Homology Model of the GABAA Receptor Examined Using Brownian Dynamics

O’Mara, Megan L.; Cromer, Brett A.; Parker, M.W.; Chung, Shin-Ho

doi:10.1529/biophysj.104.051664

Cited by 56 publications

(47 citation statements)

References 66 publications

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“…9) Homopentameric human b3 GABAR is very similar to that of insect GABAR in sensitivity to insecticides and general amino acid sequence of the TM2 region (Fig. 1).…”

Section: Structural Model For Gabar-nca Interactions With Structurallmentioning

confidence: 87%

Insecticide interactions with .GAMMA.-aminobutyric acid and nicotinic receptors: predictive aspects of structural models

Casida¹,

Tomizawa²

2008

J. Pestic. Sci.

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An insecticide binding site may be blocked by widely diverse chemicals, as with the g-aminobutyric acid receptor (GABAR), or accept only closely related compounds, as illustrated by the nicotinic acetylcholine (ACh) receptor (nAChR). The human GABAR b3-homopentamer resembles the insect receptor in sensitivity and specificity for noncompetitive antagonists (NCAs), prompting exhaustive site-directed mutagenesis (cysteine scanning), which pinpointed the critical active site as pore-facing residues Ala-2Ј, Thr-6Ј and Leu-9Ј and led to a binding site model which fits several classes of insecticidal NCAs. The nAChR agonist site was approached with an ACh binding protein (AChBP) by photoaffinity labeling and neonicotinoid docking. Chloropyridinyl chlorine contacts Ile-106/Met-116 and nitrogen is directed to Ile-118/Trp-147 via a solvent bridge(s). The guanidine/amidine plane undergoes p-stacking with Tyr-188, and the nitro/cyano pharmacophore interacts with Ser-189/Cys-190, thereby defining the binding pocket that models AChBP and possibly nAChR potency and selectivity.

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“…9) Homopentameric human b3 GABAR is very similar to that of insect GABAR in sensitivity to insecticides and general amino acid sequence of the TM2 region (Fig. 1).…”

Section: Structural Model For Gabar-nca Interactions With Structurallmentioning

confidence: 87%

Insecticide interactions with .GAMMA.-aminobutyric acid and nicotinic receptors: predictive aspects of structural models

Casida¹,

Tomizawa²

2008

J. Pestic. Sci.

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“…Modeling started from the ␣ 1 ␤ 2 ␥ 2 GABA A receptor based on the homologous nicotinic acetylcholine receptor and acetylcholine binding protein (30). This ␣-helical structure was reconstructed here as the ␤ 3 homopentamer, i.e., the two ␤ 2 subunits were directly replaced by ␤ 3 , because they have the same M2 sequence, then the two ␣ 1 subunits and one ␥ 2 subunit were replaced with ␤ 3 , by using the original ␣ 1 and ␥ 2 backbone atom positions as a guide, to make the homopentameric model.…”

Section: Methodsmentioning

confidence: 99%

“…The mutants were used to identify Cys residues undergoing disulfide cross-linking as a guide to channel pore structure (28 (6,8). Finally, modeling of the NCA-binding domain (29,30) was applied to the ␤ 3 homopentamer to determine whether the wide diversity of NCAs could fit the same site.…”

mentioning

confidence: 99%

Structural model for γ-aminobutyric acid receptor noncompetitive antagonist binding: Widely diverse structures fit the same site

Chen

Durkin

Casida

2006

Proc. Natl. Acad. Sci. U.S.A.

149

151

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Several major insecticides, including ␣-endosulfan, lindane, and fipronil, and the botanical picrotoxinin are noncompetitive antagonists (NCAs) for the GABA receptor. We showed earlier that human ␤3 homopentameric GABAA receptor recognizes all of the important GABAergic insecticides and reproduces the high insecticide sensitivity and structure-activity relationships of the native insect receptor. Despite large structural diversity, the NCAs are proposed to fit a single binding site in the chloride channel lumen lined by five transmembrane 2 segments. This hypothesis is examined with the ␤3 homopentamer by mutagenesis, pore structure studies, NCA binding, and molecular modeling. The 15 amino acids in the cytoplasmic half of the pore were mutated to cysteine, serine, or other residue for 22 mutants overall. Localization of A-1 C, A2 C, T6 C, and L9 C (index numbers for the transmembrane 2 region) in the channel lumen was established by disulfide cross-linking. Binding of two NCA radioligands ␤3 homopentamer ͉ transmembrane 2 ͉ insecticide ͉ disulfide trapping ͉ receptor model P est insect control in the past 60 years was achieved, in part, by application of Ͼ3 billion (3 ϫ 10 9 ) pounds of polychlorocycloalkane insecticides, including cyclodienes (e.g., ␣-endosulfan and dieldrin), lindane and its isomers, and others, which are now highly restricted or banned except for endosulfan and some uses of lindane (1-3). One of the replacement compounds is the phenylpyrazole fipronil. All of these insecticides and the botanical picrotoxinin (PTX) have widely diverse chemical structures but appear to act at the same nerve target. It is therefore important to understand how these compounds work in mammals and insects, or how they do not work when resistant insect strains appear.The GABA-gated chloride channel is the target for the insecticides and toxicants referred to above based on radioligand binding and electrophysiology studies (3)(4)(5)(6)(7)(8)(9)(10) (Fig. 1A). All of these compounds act in mammals and insects as noncompetitive antagonists (NCAs) to block chloride flux so the target is referred to as the GABA receptor NCA-binding site. Vertebrate GABA receptors consist of ␣, ␤, ␥, , and other subunits in various combinations, for example, ␣ 1 ␤ 2 ␥ 2 as a heteropentamer and 1 as a homopentamer (13-15). The molecular localization of the NCA site defined here (Fig. 1B) was first indicated by mutagenesis studies (16) as A2Ј (17-20), T6Ј (21, 22), and L9Ј (23, 24) in the cytoplasmic half of the transmembrane 2 domain of the channel (Fig. 2). Drosophila resistant to dieldrin (RDL) have a mutation conferring GABA receptor insensitivity identified as A2ЈS (17). The NCA target of the GABA A receptor requires a ␤ subunit, and a ␤ 3 homopentamer is sufficient for binding (9,26). Importantly, the ␤ 3 subunit from human brain, when expressed in insect Sf9 cells, assembles to form a receptor sensitive to all of the important GABAergic insecticides (9) and, surprisingly, reproduces the insecticide sensitivity and structureactivit...

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“…Conformational Mobility of 17Ј to 20Ј Region-The nAChRbased GABA A R model with an ␣-helical M2 structure places 17Ј and 20Ј in pore-facing positions (14,20) and gives average M2/M2 sulfur-sulfur distances for 17Ј, 18Ј, 19Ј, and 20Ј of 6.7, 13.6, 16.3, and 9.3 Å, respectively (Fig. 9).…”

Section: Effect Of Sulfhydryl Modification On Nca Binding Of Single Cmentioning

confidence: 99%

“…Molecular Modeling-Disulfide cross-linking interactions were examined with the ␤ 3 homopentamer model based on the homologous nAChR with ␣-helical structure throughout the transmembrane segments (3,20). All modeling was done with Maestro 6.5 (Schrödinger LLC).…”

mentioning

confidence: 99%

Spontaneous Mobility of GABAA Receptor M2 Extracellular Half Relative to Noncompetitive Antagonist Action

Chen

Durkin

Casida

2006

Journal of Biological Chemistry

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The ␥-aminobutyric acid type A receptor ␤ 3 homopentamer is spontaneously open and highly sensitive to many noncompetitive antagonists(NCAs)andZn Ј to Glu 20 Ј) has a proposed NCA interaction site at S17ЈC in an ␣ 1 subunit (6) and a Zn 2ϩ site at His 17 Ј and Glu 20 Ј in the ␤ 3 subunit (5). Disulfide trapping experiments with heteromeric receptors (␣ 1 ␤ 1 or ␣ 1 ␤ 1 ␥ 2 ) have helped to define the structure and mobility of the extracellular half of M2. The following cases of spontaneous or induced disulfide bond formation have been observed (in M2 unless stated otherwise): ␣ 1 12ЈC with several Cys mutants in the intrasubunit M3 (7); ␥ 2 14ЈC with both ␣ 1 15ЈC and ␣ 1 16ЈC and also ␥ 2 13ЈC and ␣ 1 13ЈC (8); ␣-␤ or ␤-␤ spontaneous disulfide cross-linking at 17Ј and 20Ј (9); nonadjacent ␤-␤ disulfide bridging by ␤ 1 20ЈC (10). These relationships indicate that the M2 segments can undergo significant rotational and translational movement.The ␤ 3 homopentamer is ideal for defining the open state channel structure relative to NCA action because it is self-assembling, spontaneously open, and symmetrical with high sensitivities to NCAs and Zn 2ϩ (3,11,12). To further understand M2 structure and NCA action, here we have examined the role of the extracellular half of the M2 segments in NCA and Zn 2ϩ binding involving site-directed mutagenesis with Cys, Ser, and Ala replacements in M2 and M3 using two NCA radioligands. The structural aspects were studied with SCAM (substituted Cys accessibility method) and disulfide trapping (3, 13). This investigation establishes the conformational flexibility of the extracellular half of M2 segments in the ␤ 3 homopentamer and suggests that NCAs access their binding sites both directly, through the channel pore, and indirectly, through the interface of adjacent subunits. The relationships considered lead to a model of the ␤ 3 homopentameric GABA A receptor M2 segments relative to NCA action (shown in Fig. 1). 3 Positions in the M2 segment are identified by a system of index numbers in which the absolutely conserved basic residue at the N-terminal end of M2 is numbered 0Ј, i.e. GABA A R ␤ 3 Arg 250 . Residues toward the C terminus are numbered 1Ј, 2Ј, etc., and toward the N terminus are numbered Ϫ1Ј, Ϫ2Ј, etc. EXPERIMENTAL PROCEDURES Mutagenesis

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Homology Model of the GABAA Receptor Examined Using Brownian Dynamics

Cited by 56 publications

References 66 publications

Insecticide interactions with .GAMMA.-aminobutyric acid and nicotinic receptors: predictive aspects of structural models

Insecticide interactions with .GAMMA.-aminobutyric acid and nicotinic receptors: predictive aspects of structural models

Structural model for γ-aminobutyric acid receptor noncompetitive antagonist binding: Widely diverse structures fit the same site

Spontaneous Mobility of GABAA Receptor M2 Extracellular Half Relative to Noncompetitive Antagonist Action

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