The Extracellular Linker of Muscle Acetylcholine Receptor Channels Is a Gating Control Element

Grosman, Claudio; Salamone, Frank N.; Sine, Steven M.; Auerbach, Anthony

doi:10.1085/jgp.116.3.327

Cited by 117 publications

(136 citation statements)

References 59 publications

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“…To analyze a given channel, discrete Markov models (8)(9)(10)(11)(12)(13)(14)(15)(16)) are fit to patch-clamp recordings of its current. However, it is known that different models can make identical predictions of steady-state gating statistics (17,18), so that attempts to find the best-fitting model can uncover many models that all fit equally well.…”

Section: Using Independent Open-to-closed Transitions To Simplify Aggmentioning

confidence: 99%

Using independent open-to-closed transitions to simplify aggregated Markov models of ion channel gating kinetics

Bruno

Yang

Pearson

2005

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

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Deducing plausible reaction schemes from single-channel current traces is time-consuming and difficult. The goal is to find the simplest scheme that fits the data, but there are many ways to connect even a small number of states (>2 million schemes with four open and four closed states). Many schemes make identical predictions. An exhaustive search over model space does not address the many equivalent schemes that will result. We have found a canonical form that can express all reaction schemes for binary channels. This form has the minimal number of rate constants for any rank (number of independent open-closed transitions), unlike other canonical forms such as the well established “uncoupled” scheme. Because all of the interconductance transitions in the new form are independent, we refer to it as the manifest interconductance rank (MIR) form. In the case of four open and four closed states, there are four MIR form schemes, corresponding to ranks 1-4. For many models proposed in the literature for specific ion channels, the equivalent MIR form has dramatically fewer links than the uncoupled form. By using the MIR form we prove that all rank 1 topologies with a given number of open and closed states make identical predictions in steady state, thus narrowing the search space for simple models. Moreover, we prove that fitting to canonical form preserves detailed balance. We also propose an efficient hierarchical algorithm for searching for the simplest possible model consistent with a given data set

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Section: Using Independent Open-to-closed Transitions To Simplify Aggmentioning

confidence: 99%

Using independent open-to-closed transitions to simplify aggregated Markov models of ion channel gating kinetics

Bruno

Yang

Pearson

2005

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

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“…For WT neuromuscular AChRs activated by ACh (␣ Ϸ 1,700 s Ϫ1 and ␤͞k Ϫ2 Ϸ 1), k* (Ϸ850 s Ϫ1 ) is much greater than (Ϸ70 s Ϫ1 ; see below). Thus, at the neuromuscular synapse, bursts terminate mainly by agonist dissociation from A 2 C, and b Ϸ 1͞k* Ϸ 1.2 ms (13,18).…”

Section: Resultsmentioning

confidence: 99%

“…The fast-opening constructs had mutations at position D97 [in loop 5, near the transmitter binding site (12)] in both ␣-subunits. Where noted, a second fast-opening mutation, ␣S269I, in the extracellular linker (13), was also present. The slow-closing constructs had side-chain substitutions at position L265 or S268 in the membrane domain of the ␦-subunit (9).…”

Section: Methodsmentioning

confidence: 93%

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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“…Our laboratory previously used this approach to demonstrate a glycine-induced increase in the surface exposure of six continuous residues (Arg 271 -Lys 276 ) in the GlyR M2-M3 linker domain (14). These residues lie mid-way between the binding site and the activation gate (7), and it is now well established that they experience a conformational change that is crucial for the activation of GlyRs (14 -18), ␥-aminobutyric acid, type A receptors (19 -24), and nAChRs (7,(25)(26)(27). A structural study of the Torpedo nAChR has shown recently (4) that these residues form an extramembranous extension to the M2 ␣-helix.…”

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confidence: 99%

Comparison of Taurine- and Glycine-induced Conformational Changes in the M2-M3 Domain of the Glycine Receptor

Han

Clements

Lynch

2004

Journal of Biological Chemistry

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In the ionotropic glutamate receptor, the global conformational changes induced by partial agonists are smaller than those induced by full agonists. However, in the pentameric ligand-gated ion channel receptor family, the structural basis of partial agonism is not understood. This study investigated whether full and partial agonists induce different conformation changes in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility analysis demonstrated previously that glycine binding induced an increase in surface accessibility of all residues from Arg 271 to Lys 276 in the M2-M3 domain of the homomeric ␣1 GlyR. Here we compare the surface accessibility changes induced by the full agonist, glycine, and the partial agonist, taurine. In GlyRs incorporating the A272C, S273C, L274C, or P275C mutation, the reaction rate of the cysteine-specific compound, methanethiosulfonate ethyltrimethylammonium, depended on how strongly the receptors were activated but was agonist-independent. Reaction rates could not be compared in the R271C and K276C mutant GlyRs because methanethiosulfonate ethyltrimethylammonium did not modify the extremely small currents induced by saturating taurine or equivalent low glycine concentrations. The results indicate that bound taurine and glycine molecules impose identical conformational changes to the M2-M3 domain. We therefore conclude that the higher efficacy of glycine is due to an increased ability to stabilize a common activated configuration.The glycine receptor chloride channel (GlyR) 1 mediates fast inhibitory neurotransmission in the vertebrate central nervous system (1, 2). It belongs to the family of pentameric ligandgated ion channels (LGICs) that includes the nicotinic acetylcholine receptor (nAChR) as its prototypical member (3). Each subunit incorporates a large N-terminal extracellular domain and four ␣-helical membrane-spanning domains. The second membrane-spanning (M2) domains curve radially so as to form a tapering, water-filled pore with a hydrophobic barrier (or channel gate) at either its mid-point (4) or intracellular boundary (5). The N-terminal domains contain the agonist-binding sites and a disulfide loop that is an invariant feature of LGIC receptors (6). Agonists binding in the N-terminal domain initiate conformational changes that propagate as a wave toward the channel gate (7). Different agonists induce these conformational changes with different efficacies (where efficacy is the ability of an agonist to open the channel once bound to the receptor). If the efficacy of an agonist is sufficiently low, it will behave as a partial agonist (8). The structural basis of differential agonist efficacy is not yet understood for any member of the LGIC family.Partial agonism could be caused by one, or a combination, of the following two sharply contrasting mechanisms. First, it is possible that different agonists induce different structural changes throughout the protein. A clear example of this has been characterized recently (9 -11) in the ionotropic g...

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The Extracellular Linker of Muscle Acetylcholine Receptor Channels Is a Gating Control Element

Cited by 117 publications

References 59 publications

Using independent open-to-closed transitions to simplify aggregated Markov models of ion channel gating kinetics

Using independent open-to-closed transitions to simplify aggregated Markov models of ion channel gating kinetics

A speed limit for conformational change of an allosteric membrane protein

Comparison of Taurine- and Glycine-induced Conformational Changes in the M2-M3 Domain of the Glycine Receptor

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