance (Mortensen and Smart, 2006). A high agonist sensitivity of αβ GABAA receptors is not surprising as GABA binding site is located at the α(−)/β(+) interface (Smith and Olsen, 1995).

    View all citing articles on Scopus
    View full text