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J. Biol. Chem., Vol. 279, Issue 42, 43654-43660, October 15, 2004

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Analysis of the Presence and Abundance of GABA_A Receptors Containing Two Different Types of Subunits in Murine Brain Using Point-mutated Subunits^*

Dietmar Benke, Panagiotis Fakitsas, Christian Roggenmoser, Claudia Michel, Uwe Rudolph, and Hanns Mohler¶

From the Institute of Pharmacology and Toxicology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland and the ^¶Department of Chemistry and Applied Biosciences, Federal Institute of Technology, Winterthurerstrasse 190, 8057 Zürich, Switzerland

-Aminobutyric acid, type A (GABA_A) receptors are pentameric proteins of which the majority is composed of two subunits, two subunits and one subunit. It is well documented that two different types of subunits can exist in a singles GABA_A receptor complex. However, information on the abundance of such GABA_A receptors is rather limited. Here we tested whether mice containing the His to Arg point mutation in the 1, 2, or 3 subunit at positions 101, 101, and 126, respectively, which render the respective subunits insensitive to diazepam, would be suitable to analyze this issue. Immunodepletion studies indicated that the His to Arg point mutation solely rendered those GABA_A receptors totally insensitive to diazepam binding that contain two mutated subunits in the receptor complex, whereas receptors containing one mutated and one heterologous subunit not carrying the mutation remained sensitive to diazepam binding. This feature permitted a quantitative analysis of native GABA_A receptors containing heterologous subunits by comparing the diazepam-insensitive binding sites in mutant mouse lines containing one mutated subunit with those present in mouse lines containing two different mutated subunits. The data indicate that the 11-containing receptors with 61% is the most abundant receptor subtype in brain, whereas the 12 (13%), 13 (15%), 22 (12%), 23 (2%), and 33 combinations (4%) are considerably less expressed. Only within the 1-containing receptor population does the combination of equal subunits (84% 11, 7% 12, and 8% 13) prevail, whereas in the 2-containing receptor population (46% 22, 36% 21, and 19% 23) and particularly in the 3-containing receptor population (27% 33, 56% 31, and 19% 32), the receptors with two different types of subunits predominate. This experimental approach provides the basis for a detailed analysis of the abundance of GABA_A receptors containing heterologous subunits on a brain regional level.

Received for publication, June 25, 2004 , and in revised form, August 9, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Institute of Pharmacology and Toxicology, University Zürich, Winterthurerstrasse.190, 8057 Zürich. Tel.: 41-1-635-5930; Fax: 41-1-635-6874; E-mail: benke{at}pharma.unizh.ch.

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