A single residue in the M2-M3 loop is a major determinant of coupling between binding and gating in neuronal nicotinic receptors.

Campos‐Caro, Antonio; Sala, Salvador; Ballesta, Juan J.; Vicente-Agullo, Francisco; Criado, Manuel; Sala, Francisco

doi:10.1073/pnas.93.12.6118

Cited by 87 publications

(86 citation statements)

References 33 publications

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“…The pre-TM1 region and TM2-TM3 loop of several ligandgated ion channels have been found to be critical in the coupling of agonist binding to channel gating. Certain residues of the TM2-TM3 loop in nACh receptors (33,34), glycine receptors (35), and ␥-aminobutyric acid type A receptors (36) have been found to participate in the transduction of agonist binding to channel gating. For N-methyl-D-aspartate receptors, the pre-TM1 segment was found to link agonist binding to channel gating and to affect entry into open and desensitized states (37).…”

Section: Discussionmentioning

confidence: 99%

Arginine 222 in the Pre-transmembrane Domain 1 of 5-HT3A Receptors Links Agonist Binding to Channel Gating

Zhang

Stewart

et al. 2003

Journal of Biological Chemistry

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Ligand-gated ion channels are integral membrane proteins that mediate fast synaptic transmission. Molecular biological techniques have been extensively used for determining the structure-function relationships of ligand-gated ion channels. However, the transduction mechanisms that link agonist binding to channel gating remain poorly understood. Arginine 222 (Arg-222), located at the distal end of the extracellular N-terminal domain immediately preceding the first transmembrane domain (TM1), is conserved in all 5-HT 3A receptors and ␣7-nicotinic acetylcholine receptors that have been cloned. To elucidate the possible role of Arg-222 in the function of 5-HT 3A receptors, we mutated the arginine residue to alanine (Ala) and expressed both the wildtype and the mutant receptor in human embryonic kidney 293 cells. Functional studies of expressed wild-type and mutant receptors revealed that the R222A mutation increased the apparent potency of the full agonist, serotonin (5-HT), and the partial agonist, 2-Me-5-HT, 5-and 12-fold, respectively. In addition, the mutation increased the efficacy of 2-Me-5-HT and converted it from a partial agonist to a full agonist. Furthermore, this mutation also converted the 5-HT 3 receptor antagonist/ very weak partial agonist, apomorphine, to a potent agonist. Kinetic analysis revealed that the R222A mutation increased the rate of receptor activation and desensitization but did not affect rate of deactivation. The results suggest that the pre-TM1 amino acid residue Arg-222 may be involved in the transduction mechanism linking agonist binding to channel gating in 5-HT 3A receptors.In the nervous system, serotonin type 3 (5-HT 3 ) 1 receptors can mediate fast excitatory synaptic transmission and modulate neurotransmitter release (1). To date two 5-HT 3 receptor subunits have been identified: 5-HT 3A and 5-HT 3B (2, 3). The 5-HT 3A receptor subunits can form functional channels homomerically (2), whereas the 5-HT 3B receptor subunits are nonfunctional when expressed alone (3). However, the 5-HT 3B receptor subunits can form heteromeric channels with the 5-HT 3A receptor subunits, which results in modified biophysical characteristics compared with homomerically expressed 5-HT 3A receptor subunits (3). 5-HT 3A and 5-HT 3B receptor subunits also have different distribution patterns in the nervous system. The 5-HT 3A receptor subunits are expressed in both central and peripheral neurons, whereas the 5-HT 3B receptor subunits are restricted to peripheral neurons (4). This suggests that homomeric 5-HT 3A receptors play a dominant role in 5-HT 3 receptor-mediated responses in the central nervous system. 5-HT 3 receptors belong to a superfamily of ligand-gated ion channels, which includes nicotinic acetylcholine (nACh) receptors, glycine receptors, and ␥-aminobutyric acid type A receptors (5). The subunits in this superfamily are thought to assemble as pentamers with each subunit containing a large extracellular N-terminal domain, four transmembrane domains (TM1-TM4), a large intracellular loop be...

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Section: Discussionmentioning

confidence: 99%

Arginine 222 in the Pre-transmembrane Domain 1 of 5-HT3A Receptors Links Agonist Binding to Channel Gating

Zhang

Stewart

et al. 2003

Journal of Biological Chemistry

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“…A lot of conserved lysine residues are indeed located in the receptor's extracellular part-in the region between the agonist-binding site and the ion channel. This region (especially the M2-M3 loop) has been shown to be rather important for the coupling between ligand binding and channel gating for different members of the family of ligand-gated ion channels (34)(35)(36).…”

Section: Discussionmentioning

confidence: 99%

Fixation of allosteric states of the nicotinic acetylcholine receptor by chemical cross-linking

Watty

Methfessel

Hucho

1997

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

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Receptor activity can be described in terms of ligand-induced transitions between functional states. The nicotinic acetylcholine receptor (nAChR), a prototypic ligand-gated ion channel, is an ''unconventional allosteric protein'' which exists in at least three interconvertible conformations, referred to as resting (low agonist affinity, closed channel), activated (open channel), and desensitized (high agonist affinity, closed channel). Here we show that 3,3 -dimethyl suberimidate (DMS) is an agonistic bifunctional cross-linking reagent, which irreversibly ''freezes'' the nAChR in a high agonist affinity͞closed-channel state. The monofunctional homologue methyl acetoimidate, which is also a weak cholinergic agonist, has no such irreversible effect. Glutardialdehyde, a cross-linker that is not a cholinergic effector, fixes the receptor in a low-affinity state in the absence of carbamoylcholine, but, like DMS, in a highaffinity state in its presence. Covalent cross-linking thus allows us to arrest the nAChR in defined conformational states.The nicotinic acetylcholine receptor (nAChR) is a heteropentameric transmembrane glycoprotein with the subunit stoichiometry ␣ 2 ␤␥␦. The five subunits are arranged around a central pore, which is permeable for cations in the receptor's activated state (1-3).The nAChR is an allosteric protein (4, 5) existing in different interconvertible states (4, 6). In the absence of an agonist the receptor stays in a resting or closed channel state. After binding of two agonist molecules in a positive cooperative manner, the receptor is activated and the channel opens. During longer agonist exposure the receptor desensitizes, which means that the receptor closes while acetylcholine (ACh) is still bound (7).Although the nature of allosteric transitions in oligomeric proteins is still under debate (8, 9) there is agreement that distinct allosteric states are characterized by distinct protein conformations. Because allosteric proteins are oligomeric proteins, conformational changes must be transduced from the ligand-binding sites to neighboring subunits (10). That such changes in quaternary structure accompany allosteric transitions has been shown at the atomic level by x-ray crystallography for many proteins-e.g., hemoglobin (11) and aspartate carbamoyltransferase (12, 13). These examples show that the subunit interfaces play a pivotal role in the interconversion of different allosteric states, which are characterized by different quaternary structures.For the nAChR it has been established that the two binding sites for agonists and competitive antagonists are each located at the interface between an ␣-subunit and the neighboring ␥-or ␦-subunit (14-18). Therefore one can assume that the contact sites between subunits play an important role in the function and allosteric properties for the nAChR as well.To date we know very little about the structural changes going on upon receptor activation and desensitization. Unwin and co-workers (19) examined the structure of the nAChR in the absenc...

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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.

319

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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A single residue in the M2-M3 loop is a major determinant of coupling between binding and gating in neuronal nicotinic receptors.

Cited by 87 publications

References 33 publications

Arginine 222 in the Pre-transmembrane Domain 1 of 5-HT3A Receptors Links Agonist Binding to Channel Gating

Arginine 222 in the Pre-transmembrane Domain 1 of 5-HT3A Receptors Links Agonist Binding to Channel Gating

Fixation of allosteric states of the nicotinic acetylcholine receptor by chemical cross-linking

The structural basis of function in Cys-loop receptors

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