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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 GABAA Receptor Stoichiometry and Recapitulate Receptor Kinetics But Are Insensitive to GABAA 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 GABAA 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 GABAA 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 GABAA 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 1 2 and 1 2 2 receptors. We used this method to test whether the accessory protein GABAA receptor-associated protein (GABARAP) alters GABAA 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 1 2 2S 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: GABAA receptors; macroscopic kinetics; protein concatamers; tandem subunits; GABARAP; GABAA receptor trafficking
Received Sep 5, 2005;
revised October 21, 2005;
accepted October 21, 2005.
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