Mapping the conformational wave of acetylcholine receptor channel gating

Grosman, Claudio; Zhou, Ming; Auerbach, Anthony

doi:10.1038/35001586

Cited by 312 publications

(367 citation statements)

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“…Although the precise location of the gate has been equivocal (8,13), the 4-Å density map of the nAChR (6) and recent MTS studies with glycine, ␥-aminobutyric acid, type A, and 5-HT 3 receptors implicate movement between the 6Ј and 13Ј positions (7,9,10,57). In support of this, linear free energy relationship analysis of mutant nAChRs suggested that the cytoplasmic side of M2 moves last upon activation after a wave of energy moving from the site of ligand binding down toward the cytoplasmic selectivity filter (61,62). Although linear free energy relationship analysis does not reveal information concerning the extent of movement, these studies suggest that for residues below the 11Ј site, the structure of the transition state (the highest energy state the receptor attains during gating) closely resembles that of the closed state.…”

Section: Discussionmentioning

confidence: 60%

Minimal Structural Rearrangement of the Cytoplasmic Pore during Activation of the 5-HT3A Receptor

Panicker

Cruz

Arrabit

et al. 2004

Journal of Biological Chemistry

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Ligand-gated ion channel receptors mediate the response of fast neurotransmitters by opening in less than a millisecond. Here, we investigated the activation mechanism of a serotonin-gated receptor (5-HT 3A ) by systematically introducing cysteine substitutions throughout the pore-lining M1-M2 loop and M2 transmembrane domain. We hypothesized that multiple cysteines in the narrowest region of the pore, which together can form a high affinity binding site for metal cations, would reveal changes in pore structure during gating. Using cadmium (Cd 2؉ ) as a probe, two cysteine substitutions in the cytoplasmic selectivity filter, S2 C and, to a lesser extent, G-2 C, showed high affinity inhibition with Cd 2؉ Ligand-gated ion channel (LGIC) 1 receptors are responsible for rapid chemical transmission between neurons in the nervous system (1). Alterations in the response of ligand-gated receptors have profound effects on neuronal activity in the brain. For example, mutations in nicotinic acetylcholine-gated receptors (nAChRs) and glycine-gated receptors have been linked to certain forms of epilepsy and startle disease, respectively (2-4).LGIC receptors that respond to acetylcholine, ␥-aminobutyric acid, glycine, or serotonin (5-HT) possess a conserved pair of extracellular cysteines in the N-terminal domain ("Cys loop" receptors) and are composed of five subunits, each containing four putative transmembrane domains (5). Of these four domains, the second transmembrane domain (M2) lines the majority of the water-filled pore (Fig. 1A).The rapid response of LGIC receptors is achieved by the energetic coupling of agonist binding in the extracellular Nterminal domain with a "gate" located 60 Å away in the receptor pore (M2 domain) (6). Several studies with LGIC receptors suggest that the gate is situated in the middle of the M2 (6 -12) (but see Ref. 13). The primary determinants of selectivity appear to be located below the gate, near the cytoplasmic membrane surface. Mutations in the cytoplasmic end of the M2 alter ion selectivity (14 -17), and several recent studies show that by mutating sites in this region, cation-conducting LGIC can be converted to anion-conducting channels and vice versa (18 -23). Because permeability of like-charged molecules in LGIC appears to be largely dependent on size, it is believed that the narrowest region of the open LGIC pore coincides with the channel selectivity filter, which is formed by a portion of the M1-M2 loop and the cytoplasmic side of the M2 helix (16,24). Consistent with this region being narrow, site-specific mutations in the filter alter conductivity in a manner that depends upon side-chain volume (8,24). Thus, the pore of the open receptor can be envisaged as a funnel, with a wide extracellular vestibule that tapers to a constriction at the cytoplasmic side of the membrane, where ion selectivity is determined (25).What is known about the extent of movement in the cytoplasmic selectivity filter? The 9-Å three-dimensional structures of open and closed Torpedo nAChRs derived fro...

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Section: Discussionmentioning

confidence: 60%

Minimal Structural Rearrangement of the Cytoplasmic Pore during Activation of the 5-HT3A Receptor

Panicker

Cruz

Arrabit

et al. 2004

Journal of Biological Chemistry

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“…It has been studied with single channel analysis and linear free energy relationship of gating rate constants by different mutations at each position. The results have suggested that there is a gradient change in the Φ slope factor of the linear free energy relationship, derived from the channel gating constants (opening and closing rates) of the mutations at each position, along the activation pathway [45] . Further analysis revealed that the gradient change in allosteric activation network is not continuous.…”

Section: Activation Mechanismmentioning

confidence: 99%

Allosteric activation mechanism of the cys-loop receptors

et al. 2009

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Binding of a neurotransmitter to its ionotropic receptor opens a distantly located ion channel, a process termed allosteric activation. Here we review recent advances in the molecular mechanism by which the cys-loop receptors are activated with emphasis on the best studied nicotinic acetylcholine receptors (nAChRs). With a combination of affinity labeling, mutagenesis, electrophysiology, kinetic modeling, electron microscopy (EM), and crystal structure analysis, the allosteric activation mechanism is emerging. Specifically, the binding domain and gating domain are interconnected by an allosteric activation network. Agonist binding induces conformational changes, resulting in the rotation of a β sheet of aminoterminal domain and outward movement of loop 2, loop F, and cys-loop, which are coupled to the M2-M3 linker to pull the channel to open. However, there are still some controversies about the movement of the channel-lining domain M2. Nine angstrom resolution EM structure of a nAChR imaged in the open state suggests that channel opening is the result of rotation of the M2 domain. In contrast, recent crystal structures of bacterial homologues of the cys-loop receptor family in apparently open state have implied an M2 tilting model with pore dilation and quaternary twist of the whole pentameric receptor. An elegant study of the nAChR using protonation scanning of M2 domain supports a similar pore dilation activation mechanism with minimal rotation of M2. This remains to be validated with other approaches including high resolution structure determination of the mammalian cys-loop receptors in the open state.

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“…If there is a speed limit to channel-opening, then adding a background mutation, that by itself increases the opening rate constant, to a construct that is already operating at the speed limit should have no effect. We tested this prediction by measuring b in AChRs having the mutation ␣S269I, which is in the extracellular linker and which by itself is predicted to increase the ACh-induced opening rate Ϸ25-fold and to slow the closing rate constant Ϸ4-fold (8). Burst durations were measured in constructs having only this mutation as well as in doublesubstitution constructs that carried a second mutation (␣D97A) that alone increased the opening rate constant 130-fold (Table 1) ⌽ Values.…”

Section: [4]mentioning

confidence: 99%

“…For a series of point mutations, the slope (⌽) of a log-log plot of the opening rate constant vs. the equilibrium constant is an index of the extent of progress of the perturbed site at the transition state of the reaction. In fully liganded AChRs, there is, to a first approximation, a longitudinal gradient in ⌽-values, with residues near the transmitter binding sites being open-like (⌽ Ϸ 1) and some of those in the transmembrane region being closed-like (⌽ Ϸ 0) at the transition state (8,9). These results have been interpreted as indicating that gating occurs as a reversible solitary conformational ''wave'' that links structural changes in the affinity of the binding sites with structural changes that determine ionic conductance.…”

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confidence: 99%

A speed limit for conformational change of an allosteric membrane protein

Chakrapani

Auerbach

2004

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

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Neuromuscular acetylcholine receptors are synaptic ion channels that open and close with rate constants of Ϸ48,000 s ؊1 and Ϸ1,700 s ؊1 , respectively (in adult mouse, at 24°C, ؊100 mV membrane potential). Perturbations of many different sites in the protein can change these rate constants, with those in the extracellular domain mainly affecting channel-opening and many of those in the membrane and intracellular domains mainly affecting channel-closing. We used single-channel recordings to measure the total open time per activation ( b) elicited by a low concentration of the natural transmitter, acetylcholine. b increased in constructs with mutations that increased the gating equilibrium constant by either increasing the opening or decreasing the closing rate constant. However, b did not approach the same asymptote in fast-opening and slow-closing constructs. The maximum value for the slow closers was about twice that for the fast openers. One interpretation of this difference is that there is an upper limit to the channel-opening rate constant, which we estimate to be Ϸ0.86 s ؊1 . One possibility is that this limit is the rate of conformational change in the absence of an overall activation barrier and thus reflects the kinetic prefactor for the acetylcholine receptor opening isomerization.channel gating ͉ nicotinic acetylcholine receptor ͉ energy landscape ͉ transition state A t cholinergic synapses, transmitter molecules trigger acetylcholine (ACh) receptor channels (AChRs) to switch from a stable conformation in which ion permeation is forbidden (''closed'') to one in which cations can permeate rapidly (''open''). This reversible change in structure, known as gating, involves the organized movements of many of the Ͼ2,300 residues within the five subunits that comprise this allosteric membrane protein (1-3). The driving energy for the reaction is a change in binding energy for the ligand; thus, gating must, at least, entail movements of residues at the two transmitter binding sites and in the ion conduction pathway.When activated by the natural transmitter ACh, mouse neuromuscular AChRs open rapidly (Ϸ48,000 s Ϫ1 ) and close relatively slowly (Ϸ1,700 s Ϫ1 ) (at 24°C, Ϫ100-mV membrane potential) (4). In single-molecule patch-clamp recordings with a time resolution of Ϸ25 s, the closed^open isomerization appears to be a two-state reaction with no directly detectable intermediate states. However, these states can be probed indirectly by using rate-equilibrium free energy (REFER) analyses (5-7). For a series of point mutations, the slope (⌽) of a log-log plot of the opening rate constant vs. the equilibrium constant is an index of the extent of progress of the perturbed site at the transition state of the reaction. In fully liganded AChRs, there is, to a first approximation, a longitudinal gradient in ⌽-values, with residues near the transmitter binding sites being open-like (⌽ Ϸ 1) and some of those in the transmembrane region being closed-like (⌽ Ϸ 0) at the transition state (8, 9). These results have been inte...

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Mapping the conformational wave of acetylcholine receptor channel gating

Cited by 312 publications

References 29 publications

Minimal Structural Rearrangement of the Cytoplasmic Pore during Activation of the 5-HT3A Receptor

Minimal Structural Rearrangement of the Cytoplasmic Pore during Activation of the 5-HT3A Receptor

Allosteric activation mechanism of the cys-loop receptors

A speed limit for conformational change of an allosteric membrane protein

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