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The Journal of Neuroscience, December 7, 2005, 25(49):11219-11230; doi:10.1523/JNEUROSCI.3751-05.2005

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Cellular/Molecular
Tandem Subunits Effectively Constrain GABA_A Receptor Stoichiometry and Recapitulate Receptor Kinetics But Are Insensitive to GABA_A Receptor-Associated Protein

Andrew J. Boileau,¹ Robert A. Pearce,² and Cynthia Czajkowski¹

Departments of ¹Physiology and ²Anesthesiology, University of Wisconsin-Madison, Madison, Wisconsin 53711

GABAergic synapses likely contain multiple GABA_A receptor subtypes, making postsynaptic currents difficult to dissect. However, even in heterologous expression systems, analysis of receptors composed of , , and subunits can be confounded by receptors expressed from and subunits alone. To produce recombinant GABA_A receptors containing fixed subunit stoichiometry, we coexpressed individual subunits with a "tandem" 1 subunit linked to a 2 subunit. Cotransfection of the 2 subunit with -tandem subunits in human embryonic kidney 293 cells produced currents that were similar in their macroscopic kinetics, single-channel amplitudes, and pharmacology to overexpression of the subunit with nonlinked 1 and 2 subunits. Similarly, expression of subunits together with -tandem subunits produced receptors having physiological and pharmacological characteristics that closely matched cotransfection of with subunits. In this first description of tandem GABA_A subunits measured with patch-clamp and rapid agonist application techniques, we conclude that incorporation of -tandem subunits can be used to fix stoichiometry and to establish the intrinsic kinetic properties of 12 and 122 receptors. We used this method to test whether the accessory protein GABA_A receptor-associated protein (GABARAP) alters GABA_A receptor properties directly or influences subunit composition. In recombinant receptors with fixed stoichiometry, coexpression of GABARAP-enhanced green fluorescent protein (EGFP) fusion protein had no effect on desensitization, deactivation, or diazepam potentiation of GABA-mediated currents. However, in 122S transfections in which stoichiometry was not fixed, GABARAP-EGFP altered desensitization, deactivation, and diazepam potentiation of GABA-mediated currents. The data suggest that GABARAP does not alter receptor kinetics directly but by facilitating surface expression of receptors.

Key words: GABA_A receptors; macroscopic kinetics; protein concatamers; tandem subunits; GABARAP; GABA_A receptor trafficking

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Received Sep 5, 2005; revised October 21, 2005; accepted October 21, 2005.

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