scholarly journals Loop C and the mechanism of acetylcholine receptor–channel gating

2013 ◽  
Vol 141 (4) ◽  
pp. 467-478 ◽  
Author(s):  
Prasad Purohit ◽  
Anthony Auerbach
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Single Channel ◽  
Communication Link ◽  
Channel Opening ◽  
State Models ◽  
Open Question ◽  
Acetylcholine Receptor Channel ◽  
Global Transition ◽  
Receptor Channel

Agonist molecules at the two neuromuscular acetylcholine (ACh) receptor (AChR) transmitter-binding sites increase the probability of channel opening. In one hypothesis for AChR activation (“priming”), the capping of loop C at each binding site transfers energy independently to the distant gate over a discrete structural pathway. We used single-channel analyses to examine the experimental support for this proposal with regard to brief unliganded openings, the effects of loop-C modifications, the effects of mutations to residues either on or off the putative pathway, and state models for describing currents at low [ACh]. The results show that (a) diliganded and brief unliganded openings are generated by the same essential, global transition; (b) the radical manipulation of loop C does not prevent channel opening but impairs agonist binding; (c) both on- and off-pathway mutations alter gating by changing the relative stability of the open-channel conformation by local interactions rather than by perturbing a specific site–gate communication link; and (d) it is possible to estimate directly the rate constants for agonist dissociation from and association to both the low and high affinity forms of the AChR-binding site by using a cyclic kinetic model. We conclude that the mechanism of energy transfer between the binding sites and the gate remains an open question.

scholarly journals The Role of Loop 5 in Acetylcholine Receptor Channel Gating

2003 ◽  
Vol 122 (5) ◽  
pp. 521-539 ◽  
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.

scholarly journals Action of nicotine and analogs on acetylcholine receptors having mutations of transmitter-binding site residue αG153

2012 ◽  
Vol 141 (1) ◽  
pp. 95-104 ◽  
Author(s):  
Snehal Jadey ◽  
Prasad Purohit ◽  
Anthony Auerbach
Keyword(s):  
Binding Site ◽  
Acetylcholine Receptors ◽  
Single Channel ◽  
Exceptional Case ◽  
Primary Target ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Acetylcholine Receptor Channel ◽  
Receptor Channel

A primary target for nicotine is the acetylcholine receptor channel (AChR). Some of the ability of nicotine to activate differentially AChR subtypes has been traced to a transmitter-binding site amino acid that is glycine in lower affinity and lysine in higher affinity AChRs. We studied the effects of mutations of this residue (αG153) in neuromuscular AChRs activated by nicotine and eight other agonists including nornicotine and anabasine. All of the mutations increased the unliganded gating equilibrium constant. The affinity of the resting receptor (Kd) and the net binding energy from the agonist for gating (ΔGB) were estimated by cross-concentration fitting of single-channel currents. In all but one of the agonist/mutant combinations there was a moderate decrease in Kd and essentially no change in ΔGB. The exceptional case was nicotine plus lysine, which showed a large, >8,000-fold decrease in Kd but no change in ΔGB. The extraordinary specificity of this combination leads us to speculate that AChRs with a lysine at position αG153 may be exposed to a nicotine-like compound in vivo.

scholarly journals K+ Occupancy of the N-Methyl-d-Aspartate Receptor Channel Probed by Mg2+ Block

2001 ◽  
Vol 117 (3) ◽  
pp. 287-298 ◽  
Author(s):  
Yongling Zhu ◽  
Anthony Auerbach
Keyword(s):  
Binding Sites ◽  
Voltage Dependence ◽  
Single Channel ◽  
Extracellular Solution ◽  
Aspartate Receptor ◽  
Apparent Decrease ◽  
Internal Site ◽  
Equilibrium Dissociation ◽  
Kinetics Of ◽  
Receptor Channel

The single-channel kinetics of extracellular Mg2+ block was used to probe K+ binding sites in the permeation pathway of rat recombinant NR1/NR2B NMDA receptor channels. K+ binds to three sites: two that are external and one that is internal to the site of Mg2+ block. The internal site is ∼0.84 through the electric field from the extracellular surface. The equilibrium dissociation constant for this site for K+ is 304 mM at 0 mV and with Mg2+ in the pore. The occupancy of any one of the three sites by K+ effectively prevents the association of extracellular Mg2+. Occupancy of the internal site also prevents Mg2+ permeation and increases (by approximately sevenfold) the rate constant for Mg2+ dissociation back to the extracellular solution. Under physiological intracellular ionic conditions and at −60 mV, there is ∼1,400-fold apparent decrease in the affinity of the channel for extracellular Mg2+ and ∼2-fold enhancement of the apparent voltage dependence of Mg2+ block caused by the voltage dependence of K+ occupancy of the external and internal sites.

scholarly journals Energy Changes for Small Molecules at the Acetylcholine Receptor-Channel Transmitter Binding Site

2010 ◽  
Vol 98 (3) ◽  
pp. 132a
Author(s):  
Snehal V. Jadey ◽  
Prasad Purohit ◽  
Timothy Gregg ◽  
Anthony Auerbach
Keyword(s):  
Small Molecules ◽  
Binding Site ◽  
Acetylcholine Receptor ◽  
Acetylcholine Receptor Channel ◽  
Receptor Channel

scholarly journals Activation and inactivation of single calcium channels in snail neurons.

1984 ◽  
Vol 83 (5) ◽  
pp. 751-769 ◽  
Author(s):  
A M Brown ◽  
H D Lux ◽  
D L Wilson
Keyword(s):  
Waiting Time ◽  
Single Channel ◽  
Waiting Times ◽  
State Model ◽  
Ca Channel ◽  
Snail Neurons ◽  
Channel Opening ◽  
Three State Model ◽  
State Models ◽  
Ca Currents

Activation and inactivation properties of Ca currents were investigated by studying the behavior of single Ca channels in snail neurons. The methods described in the previous paper were used. In addition, a zero-phase digital filter has been incorporated to improve the analysis of latencies to first opening, or waiting times. It was found that a decrease in the probability of single channel opening occurred with time. This was especially marked at 29 degrees C and paralleled the inactivation observed in macroscopic currents. The fact that a single channel was observed means that there is a significant amount of reopening from the "inactivated" state. Small depolarizations at 18 degrees C showed little inactivation. From these measurements, histograms of single channel open, closed, and waiting times were analyzed to estimate the rate constants of a three-state model of activation. Two serious discrepancies with the model were found. First, waiting time distributions at -20 mV were slower than those predicted by parameters obtained from an analysis of the single channel closed times. Second, it was shown that the time and the magnitude of the peak of the waiting time histogram were inconsistent with a three-state model. It is concluded that a minimum of four states are involved in activation. Some four-state models may be eliminated from further consideration. However, a comprehensive model of Ca channel kinetics must await further measurements.

scholarly journals Mechanisms of Barbiturate Inhibition of Acetylcholine Receptor Channels

1997 ◽  
Vol 109 (3) ◽  
pp. 401-414 ◽  
Author(s):  
James P. Dilger ◽  
Rebecca Boguslavsky ◽  
Martin Barann ◽  
Tamir Katz ◽  
Ana Maria Vidal
Keyword(s):  
Binding Site ◽  
Acetylcholine Receptor ◽  
Single Channel ◽  
Inhibitory Effects ◽  
Channel Current ◽  
State Dependent ◽  
Rapid Perfusion ◽  
Kinetics Of ◽  
Kinetics Of Inhibition ◽  
Acetylcholine Receptor Channel

We used patch clamp techniques to study the inhibitory effects of pentobarbital and barbital on nicotinic acetylcholine receptor channels from BC3H-1 cells. Single channel recording from outside-out patches reveals that both drugs cause acetylcholine-activated channel events to occur in bursts. The mean duration of gaps within bursts is 2 ms for 0.1 mM pentobarbital and 0.05 ms for 1 mM barbital. In addition, 1 mM barbital reduces the apparent single channel current by 15%. Both barbiturates decrease the duration of openings within a burst but have only a small effect on the burst duration. Macroscopic currents were activated by rapid perfusion of 300 μM acetylcholine to outside-out patches. The concentration dependence of peak current inhibition was fit with a Hill function; for pentobarbital, Ki = 32 μM, n = 1.09; for barbital, Ki = 1900 μM, n = 1.24. Inhibition is voltage independent. The kinetics of inhibition by pentobarbital are at least 30 times faster than inhibition by barbital (3 ms vs. <0.1 ms at the Ki). Pentobarbital binds ≥10-fold more tightly to open channels than to closed channels; we could not determine whether the binding of barbital is state dependent. Experiments performed with both barbiturates reveal that they do not compete for a single binding site on the acetylcholine receptor channel protein, but the binding of one barbiturate destabilizes the binding of the other. These results support a kinetic model in which barbiturates bind to both open and closed states of the AChR and block the flow of ions through the channel. An additional, lower-affinity binding site for pentobarbital may explain the effects seen at >100 μM pentobarbital.

scholarly journals A Functionally Conserved Mechanism of Modulation via a Vestibule Site in Pentameric Ligand-Gated Ion Channels

2019 ◽  
Author(s):  
Marijke Brams ◽  
Cedric Govaerts ◽  
Kumiko Kambara ◽  
Kerry Price ◽  
Radovan Spurny ◽  
...  
Keyword(s):  
Ion Channels ◽  
Binding Site ◽  
Binding Sites ◽  
Allosteric Modulators ◽  
Channel Opening ◽  
Single Domain Antibodies ◽  
Allosteric Binding Sites ◽  
Target Binding ◽  
Gated Ion Channels ◽  
Ligand Gated Ion Channels

ABSTRACTPentameric ligand-gated ion channels (pLGICs) belong to a class of ion channels involved in fast synaptic signaling in the central and peripheral nervous systems. Molecules acting as allosteric modulators target binding sites that are remote from the neurotransmitter binding site, but functionally affect coupling of ligand binding to channel opening. Here, we investigated an allosteric binding site in the ion channel vestibule, which has converged from a series of studies on prokaryote and eukaryote channel homologs. We discovered single domain antibodies, called nanobodies, which are functionally active as allosteric modulators, and solved co-crystal structures of the prokaryote channel ELIC bound either to a positive (PAM) or a negative (NAM) allosteric modulator. We extrapolate the functional importance of the vestibule binding site to eukaryote ion channels, suggesting a conserved mechanism of allosteric modulation. This work identifies key elements of allosteric binding sites and extends drug design possibilities in pLGICs using nanobodies.

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