Arg-274 and Leu-277 of the γ-Aminobutyric Acid Type A Receptor α2 Subunit Define Agonist Efficacy and Potency

O'Shea, Sean M.; Harrison, Neil L.

doi:10.1074/jbc.m001299200

Cited by 40 publications

(48 citation statements)

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“…Furthermore, we investigated the role of other basic residues located in TM2-3L of the ␤ 2 subunit (Arg 269 and Lys 279 ) and found that only the basic residue at position 274 is sensitive to a charge reversal mutation, suggesting that it is uniquely involved in electrostatic interactions important for receptor activation. This study also confirms the importance of TM2-3L of the GABA A -R ␤ 2 subunit in receptor activation, as previously reported for the ␣ 1 , ␣ 2 , ␤ 2 , and ␤ 3 subunits (12,14,18,25). An interesting difference between the ␣ 1 and ␤ 2 subunits was observed here because ␤ 2 (L272A) failed to alter receptor function, whereas the ␣ 1 ortholog (L277A) reduces GABA sensitivity dramatically (EC 50 ϭ 278 M) (26).…”

Section: Characterization Of Mutations In Loops 2 and 7 Of Thesupporting

confidence: 75%

“…Previous reports have suggested that TM2-3L of the GABA A -R ␣ subunit is involved in receptor activation (12,18). To determine whether this domain in the GABA A -R ␤ 2 subunit plays a similar role, we created point mutations R269A, R269D, L272A, K274D, and K279D at positions corresponding to mutations in the GABA A -R ␣ subunit that are important for receptor function (12,18). The mutant receptors were then characterized by determining GABA concentration-response curves.…”

Section: Characterization Of Mutations In the Tm2-tm3mentioning

confidence: 99%

“…Determination of Agonist Efficacy in Mutants-The potency of the agonist (measured here as the EC 50 for GABA) can be influenced at either step in the simplified receptor activation pathway: agonist binding or isomerization from the closed to open state (18,19). With high efficacy agonists (E Ͼ 10), slight reductions in the ability of the agonist-bound receptor to isomerize from the closed to open state typically produce a reduction in sensitivity to agonist, but little reduction in maximal response.…”

Section: Characterization Of Mutations In the Tm2-tm3mentioning

confidence: 99%

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Charged Residues in the β2 Subunit Involved in GABAA Receptor Activation

Kash¹,

Dizon

Trudell

et al. 2004

Journal of Biological Chemistry

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Fast synaptic inhibition in the mammalian central nervous system is mediated primarily via activation of the ␥-aminobutyric acid type A receptor (GABA A -R). Upon agonist binding, the receptor undergoes a structural transition from the closed to the open state. This transition, known as gating, is thought to be associated with a sequence of conformational changes originating at the agonist-binding site, ultimately resulting in opening of the channel. Using site-directed mutagenesis and several different GABA A -R agonists, we identified a number of highly conserved charged residues in the GABA A -R ␤ 2 subunit that appear to be involved in receptor activation. We then used charge reversal double mutants and disulfide trapping to investigate the interactions between these flexible loops within the ␤ 2 subunit. The results suggest that interactions between an acidic residue in loop 7 (Asp 146 ) and a basic residue in pretransmembrane domain-1 (Lys 215 ) are involved in coupling agonist binding to channel gating.Fast synaptic inhibition is a major determinant of network dynamics in the central nervous system (1). In the mammalian brain, this is mediated primarily via activation of the ␥-aminobutyric acid type A receptor (GABA A -R) 1 (2, 3). Each GABA A -R is composed of five subunits arranged around a central ion-conducting pore (4), with each subunit consisting of a large intracellular domain, four transmembrane domains (TM1-TM4), and a larger N-terminal extracellular domain (2). Experimental evidence suggests that the GABA-binding site lies within an asymmetric pocket formed at the interface between the ␣ and ␤ receptor subunits (5-8). The channel "gate" in the GABA A -R is believed to be formed by charged residues in the TM1-TM2 loop (9 -11). The binding of agonist triggers a complex structural transition that results in the opening of the gate, allowing ions to flow through the channel. The mechanisms by which this occurs remain poorly defined.The nature of the coupling between binding and channel opening in this receptor family has been recently investigated in several laboratories. Interactions between charged residues in the flexible loops 2 and 7 in the extracellular domain and those in the short linker between the second and third transmembrane domains (TM2-3L) of the GABA A -R ␣ 1 subunit have been implicated in the process of receptor activation (12). In addition, a recent report suggests that the pre-TM1 region is critical for receptor activation in the closely related serotonin (5-HT 3 ) receptor (13). In this study, we used site-directed mutagenesis and a number of GABA A -R agonists of varying efficacies to examine the contribution of the corresponding flexible loops in the GABA A -R ␤ 2 subunit to receptor activation. Based on previous studies (12-14) and the presence of highly conserved charged residues (see Fig. 1), we hypothesized that interactions between charged residues in these domains are crucial for coupling agonist binding to channel gating. We tested this hypothesis using a charge revers...

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Section: Characterization Of Mutations In Loops 2 and 7 Of Thesupporting

confidence: 75%

Section: Characterization Of Mutations In the Tm2-tm3mentioning

confidence: 99%

Section: Characterization Of Mutations In the Tm2-tm3mentioning

confidence: 99%

See 1 more Smart Citation

Charged Residues in the β2 Subunit Involved in GABAA Receptor Activation

Kash¹,

Dizon

Trudell

et al. 2004

Journal of Biological Chemistry

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“…Despite these difficulties, techniques have been developed that can potentially identify binding or gating changes using EC 50 and peak current measurements. For example, the relative efficacy of a partial agonist has been used as an internal control for receptor expression, and changes in relative efficacy have thus been interpreted as being caused by gating changes (Maksay et al, 2000;O'Shea and Harrison, 2000;Wagner and Czajkowski, 2001). In contrast, evidence for binding changes has included: (1) a change in the potency of GABA that is not accompanied by a change in the potency of a modulator that opens the channel but does not bind at the GABA-binding site, and (2) large changes in GABA potency in the absence of large changes in maximal cur- rent (Amin and Weiss, 1993).…”

Section: Discussionmentioning

confidence: 99%

An Arginine Involved in GABA Binding and Unbinding But Not Gating of the GABA_AReceptor

Wagner

Czajkowski²,

Jones³

2004

J. Neurosci.

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GABA A receptor function can be conceptually divided into interactions between ligand and receptor (binding) and the opening and closing of the ligand-bound channel (gating). The relationship between binding, gating, and receptor structure remains unclear. Studies of mutations have identified many amino acid residues that contribute to the GABAbinding site. Most of these studies assayed changes in GABA dose-response curves, which are macroscopic measures that depend on the interplay of many processes and cannot resolve individual microscopic transitions. Understanding the microscopic basis of binding and gating is critical, because kinetic transition rates predict how receptors will behave at synapses. Furthermore, microscopic rates are directly related to the molecular interactions underlying receptor function.Here, we focused on a residue (␤ 2 -R207) previously identified as lining the GABA-binding site that, when mutated to cysteine, greatly reduces apparent GABA affinity and was predicted to affect both binding and gating. To better understand the role of ␤ 2 -R207, we expressed ␣ 1 ␤ 2 and ␣ 1 ␤ 2 -R207C receptors in human embryonic kidney 293 cells and studied receptor kinetics using fast solution applications. The mutation accelerated deactivation by 10-fold, without altering desensitization in the presence of saturating GABA. Maximum open probability and single-channel open times were also unaltered by the mutation, but the GABA-binding rate was reduced eightfold. Therefore, the effects of this mutation in a predicted binding site residue are solely attributable to changes in GABA-binding and unbinding kinetics, with no changes in channel gating. Because ␤ 2 -R207 stabilizes GABA in the binding pocket, it may directly contact the GABA molecule.

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“…Studies have shown that mutations in this region disrupt activation in nACh, 5-HT 3 , GABA and Gly receptors (Campos-Caro et al 1996 ;Deane & Lummis, 2001 ;Grosman et al 2000aGrosman et al , 2000bKusama et al 1994 ;Lewis et al 1998 ;Lynch et al 1997 ;O'Shea & Harrison, 2000 ;Rajendra et al 1995 ;Rovira et al 1998Rovira et al , 1999Saul et al 1999 ;Sigel et al 1999). The structure of this loop has been examined by a range of techniques, including NMR and electron microscopy, and the data suggest that there are differences between cation-and anion-selective receptors.…”

Section: The M2-m3 Loopmentioning

confidence: 99%

The structural basis of function in Cys-loop receptors

Thompson

Lester

Lummis

2010

Quart. Rev. Biophys.

326

398

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Abstract. Cys-loop receptors are membrane-spanning neurotransmitter-gated ion channels that are responsible for fast excitatory and inhibitory transmission in the peripheral and central nervous systems. The best studied members of the Cys-loop family are nACh, 5-HT 3 , GABA A and glycine receptors. All these receptors share a common structure of five subunits, pseudo-symmetrically arranged to form a rosette with a central ion-conducting pore. Some are cation selective (e.g. nACh and 5-HT 3 ) and some are anion selective (e.g. GABA A and glycine). Each receptor has an extracellular domain (ECD) that contains the ligand-binding sites, a transmembrane domain (TMD) that allows ions to pass across the membrane, and an intracellular domain (ICD) that plays a role in channel conductance and receptor modulation. Cys-loop receptors are the targets for many currently used clinically relevant drugs (e.g. benzodiazepines and anaesthetics). Understanding the molecular mechanisms of these receptors could therefore provide the catalyst for further development in this field, as well as promoting the development of experimental techniques for other areas of neuroscience.In this review, we present our current understanding of Cys-loop receptor structure and function. The ECD has been extensively studied. Research in this area has been stimulated in recent years by the publication of high-resolution structures of nACh receptors and related proteins, which have permitted the creation of many Cys loop receptor homology models of this region. Here, using the 5-HT 3 receptor as a typical member of the family, we describe how homology modelling and ligand docking can provide useful but not definitive information about ligand interactions. We briefly consider some of the many Cys-loop receptors modulators. We discuss the current understanding of the structure of the TMD, and how this links to the ECD to allow channel gating, and consider the roles of the ICD, whose structure is poorly understood. We also describe some of the current methods that are beginning to reveal the differences between different receptor states, and may ultimately show structural details of transitions between them.

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Arg-274 and Leu-277 of the γ-Aminobutyric Acid Type A Receptor α2 Subunit Define Agonist Efficacy and Potency

Cited by 40 publications

References 40 publications

Charged Residues in the β2 Subunit Involved in GABAA Receptor Activation

Charged Residues in the β2 Subunit Involved in GABAA Receptor Activation

An Arginine Involved in GABA Binding and Unbinding But Not Gating of the GABA_AReceptor

The structural basis of function in Cys-loop receptors

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Arg-274 and Leu-277 of the γ-Aminobutyric Acid Type A Receptor α2 Subunit Define Agonist Efficacy and Potency

Cited by 40 publications

References 40 publications

Charged Residues in the β2 Subunit Involved in GABAA Receptor Activation

Charged Residues in the β2 Subunit Involved in GABAA Receptor Activation

An Arginine Involved in GABA Binding and Unbinding But Not Gating of the GABAAReceptor

The structural basis of function in Cys-loop receptors

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About

An Arginine Involved in GABA Binding and Unbinding But Not Gating of the GABA_AReceptor