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The Journal of Neuroscience, March 26, 2008, 28(13):3490-3499; doi:10.1523/JNEUROSCI.5727-07.2008

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Cellular/Molecular
Structural Mechanisms Underlying Benzodiazepine Modulation of the GABA_A Receptor

Susan M. Hanson and Cynthia Czajkowski

Department of Physiology, University of Wisconsin–Madison, Madison, Wisconsin 53711

Correspondence should be addressed to Cynthia Czajkowski, Department of Physiology, University of Wisconsin–Madison, 601 Science Drive, Madison, WI 53711. Email: czajkowski{at}physiology.wisc.edu

Many clinically important drugs target ligand-gated ion channels; however, the mechanisms by which these drugs modulate channel function remain elusive. Benzodiazepines (BZDs), anesthetics, and barbiturates exert their CNS actions by binding to GABA_A receptors and modulating their function. The structural mechanisms by which BZD binding is transduced to potentiation or inhibition of GABA-induced current (I_GABA) are essentially unknown. Here, we explored the role of the ₂Q182-R197 region (Loop F/9) in the modulation of I_GABA by positive (flurazepam, zolpidem) and negative [3-carbomethoxy-4-ethyl-6,7-dimethoxy-β-carboline (DMCM)] BZD ligands. Each residue was individually mutated to cysteine, coexpressed with wild-type ₁ and β₂ subunits in Xenopus oocytes, and analyzed using two-electrode voltage clamp. Individual mutations differentially affected BZD modulation of I_GABA. Mutations affecting positive modulation span the length of this region, whereas ₂W183C at the beginning of Loop F was the only mutation that adversely affected DMCM inhibition. Radioligand binding experiments demonstrate that mutations in this region do not alter BZD binding, indicating that the observed changes in modulation result from changes in BZD efficacy. Flurazepam and zolpidem significantly slowed covalent modification of ₂R197C, whereas DMCM, GABA, and the allosteric modulator pentobarbital had no effects, demonstrating that ₂Loop F is a specific transducer of positive BZD modulator binding. Therefore, ₂Loop F plays a key role in defining BZD efficacy and is part of the allosteric pathway allowing positive BZD modulator-induced structural changes at the BZD binding site to propagate through the protein to the channel domain.

Key words: GABA; GABA_A receptor; benzodiazepine; efficacy; allosteric modulation; Loop F; zolpidem

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Received Oct. 3, 2007; revised Jan. 31, 2008; accepted Feb. 20, 2008.

Correspondence should be addressed to Cynthia Czajkowski, Department of Physiology, University of Wisconsin–Madison, 601 Science Drive, Madison, WI 53711. Email: czajkowski{at}physiology.wisc.edu

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