Acetylcholine Receptor Gating: Movement in the α-Subunit Extracellular Domain

Acetylcholine receptor channel gating is a brownian conformational cascade in which nanometer-sized domains (“Φ blocks”) move in staggering sequence to link an affinity change at the transmitter binding sites with a conductance change in the pore. In the α-subunit, the first Φ-block to move during channel opening is comprised of residues near the transmitter binding site and the second is comprised of residues near the base of the extracellular domain. We used the rate constants estimated from single-channel currents to infer the gating dynamics of Y127 and K145, in the inner and outer sheet of the β-core of the α-subunit. Y127 is at the boundary between the first and second Φ blocks, at a subunit interface. αY127 mutations cause large changes in the gating equilibrium constant and with a characteristic Φ-value (Φ = 0.77) that places this residue in the second Φ-block. We also examined the effect on gating of mutations in neighboring residues δI43 (Φ = 0.86), εN39 (complex kinetics), αI49 (no effect) and in residues that are homologous to αY127 on the ε, β, and δ subunits (no effect). The extent to which αY127 gating motions are coupled to its neighbors was estimated by measuring the kinetic and equilibrium constants of constructs having mutations in αY127 (in both α subunits) plus residues αD97 or δI43. The magnitude of the coupling between αD97 and αY127 depended on the αY127 side chain and was small for both H (0.53 kcal/mol) and C (−0.37 kcal/mol) substitutions. The coupling across the single α–δ subunit boundary was larger (0.84 kcal/mol). The Φ-value for K145 (0.96) indicates that its gating motion is correlated temporally with the motions of residues in the first Φ-block and is not synchronous with those of αY127. This suggests that the inner and outer sheets of the α-subunit β-core do not rotate as a rigid body.

Download Full-text

Mutation in the M1 Domain of the Acetylcholine Receptor α Subunit Decreases the Rate of Agonist Dissociation

The Journal of General Physiology ◽

10.1085/jgp.109.6.757 ◽

1997 ◽

Vol 109 (6) ◽

pp. 757-766 ◽

Cited By ~ 108

Author(s):

Hai-Long Wang ◽

Anthony Auerbach ◽

Nina Bren ◽

Kinji Ohno ◽

Andrew G. Engel ◽

...

Keyword(s):

Kinetic Analysis ◽

Acetylcholine Receptor ◽

Rate Constants ◽

Single Channel ◽

Receptor Activation ◽

Congenital Myasthenic Syndrome ◽

Α Subunit ◽

Apparent Affinity ◽

Amino Terminal ◽

Channel Opening

We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) α subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing αN217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC50 to lower agonist concentrations for αN217K. The apparent affinity of ACh binding, measured by competition against the rate of 125I-α-bungarotoxin binding, is also enhanced 20-fold by αN217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the β, ε, or δ subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.

Download Full-text

The Role of Loop 5 in Acetylcholine Receptor Channel Gating

The Journal of General Physiology ◽

10.1085/jgp.200308885 ◽

2003 ◽

Vol 122 (5) ◽

pp. 521-539 ◽

Cited By ~ 51

Author(s):

Sudha Chakrapani ◽

Timothy D. Bailey ◽

Anthony Auerbach

Keyword(s):

Binding Site ◽

Acetylcholine Receptor ◽

Single Channel ◽

Channel Gating ◽

Α Subunit ◽

Acetylcholine Binding Protein ◽

Relationship Analysis ◽

Subunit Interface ◽

Acetylcholine Receptor Channel ◽

Receptor Channel

Nicotinic acetylcholine receptor channel (AChR) gating is an organized sequence of molecular motions that couples a change in the affinity for ligands at the two transmitter binding sites with a change in the ionic conductance of the pore. Loop 5 (L5) is a nine-residue segment (mouse α-subunit 92–100) that links the β4 and β5 strands of the extracellular domain and that (in the α-subunit) contains binding segment A. Based on the structure of the acetylcholine binding protein, we speculate that in AChRs L5 projects from the transmitter binding site toward the membrane along a subunit interface. We used single-channel kinetics to quantify the effects of mutations to αD97 and other L5 residues with respect to agonist binding (to both open and closed AChRs), channel gating (for both unliganded and fully-liganded AChRs), and desensitization. Most αD97 mutations increase gating (up to 168-fold) but have little or no effect on ligand binding or desensitization. Rate-equilibrium free energy relationship analysis indicates that αD97 moves early in the gating reaction, in synchrony with the movement of the transmitter binding site (Φ = 0.93, which implies an open-like character at the transition state). αD97 mutations in the two α-subunits have unequal energetic consequences for gating, but their contributions are independent. We conclude that the key, underlying functional consequence of αD97 perturbations is to increase the unliganded gating equilibrium constant. L5 emerges as an important and early link in the AChR gating reaction which, in the absence of agonist, serves to increase the relative stability of the closed conformation of the protein.

Download Full-text

Gating Dynamics of the Acetylcholine Receptor Extracellular Domain

The Journal of General Physiology ◽

10.1085/jgp.200309004 ◽

2004 ◽

Vol 123 (4) ◽

pp. 341-356 ◽

Cited By ~ 109

Author(s):

Sudha Chakrapani ◽

Timothy D. Bailey ◽

Anthony Auerbach

Keyword(s):

Conformational Change ◽

Acetylcholine Receptors ◽

Single Channel ◽

Extracellular Domain ◽

Mouse Muscle ◽

Α Subunit ◽

Acetylcholine Binding Protein ◽

Almost All ◽

Loop 2 ◽

Sandwich Core

We used single-channel recording and model-based kinetic analyses to quantify the effects of mutations in the extracellular domain (ECD) of the α-subunit of mouse muscle–type acetylcholine receptors (AChRs). The crystal structure of an acetylcholine binding protein (AChBP) suggests that the ECD is comprised of a β-sandwich core that is surrounded by loops. Here we focus on loops 2 and 7, which lie at the interface of the AChR extracellular and transmembrane domains. Side chain substitutions in these loops primarily affect channel gating by either decreasing or increasing the gating equilibrium constant. Many of the mutations to the β-core prevent the expression of functional AChRs, but of the mutants that did express almost all had wild-type behavior. Rate-equilibrium free energy relationship analyses reveal the presence of two contiguous, distinct synchronously-gating domains in the α-subunit ECD that move sequentially during the AChR gating reaction. The transmitter-binding site/loop 5 domain moves first (Φ = 0.93) and is followed by the loop 2/loop 7 domain (Φ = 0.80). These movements precede that of the extracellular linker (Φ = 0.69). We hypothesize that AChR gating occurs as the stepwise movements of such domains that link the low-to-high affinity conformational change in the TBS with the low-to-high conductance conformational change in the pore.

Download Full-text

A Transmembrane Binding Site at a Subunit Interface for Torpedo Nicotinic Acetylcholine Receptor Potentiators And Inhibitors

Biophysical Journal ◽

10.1016/j.bpj.2009.12.708 ◽

2010 ◽

Vol 98 (3) ◽

pp. 131a

Author(s):

Ayman K. Hamouda ◽

Deidre Stewart ◽

S. Shaukat Husain ◽

Jonathan B. Cohen

Keyword(s):

Binding Site ◽

Acetylcholine Receptor ◽

Nicotinic Acetylcholine Receptor ◽

Nicotinic Acetylcholine ◽

Subunit Interface ◽

A Subunit

Download Full-text

Mechanism of calcium potentiation of the α7 nicotinic acetylcholine receptor

The Journal of General Physiology ◽

10.1085/jgp.202012606 ◽

2020 ◽

Vol 152 (9) ◽

Author(s):

Kathiresan Natarajan ◽

Nuriya Mukhtasimova ◽

Jeremías Corradi ◽

Matías Lasala ◽

Cecilia Bouzat ◽

...

Keyword(s):

Acetylcholine Receptor ◽

Nicotinic Acetylcholine Receptor ◽

Single Channel ◽

Structural Motif ◽

Site Mutation ◽

Α7 Nicotinic Acetylcholine Receptor ◽

Nicotinic Acetylcholine ◽

Low Concentrations ◽

Channel Opening ◽

Dynamics Simulations

The α7 nicotinic acetylcholine receptor (nAChR) is among the most abundant types of nAChR in the brain, yet the ability of nerve-released ACh to activate α7 remains enigmatic. In particular, a major population of α7 resides in extra-synaptic regions where the ACh concentration is reduced, owing to dilution and enzymatic hydrolysis, yet ACh shows low potency in activating α7. Using high-resolution single-channel recording techniques, we show that extracellular calcium is a powerful potentiator of α7 activated by low concentrations of ACh. Potentiation manifests as robust increases in the frequency of channel opening and the average duration of the openings. Molecular dynamics simulations reveal that calcium binds to the periphery of the five ligand binding sites and is framed by a pair of anionic residues from the principal and complementary faces of each site. Mutation of residues identified by simulation prevents calcium from potentiating ACh-elicited channel opening. An anionic residue is conserved at each of the identified positions in all vertebrate species of α7. Thus, calcium associates with a novel structural motif on α7 and is an obligate cofactor in regions of limited ACh concentration.

Download Full-text

Desensitization of Mouse Nicotinic Acetylcholine Receptor Channels

The Journal of General Physiology ◽

10.1085/jgp.112.2.181 ◽

1998 ◽

Vol 112 (2) ◽

pp. 181-197 ◽

Cited By ~ 118

Author(s):

Anthony Auerbach ◽

Gustav Akk

Keyword(s):

Acetylcholine Receptor ◽

Rate Constant ◽

Binding Sites ◽

Single Channel ◽

Ion Permeation ◽

Activation Gate ◽

The Status ◽

Single Channel Currents ◽

Cyclic Models ◽

Channel Currents

The rate constants of acetylcholine receptor channels (AChR) desensitization and recovery were estimated from the durations and frequencies of clusters of single-channel currents. Diliganded-open AChR desensitize much faster than either unliganded- or diliganded-closed AChR, which indicates that the desensitization rate constant depends on the status of the activation gate rather than the occupancy of the transmitter binding sites. The desensitization rate constant does not change with the nature of the agonist, the membrane potential, the species of permeant cation, channel block by ACh, the subunit composition (ε or γ), or several mutations that are near the transmitter binding sites. The results are discussed in terms of cyclic models of AChR activation, desensitization, and recovery. In particular, a mechanism by which activation and desensitization are mediated by two distinct, but interrelated, gates in the ion permeation pathway is proposed.

Download Full-text

Improved resolution of single channel dwell times reveals mechanisms of binding, priming, and gating in muscle AChR

The Journal of General Physiology ◽

10.1085/jgp.201611584 ◽

2016 ◽

Vol 148 (1) ◽

pp. 43-63 ◽

Cited By ~ 26

Author(s):

Nuriya Mukhtasimova ◽

Corrie J.B. daCosta ◽

Steven M. Sine

Keyword(s):

Rate Constants ◽

Single Channel ◽

Partial Agonist ◽

Equilibrium Constants ◽

Channel Gating ◽

Kinetic Scheme ◽

Step Response ◽

Forward Rate ◽

Gain Of Function ◽

Single Channel Currents

The acetylcholine receptor (AChR) from vertebrate skeletal muscle initiates voluntary movement, and its kinetics of activation are crucial for maintaining the safety margin for neuromuscular transmission. Furthermore, the kinetic mechanism of the muscle AChR serves as an archetype for understanding activation mechanisms of related receptors from the Cys-loop superfamily. Here we record currents through single muscle AChR channels with improved temporal resolution approaching half an order of magnitude over our previous best. A range of concentrations of full and partial agonists are used to elicit currents from human wild-type and gain-of-function mutant AChRs. For each agonist–receptor combination, rate constants are estimated from maximum likelihood analysis using a kinetic scheme comprised of agonist binding, priming, and channel gating steps. The kinetic scheme and rate constants are tested by stochastic simulation, followed by incorporation of the experimental step response, sampling rate, background noise, and filter bandwidth. Analyses of the simulated data confirm all rate constants except those for channel gating, which are overestimated because of the established effect of noise on the briefest dwell times. Estimates of the gating rate constants were obtained through iterative simulation followed by kinetic fitting. The results reveal that the agonist association rate constants are independent of agonist occupancy but depend on receptor state, whereas those for agonist dissociation depend on occupancy but not on state. The priming rate and equilibrium constants increase with successive agonist occupancy, and for a full agonist, the forward rate constant increases more than the equilibrium constant; for a partial agonist, the forward rate and equilibrium constants increase equally. The gating rate and equilibrium constants also increase with successive agonist occupancy, but unlike priming, the equilibrium constants increase more than the forward rate constants. As observed for a full and a partial agonist, the gain-of-function mutation affects the relationship between rate and equilibrium constants for priming but not for channel gating. Thus, resolving brief single channel currents distinguishes priming from gating steps and reveals how the corresponding rate and equilibrium constants depend on agonist occupancy.

Download Full-text

K Channel Subconductance Levels Result from Heteromeric Pore Conformations

The Journal of General Physiology ◽

10.1085/jgp.200509253 ◽

2005 ◽

Vol 126 (2) ◽

pp. 87-103 ◽

Cited By ~ 44

Author(s):

Mark L. Chapman ◽

Antonius M.J. VanDongen

Keyword(s):

Single Channel ◽

K Channel ◽

K Channels ◽

Multiple Sensors ◽

Channel Opening ◽

Single Channel Analysis ◽

Binary Character ◽

A Subunit ◽

Subconductance States ◽

Voltage Sensing Domain

Voltage-gated K channels assemble from four identical subunits symmetrically arranged around a central permeation pathway. Each subunit harbors a voltage-sensing domain. The sigmoidal nature of the activation kinetics suggests that multiple sensors need to undergo a conformational change before the channel can open. Following activation, individual K channels alternate stochastically between two main permeation states, open and closed. This binary character of single channel behavior suggests the presence of a structure in the permeation pathway that can exist in only two conformations. However, single channel analysis of drk1 (Kv2.1) K channels demonstrated the existence of four additional, intermediate conductance levels. These short-lived subconductance levels are visited when the channel gate moves between the closed and fully open state. We have proposed that these sublevels arise from transient heteromeric pore conformations, in which some, but not all, subunits are in the “open” state. A minimal model based on this hypothesis relates specific subconductance states with the number of activated subunits (Chapman et al., 1997). To stringently test this hypothesis, we constructed a tandem dimer that links two K channel subunits with different activation thresholds. Activation of this dimer by strong depolarizations resulted in the characteristic binary open–close behavior. However, depolarizations to membrane potentials in between the activation thresholds of the two parents elicited highly unusual single channel gating, displaying frequent visits to two subconductance levels. The voltage dependence and kinetics of the small and large sublevels associate them with the activation of one and two subunits, respectively. The data therefore support the hypothesis that subconductance levels result from heteromeric pore conformations. In this model, both sensor movement and channel opening have a subunit basis and these processes are allosterically coupled.

Download Full-text

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

The Journal of General Physiology ◽

10.1085/jgp.116.3.327 ◽

2000 ◽

Vol 116 (3) ◽

pp. 327-340 ◽

Cited By ~ 84

Author(s):

Claudio Grosman ◽

Frank N. Salamone ◽

Steven M. Sine ◽

Anthony Auerbach

Keyword(s):

Acetylcholine Receptors ◽

Single Channel ◽

Channel Gating ◽

Congenital Myasthenic Syndrome ◽

Control Element ◽

Dependent Manner ◽

Α Subunit ◽

Transmembrane Segments ◽

Channel Opening ◽

Myasthenic Syndrome

We describe the functional consequences of mutations in the linker between the second and third transmembrane segments (M2–M3L) of muscle acetylcholine receptors at the single-channel level. Hydrophobic mutations (Ile, Cys, and Phe) placed near the middle of the linker of the α subunit (αS269) prolong apparent openings elicited by low concentrations of acetylcholine (ACh), whereas hydrophilic mutations (Asp, Lys, and Gln) are without effect. Because the gating kinetics of the αS269I receptor (a congenital myasthenic syndrome mutant) in the presence of ACh are too fast, choline was used as the agonist. This revealed an ∼92-fold increased gating equilibrium constant, which is consistent with an ∼10-fold decreased EC50 in the presence of ACh. With choline, this mutation accelerates channel opening ∼28-fold, slows channel closing ∼3-fold, but does not affect agonist binding to the closed state. These ratios suggest that, with ACh, αS269I acetylcholine receptors open at a rate of ∼1.4 × 106 s−1 and close at a rate of ∼760 s−1. These gating rate constants, together with the measured duration of apparent openings at low ACh concentrations, further suggest that ACh dissociates from the diliganded open receptor at a rate of ∼140 s−1. Ile mutations at positions flanking αS269 impair, rather than enhance, channel gating. Inserting or deleting one residue from this linker in the α subunit increased and decreased, respectively, the apparent open time approximately twofold. Contrary to the αS269I mutation, Ile mutations at equivalent positions of the β, ε, and δ subunits do not affect apparent open-channel lifetimes. However, in β and ε, shifting the mutation one residue to the NH2-terminal end enhances channel gating. The overall results indicate that this linker is a control element whose hydrophobicity determines channel gating in a position- and subunit-dependent manner. Characterization of the transition state of the gating reaction suggests that during channel opening the M2–M3L of the α subunit moves before the corresponding linkers of the β and ε subunits.

Download Full-text