Charged Residues in the β2 Subunit Involved in GABAA Receptor Activation

Kash, Thomas L.; Dizon, Maria Johanna F.; Trudell, James R.; Harrison, Neil L.

doi:10.1074/jbc.m311441200

Cited by 68 publications

(98 citation statements)

References 34 publications

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“…Previous studies also support this interpretation; cysteine mutations of the residues equivalent to Arg-257 in the rat GABA A receptor ␣ 1 and ␤ 2 subunits (Arg-119 and Lys-215, respectively) yield receptors with similar GABA EC 50 values as wild type receptors (17), and even reversing the charge in K215D mutant receptors only increased GABA EC 50 values ϳ10-fold (19).…”

Section: Journal Of Biological Chemistry 25627supporting

confidence: 53%

Transducing Agonist Binding to Channel Gating Involves Different Interactions in 5-HT3 and GABAC Receptors

Price

Millen

Lummis

2007

Journal of Biological Chemistry

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5-Hydroxytryptamine (5-HT) 3 and ␥-aminobutyric acid, type C (GABA C ) receptors are members of the Cys-loop superfamily of neurotransmitter receptors, which also includes nicotinic acetylcholine, GABA A , and glycine receptors. The details of how agonist binding to these receptors results in channel opening is not fully understood but is known to involve charged residues at the extracellular/transmembrane interface. Here we have examined the roles of such residues in 5-HT 3 and GABA C receptors. Charge reversal experiments combined with data from activation by the partial agonist ␤-alanine show that in GABA C receptors there is a salt bridge between Glu-92 (in loop 2) and Arg-258 (in the pre-M1 region), which is involved in receptor gating. The equivalent residues in the 5-HT 3 receptor are important for receptor expression, but charge reversal experiments do not restore function, indicating that there is not a salt bridge here. There is, however, an interaction between Glu-215 (loop 9) and Arg-246 (pre-M1) in the 5-HT 3 receptor, although the coupling energy determined from mutant cycle analysis is lower than might be expected for a salt bridge. Overall the data show that charged residues at the extracellular/transmembrane domain interfaces in 5-HT 3 and GABA C receptors are important and that specific, but not equivalent, molecular interactions between them are involved in the gating process. Thus, we propose that the molecular details of interactions in the transduction pathway between the binding site and the pore can differ between different Cys-loop receptors.There have been many biochemical and functional studies on different members of the Cys-loop family of ligand-gated ion channels, but the prototypic member of this family, the neuromuscular nicotinic acetylcholine (nACh) 2 receptor, is still the best understood. This is the only receptor for which reasonably high resolution structural information (4 Å) is available from cryo-electron microscopy studies (1), and further knowledge has come from x-ray crystal structures of acetylcholine binding proteins (AChBP), which are homologous to the extracellular domain of this receptor (2, 3). Cys-loop receptors are composed of five pseudo-symmetrically arranged subunits surrounding a central ion-conducting pore. Each subunit is composed of an extracellular, a transmembrane, and an intracellular domain. The extracellular domain (ECD) contains the ligand binding site, which is formed at the interface of two adjacent subunits by the convergence of three amino acid loops (A-C) from one (the principal) subunit and three ␤-strands (D-F) from the adjacent (or complementary) subunit. The transmembrane domain (TMD) contains four membrane spanning ␣-helices (M1-M4) and a short C terminus. M2 from each subunit lines the pore and contains regions responsible for channel gating and ion selectivity. A large loop between M3-M4 forms the intracellular domain and is involved in channel conductance and modulation.Structural details extrapolated from AChBP have greatly enhanced o...

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Section: Journal Of Biological Chemistry 25627supporting

confidence: 53%

Transducing Agonist Binding to Channel Gating Involves Different Interactions in 5-HT3 and GABAC Receptors

Price

Millen

Lummis

2007

Journal of Biological Chemistry

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“…Indeed, this has been suggested by previous studies seeking to explain how channel gating is affected by charged residues in the transition zone (37,38). For example, in the GABA A R ␤2 subunit, the positively charged Lys, which is equivalent to the 217 residue in the GlyR ␣1 subunit, has been shown to interact with three negatively charged residues in the vicinity of the positively charged Arg, equivalent to the 143 residue in GlyR ␣1 subunit (27). These three negatively charged residues might shield the two positively charged residues from direct interaction and ensure proper folding and subsequent surface expression of the protein.…”

Section: Mutations Of Charged Residues In the Transition Zone Blockmentioning

confidence: 90%

Incompatibility between a Pair of Residues from the Pre-M1 Linker and Cys-loop Blocks Surface Expression of the Glycine Receptor

Shan

Lynch

2012

Journal of Biological Chemistry

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Background: Structural basis determining glycine receptor surface expression is barely known. Results: A pair of positively charged residues from the pre-M1 linker and Cys-loop blocks glycine receptor surface expression. Conclusion: Compatibility of residues, in close proximity to each other, is essential for glycine receptor surface expression. Significance: We provide a novel mechanism, i.e. residue incompatibility, for explaining mutation-induced reduction in channel surface expression.

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“…Electrostatic interactions are thought to occur between residues in extracellular loops 2, 7 (the conserved cys-loop), and 9, with amino acids in the pre-TM1 region, the TM2-3 extracellular loop, and post-TM4 residues (20) linking ligand binding to channel opening. In the GABA A receptor specific electrostatic interactions between D57 and D149 residues in loops 2 and 7 with K276 in the TM2-3 linker region affect gating (21); later work also implicated a residue in the pre-TM1 region (22). However, in the homomeric α1 GlyR, direct electrostatic interactions between D53 or E57 of loop 2, or D148 of loop 7, with K276 in the TM2-3 linker were not observed (23).…”

Section: Discussionmentioning

confidence: 99%

Disruption of an intersubunit electrostatic bond is a critical step in glycine receptor activation

Todorovic

Welsh

Bertaccini

et al. 2010

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

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Proper regulation of neurotransmission requires that ligandactivated ion channels remain closed until agonist binds. How channels then open remains poorly understood. Glycine receptor (GlyR) gating is initiated by agonist binding at interfaces between adjacent subunits in the extracellular domain. Aspartate-97, located at the α1 GlyR interface, is a conserved residue in the cys-loop receptor superfamily. The mutation of D97 to arginine (D97R) causes spontaneous channel opening, with open and closed dwell times similar to those of maximally activated WT GlyR. Using a model of the N-terminal domain of the α1 GlyR, we hypothesized that an arginine-119 residue was forming intersubunit electrostatic bonds with D97. The D97R/R119E charge reversal restored this interaction, stabilizing channels in their closed states. Cysteine substitution shows that this link occurs between adjacent subunits. This intersubunit electrostatic interaction among GlyR subunits thus contributes to the stabilization of the closed channel state, and its disruption represents a critical step in GlyR activation.cysteine substitution | electrophysiology | mutagenesis | Xenopus oocytes G lycine receptors (GlyR) are pentameric anion-conducting members of the cys-loop receptor superfamily, with their subunits arranged around a central ion pore. Each subunit consists of a large extracellular N-terminal ligand binding domain and four transmembrane segments (TM1-TM4); TM2 of each subunit lines the pore (1). When glycine binds to initiate channel opening it interacts with specific amino acids located at intersubunit interfaces, and spontaneous openings do not occur in the absence of neurotransmitter (2). Six loops of amino acids located on adjacent subunits constitute the known glycine binding site. On the plus (+) side of the interface on one subunit are loops A-C, whereas loops D-F are located on the minus (−) side of an adjacent subunit (3). In the related nicotinic acetylcholine receptor (nAChR), signal transduction after agonist binding is described as a "Brownian conformational wave" that travels down the interface between subunits (4). Auerbach and colleagues used ϕ analysis to show that the binding pocket region of the N-terminal domain is the first to move after ligand binds. Loops 2 and 7 (the cys-loop) of the Nterminal domain interact with the extracellular end of TM1 and the TM2-3 linker region to transmit binding signals to the channel gate (5-7). In the α1 GlyR subunit, D148 in loop 7 forms an electrostatic bridge with K276 in the TM2-3 linker (8).Our previous work demonstrated that mutation of D97 in loop A results in spontaneous channel opening (9). This D97 residue is conserved within the cys-loop receptor superfamily (Fig. 1), suggesting its critical role in channel function. In this article we report on an electrostatic interaction between specific charged amino acid residues at the interfaces of adjacent subunits that contribute to the stabilization of the closed channel state in the absence of neurotransmitter. Disruption of these ...

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Charged Residues in the β2 Subunit Involved in GABAA Receptor Activation

Cited by 68 publications

References 34 publications

Transducing Agonist Binding to Channel Gating Involves Different Interactions in 5-HT3 and GABAC Receptors

Transducing Agonist Binding to Channel Gating Involves Different Interactions in 5-HT3 and GABAC Receptors

Incompatibility between a Pair of Residues from the Pre-M1 Linker and Cys-loop Blocks Surface Expression of the Glycine Receptor

Disruption of an intersubunit electrostatic bond is a critical step in glycine receptor activation

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