Comparative modeling of a GABAA alpha1 receptor using three crystal structures as templates

Trudell, James R.; Bertaccini, Edward

doi:10.1016/j.jmgm.2004.03.004

Cited by 41 publications

(46 citation statements)

References 71 publications

(207 reference statements)

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“…The previously identified areas of the distal M2 domain and the M2-M3 loop are adjacent and continuous, suggesting that they form a single transduction domain (Bera et al, 2002;Miyazawa et al, 2003;Trudell and Bertaccini, 2004). Structurally, previous studies indicate that, although hydropathy analysis indicates that M2 ends as indicated in our study, sequence analysis and structural modeling indicate that the M2 ␣-helix extends further, to at least ␥P288.…”

Section: M2-m3 Loop Residues Relevant For the Coupling Of Benzodiazepsupporting

confidence: 64%

“…Given that it is physically connected to both the N terminus and the M2 gate region, M1 seemed a likely candidate for the transduction of binding to gating. Additional support comes from a GABA A receptor model (Trudell and Bertaccini, 2004), indicating that the N-terminal region of M1 before the conserved P243 lines the receptor pore by intercalating between M2 channel-lining domains. The ␥-␦ M1q chimera, which extends the N-terminal ␥2L subunit sequence through the extracellular end of M1 in a ␣1␤3␦ receptor, exhibited robust diazepam potentiation of GABA-evoked currents, in contrast to the nonresponsive ␥-␦ M1e chimera, which contained only the ␥2L subunit N-terminal binding domain.…”

Section: Benzodiazepine Transduction Mediated By Domainmentioning

confidence: 99%

“…Recently, the M2-M3 loop has been implicated in LGIC gating [nAChR (Bera et al, 2002); GABA A receptor (O'Shea and Harrison, 2000;Kash et al, 2003Kash et al, , 2004Trudell and Bertaccini, 2004); glycine receptor (Lynch et al, 2001)]. The N-terminal portion of the M2-M3 loop in GABA A and glycine receptors has been shown to undergo a conformational change during gating [GABA (Bera et al, 2002); glycine (Lynch et al, 1997)], suggesting that it may interact with other receptor areas.…”

Section: Benzodiazepine Transduction Mediated By Domainmentioning

confidence: 99%

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Structural Determinants of Benzodiazepine Allosteric Regulation of GABA_AReceptor Currents

Jones-Davis¹,

Song²,

Gallagher³

et al. 2005

J. Neurosci.

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Benzodiazepine enhancement of GABA A receptor current requires a ␥ subunit, and replacement of the ␥ subunit by the ␦ subunit abolishes benzodiazepine enhancement. Although it has been demonstrated that benzodiazepines bind to GABA A receptors at the junction between ␣ and ␥ subunits, the structural basis for the coupling of benzodiazepine binding to allosteric enhancement of the GABA A receptor current is unclear. To determine the structural basis for this coupling, the present study used a chimera strategy, using ␥2L-␦ GABA A receptor subunit chimeras coexpressed with ␣1 and ␤3 subunits in human embryonic kidney 293T cells. Different domains of the ␥2L subunit were replaced by ␦ subunit sequence, and diazepam sensitivity was determined. Chimeric subunits revealed two areas of interest: domain 1 in transmembrane domain 1 (M1) and domain 2 in the C-terminal portion of transmembrane domain 2 (M2) and the M2-M3 extracellular loop. In those domains, site-directed mutagenesis demonstrated that the following two groups of residues were involved in benzodiazepine transduction of current enhancement: residues Y235, F236, T237 in M1; and S280, T281, I282 in M2 as well as the entire M2-M3 loop. These results suggest that a pocket of residues may transduce benzodiazepine binding to increased gating. Benzodiazepine transduction involves a group of residues that connects the N terminus and M1, and another group of residues that may facilitate an interaction between the N terminus and the M2 and M2-M3 loop domains.

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Section: M2-m3 Loop Residues Relevant For the Coupling Of Benzodiazepsupporting

confidence: 64%

Section: Benzodiazepine Transduction Mediated By Domainmentioning

confidence: 99%

Section: Benzodiazepine Transduction Mediated By Domainmentioning

confidence: 99%

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Structural Determinants of Benzodiazepine Allosteric Regulation of GABA_AReceptor Currents

Jones-Davis¹,

Song²,

Gallagher³

et al. 2005

J. Neurosci.

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“…2D). First, K i was calculated from the half-maximum concentration IC 50 as determined from fitting to Equation 12 and from the Cheng-Prusoff equation (58), Equation 13,…”

Section: ␤2m1mentioning

confidence: 99%

Extracellular Domain Nicotinic Acetylcholine Receptors Formed by α4 and β2 Subunits

Person

Bills

Liu

et al. 2005

Journal of Biological Chemistry

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Models of the extracellular ligand-binding domain of nicotinic acetylcholine receptors (nAChRs), which are pentameric integral membrane proteins, are attractive for structural studies because they potentially are water-soluble and better candidates for x-ray crystallography and because their smaller size is more amenable for NMR spectroscopy. The complete N-terminal extracellular domain is a promising foundation for such models, based on previous studies of ␣7 and muscle-type subunits. Specific design requirements leading to high structural fidelity between extracellular domain nAChRs and full-length nAChRs, however, are not well understood. To study these requirements in heteromeric nAChRs, the extracellular domains of ␣4 and ␤2 subunits with or without the first transmembrane domain (M1) were expressed in Xenopus oocytes and compared with ␣4␤2 nAChRs based on ligand binding and subunit assembly properties. Ligand affinities of detergent-solubilized, extracellular domain ␣4␤2 nAChRs formed from subunits with M1 were nearly identical to affinities of ␣4␤2 nAChRs when measured with [ 3 H]epibatidine, cytisine, nicotine, and acetylcholine. Velocity sedimentation suggested that these extracellular domain nAChRs predominantly formed pentamers. The yield of these extracellular domain nAChRs was about half the yield of ␣4␤2 nAChRs. In contrast, [3 H]epibatidine binding was not detected from the extracellular domain ␣4 and ␤2 subunits without M1, implying no detectable expression of extracellular domain nAChRs from these subunits. These results suggest that M1 domains on both ␣4 and ␤2 play an important role for efficient expression of extracellular domain ␣4␤2 nAChRs that are high fidelity structural models of full-length ␣4␤2 nAChRs. Nicotinic acetylcholine receptors (nAChRs)2 are ligand-gated ion channels expressed mainly in the nervous system and at the neuromuscular junction (1-3), although they also are expressed elsewhere (4). They are members of the superfamily of nicotinoid receptors, which includes ␥-aminobutyric acid (GABA) type A and C, glycine, and serotonin 5-HT3 receptors. They contain five homologous subunits, with subunits designated ␣1-␣10, ␤1-␤4, ␦, ␥, and ⑀. Each subunit is an integral membrane protein with a large, ligand-binding N-terminal extracellular domain, four transmembrane domains (M1-M4), and a cytoplasmic loop between M3 and M4. The study of structure and dynamics of nAChRs has been motivated by their roles in normal and pathologic neurophysiology, including roles in addiction, neurodegeneration, epilepsy, myasthenia gravis, and congenital myasthenic syndromes (5).Knowledge of the structure of nAChRs has come from biochemical, electrophysiological, and imaging methods (6 -8). These methods, however, do not provide comprehensive structure at atomic resolution. A significant advance toward such information has come recently from the crystallographic structure of acetylcholine-binding protein (AChBP), a water-soluble pentamer that has been the basis for homology modeling of the extracellular...

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“…Recently, GABA A receptor homology models (Cromer et al, 2002;Trudell and Bertaccini, 2004;Ernst et al, 2005) were constructed based on the acetylcholine binding protein and Torpedo ACh receptor structures (Brejc et al, 2001;Miyazawa et al, 2003;Celie et al, 2004;Unwin, 2005). Homology models depend on the correct alignment of subunit sequences from different superfamily members.…”

Section: Introductionmentioning

confidence: 99%

State-Dependent Cross-Linking of the M2 and M3 Segments: Functional Basis for the Alignment of GABA_Aand Acetylcholine Receptor M3 Segments

Jansen¹,

Akabas²

2006

J. Neurosci.

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Construction of a GABA A receptor homology model based on the acetylcholine (ACh) receptor structure is complicated by the low sequence similarity between GABA A and ACh M3 transmembrane segments that creates significant uncertainty in their alignment. We determined the orientation of the GABA A M2 and M3 transmembrane segments using disulfide cross-linking. The M2 residues ␣1M266 (11Ј) and ␣1T267 (12Ј) were mutated to cysteine in either wild type or single M3 cysteine mutant (␣1V297C, ␣1A300C to ␣1A305C) backgrounds. We assayed spontaneous and induced disulfide bond formation. Reduction with DTT significantly potentiated GABAinduced currents in ␣1T267C-L301C and ␣1T267C-F304C. Copper phenanthroline-induced oxidation inhibited GABA-induced currents in these mutants and in ␣1T267C-A305C. Intrasubunit disulfide bonds formed between these Cys pairs, implying that the ␣-carbon separation was at most 5.6 Å. The reactive ␣1M3 residues (L301, F304, A305) lie on the same face of an ␣-helix. The unresponsive ones (A300, I302, E303) lie on the opposite face. In the resting state, the reactive side of ␣1M3 faces M2-␣1T267. In conjunction with the ACh structure, our data indicate that alignment of GABA A and ACh M3 requires a single gap in the GABA A M2-M3 loop. In the presence of GABA, oxidation of ␣1T267C-L301C and ␣1T267C-F304C had no effect, but oxidation of ␣1T267C-A305C caused a significant increase in spontaneous channel opening. We infer that, as the channel opens, the distance and/or orientation between M2-␣1T267 and M3-␣1A305 changes such that the disulfide bond stabilizes the open state. This begins to define the conformational motion that M2 undergoes during channel opening.

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Comparative modeling of a GABAA alpha1 receptor using three crystal structures as templates

Cited by 41 publications

References 71 publications

Structural Determinants of Benzodiazepine Allosteric Regulation of GABA_AReceptor Currents

Structural Determinants of Benzodiazepine Allosteric Regulation of GABA_AReceptor Currents

Extracellular Domain Nicotinic Acetylcholine Receptors Formed by α4 and β2 Subunits

State-Dependent Cross-Linking of the M2 and M3 Segments: Functional Basis for the Alignment of GABA_Aand Acetylcholine Receptor M3 Segments

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Comparative modeling of a GABAA alpha1 receptor using three crystal structures as templates

Cited by 41 publications

References 71 publications

Structural Determinants of Benzodiazepine Allosteric Regulation of GABAAReceptor Currents

Structural Determinants of Benzodiazepine Allosteric Regulation of GABAAReceptor Currents

Extracellular Domain Nicotinic Acetylcholine Receptors Formed by α4 and β2 Subunits

State-Dependent Cross-Linking of the M2 and M3 Segments: Functional Basis for the Alignment of GABAAand Acetylcholine Receptor M3 Segments

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Resources

About

Structural Determinants of Benzodiazepine Allosteric Regulation of GABA_AReceptor Currents

Structural Determinants of Benzodiazepine Allosteric Regulation of GABA_AReceptor Currents

State-Dependent Cross-Linking of the M2 and M3 Segments: Functional Basis for the Alignment of GABA_Aand Acetylcholine Receptor M3 Segments