Zn<sup>2+</sup> inhibition of recombinant GABA<sub>A</sub> receptors: an allosteric, state‐dependent mechanism determined by the γ‐subunit

Gingrich, Kevin J.; Burkat, Paul M.

doi:10.1111/j.1469-7793.1998.609bv.x

Cited by 47 publications

(34 citation statements)

References 43 publications

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“…In agreement with previous results (Draguhn et al, 1990;Gingrich and Burkat, 1998), currents mediated by the wild-type ␣ 1 ␤ 3 ␥ 2 receptors were only weakly reduced (86 Ϯ 4% of control; n ϭ 6; total of four transfections), whereas currents mediated by ␣ 1 ␤ 3 receptor were reduced to 7 Ϯ 3% (n ϭ 6; total of three transfections) (Fig. 5F ) in the presence of Zn 2ϩ .…”

Section: Amino Acid Sequence ␣ 1 (80-100) Mediates Binding To ␥ 2 Subsupporting

confidence: 80%

Alternate Use of Distinct Intersubunit Contacts Controls GABA_AReceptor Assembly and Stoichiometry

et al. 2001

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GABA A receptors are the major inhibitory transmitter receptors in the CNS. Recombinant GABA A receptors composed of ␣ 1 ␤ 3 ␥ 2 subunits have been demonstrated to assemble as pentamers consisting of two ␣ 1 , two ␤ 3 , and one ␥ 2 subunit. Using truncated and chimeric ␣ 1 subunits, we identified the ␣ 1 (80-100) sequence as a major binding site for ␥ 2 subunits. In addition, we demonstrated its direct interaction with ␥ 2 (91-104), a sequence that previously has been identified to form the contact to ␣ 1 subunits. The observation that the amino acid residues ␣ 1 P96 and ␣ 1 H101, which can be photolabeled by [ 3 H]flunitrazepam, are located within or adjacent to the ␣ 1 (80-100) sequence, indicates that the benzodiazepine binding site of GABA A receptors is located close to this intersubunit contact. The observation that ␣ 1 (80-100) interacts with ␥ 2 but not with ␤ 3 subunits indicates the existence of an additional ␤ 3 binding site on ␣ 1 subunits. The preferred alternate use of the ␥ 2 and ␤ 3 binding sites in two different ␣ 1 subunits of the same receptor ensures the incorporation of only a single ␥ 2 subunit and thus, determines subunit stoichiometry of ␣ 1 ␤ 3 ␥ 2 receptors. Distinct binding sites and their alternate use can therefore explain how subunits of hetero-oligomeric transmembrane proteins assemble into a defined protein complex.

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Section: Amino Acid Sequence ␣ 1 (80-100) Mediates Binding To ␥ 2 Subsupporting

confidence: 80%

Alternate Use of Distinct Intersubunit Contacts Controls GABA_AReceptor Assembly and Stoichiometry

et al. 2001

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“…To test this possibility, we measured the Zn 2ϩ sensitivity of ␣ 1 ␤ 2 ␥ 2 W183C and ␣ 1 ␤ 2 ␥ 2 R197C receptors. GABA A Rs composed of ␣ 1 ␤ 2 subunits are more sensitive to Zn 2ϩ blockade than ␣ 1 ␤ 2 ␥ 2 receptors; thus, Zn 2ϩ sensitivity of I GABA can be used to assess ␥ 2 subunit expression (Draguhn et al, 1990;Gingrich and Burkat, 1998). When 30 M ZnCl 2 was coapplied with 1 mM GABA, I GABA was reduced by 84 Ϯ 5% in ␣ 1 ␤ 2 receptors but only by 6 Ϯ 2% in ␣ 1 ␤ 2 ␥ 2 receptors (Fig.…”

Section: Resultsmentioning

confidence: 99%

Structural Mechanisms Underlying Benzodiazepine Modulation of the GABA_AReceptor

Hanson¹,

Czajkowski²

2008

J. Neurosci.

106

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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 GABAinduced current (I GABA ) are essentially unknown. Here, we explored the role of the ␥ 2 Q182-R197 region (Loop F/9) in the modulation of I GABA by positive (flurazepam, zolpidem) and negative [3-carbomethoxy-4-ethyl-6,7-dimethoxy-␤-carboline (DMCM)] BZD ligands. Each residue was individually mutated to cysteine, coexpressed with wild-type ␣ 1 and ␤ 2 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 ␥ 2 W183C 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 ␥ 2 R197C, whereas DMCM, GABA, and the allosteric modulator pentobarbital had no effects, demonstrating that ␥ 2 Loop F is a specific transducer of positive BZD modulator binding. Therefore, ␥ 2 Loop 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.

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“…Cells were exposed to 1 mM GABA, approximating the peak GABA concentration at a synapse, for 400 msec. Previous studies have shown that neurons with endogenous GABA A receptors (Mellor and Randall, 1997;Zhu and Vicini, 1997) as well as cells expressing recombinant GABA A receptors (Gingrich and Burkat, 1998;Haas and Macdonald, 1999;Bianchi and Macdonald, 2002;Scheller and Forman, 2002) with ␣, ␤, and ␥ subunits have fast (Ͻ20 msec), intermediate (ϳ200 msec), slow (ϳ 1000 msec), and ultraslow (ϳ10,000 msec) components of desensitization when GABA is applied for several seconds. With the briefer (400 msec) GABA applications used in this study, only the fast and intermediate phases of desensitization could be resolved.…”

Section: Resultsmentioning

confidence: 99%

The Juvenile Myoclonic Epilepsy GABA_AReceptor α1 Subunit Mutation A322D Produces Asymmetrical, Subunit Position-Dependent Reduction of Heterozygous Receptor Currents and α1 Subunit Protein Expression

Gallagher¹,

Song²,

Arain³

et al. 2004

J. Neurosci.

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Individuals with autosomal dominant juvenile myoclonic epilepsy are heterozygous for a GABA A receptor ␣1 subunit mutation (␣1A322D). GABA A receptor ␣␤␥ subunits are arranged around the pore in a ␤-␣-␤-␣-␥ sequence (counterclockwise from the synaptic cleft). Therefore, each ␣1 subunit has different adjacent subunits, and heterozygous expression of ␣1(A322D), ␤, and ␥ subunits could produce receptors with four different subunit arrangements:Expression of a 1:1 mixture of wild-type and ␣1(A322D) subunits with ␤2S and ␥2S subunits (heterozygous transfection) produced smaller currents than wild type and much larger currents than homozygous mutant transfections. Western blot and biotinylation assays demonstrated that the amount of total and surface ␣1 subunit from heterozygous transfections was also intermediate between those of wild-type and homozygous mutant transfections. ␣1(A322D) mutations were then made in covalently tethered triplet (␥2S-␤2S-␣1) and tandem (␤2S-␣1) concatamers to target selectively ␣1(A322D) to each of the asymmetric ␣1 subunits. Coexpression of mutant and wild-type concatamers resulted in expression of either Het ␤␣␤ or Het ␤␣␥ receptors. Het ␤␣␤ currents were smaller than wild type and much larger than Het ␤␣␥ and homozygous currents. Furthermore, Het ␤␣␤ transfections contained less ␤-␣ concatamer than wild type but more than both Het ␤␣␥ and homozygous mutant transfections. Thus, whole-cell currents and protein expression of heterozygous ␣1(A322D)␤2S␥2S receptors depended on the position of the mutant ␣1 subunit, and GABA A receptor currents in heterozygous individuals likely result primarily from wild-type and Het ␤␣␤ receptors with little contribution from Het ␤␣␥ and homozygous receptors.

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Zn²⁺ inhibition of recombinant GABA_A receptors: an allosteric, state‐dependent mechanism determined by the γ‐subunit

Cited by 47 publications

References 43 publications

Alternate Use of Distinct Intersubunit Contacts Controls GABA_AReceptor Assembly and Stoichiometry

Alternate Use of Distinct Intersubunit Contacts Controls GABA_AReceptor Assembly and Stoichiometry

Structural Mechanisms Underlying Benzodiazepine Modulation of the GABA_AReceptor

The Juvenile Myoclonic Epilepsy GABA_AReceptor α1 Subunit Mutation A322D Produces Asymmetrical, Subunit Position-Dependent Reduction of Heterozygous Receptor Currents and α1 Subunit Protein Expression

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Zn2+ inhibition of recombinant GABAA receptors: an allosteric, state‐dependent mechanism determined by the γ‐subunit

Cited by 47 publications

References 43 publications

Alternate Use of Distinct Intersubunit Contacts Controls GABAAReceptor Assembly and Stoichiometry

Alternate Use of Distinct Intersubunit Contacts Controls GABAAReceptor Assembly and Stoichiometry

Structural Mechanisms Underlying Benzodiazepine Modulation of the GABAAReceptor

The Juvenile Myoclonic Epilepsy GABAAReceptor α1 Subunit Mutation A322D Produces Asymmetrical, Subunit Position-Dependent Reduction of Heterozygous Receptor Currents and α1 Subunit Protein Expression

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Product

Resources

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Zn²⁺ inhibition of recombinant GABA_A receptors: an allosteric, state‐dependent mechanism determined by the γ‐subunit

Alternate Use of Distinct Intersubunit Contacts Controls GABA_AReceptor Assembly and Stoichiometry

Alternate Use of Distinct Intersubunit Contacts Controls GABA_AReceptor Assembly and Stoichiometry

Structural Mechanisms Underlying Benzodiazepine Modulation of the GABA_AReceptor

The Juvenile Myoclonic Epilepsy GABA_AReceptor α1 Subunit Mutation A322D Produces Asymmetrical, Subunit Position-Dependent Reduction of Heterozygous Receptor Currents and α1 Subunit Protein Expression