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 Naturally Occurring Mutations at the Acetylcholine Receptor Binding Site Independently Alter ACh Binding and Channel Gating

2002 ◽  
Vol 120 (4) ◽  
pp. 483-496 ◽  
Author(s):  
Steven M. Sine ◽  
Xing-Ming Shen ◽  
Hai-Long Wang ◽  
Kinji Ohno ◽  
Won-Yong Lee ◽  
...  
Keyword(s):  
Binding Site ◽  
Acetylcholine Receptor ◽  
Rate Constants ◽  
Structural Model ◽  
Single Channel ◽  
Channel Gating ◽  
Congenital Myasthenic Syndrome ◽  
Protein Chain ◽  
Α Subunit ◽  
Closed State

By defining functional defects in a congenital myasthenic syndrome (CMS), we show that two mutant residues, located in a binding site region of the acetylcholine receptor (AChR) epsilon subunit, exert opposite effects on ACh binding and suppress channel gating. Single channel kinetic analysis reveals that the first mutation, εN182Y, increases ACh affinity for receptors in the resting closed state, which promotes sequential occupancy of the binding sites and discloses rate constants for ACh occupancy of the nonmutant αδ site. Studies of the analogous mutation in the δ subunit, δN187Y, disclose rate constants for ACh occupancy of the nonmutant αε site. The second CMS mutation, εD175N, reduces ACh affinity for receptors in the resting closed state; occupancy of the mutant site still promotes gating because a large difference in affinity is maintained between closed and open states. εD175N impairs overall gating, however, through an effect independent of ACh occupancy. When mapped on a structural model of the AChR binding site, εN182Y localizes to the interface with the α subunit, and εD175 to the entrance of the ACh binding cavity. Both εN182Y and εD175 show state specificity in affecting closed relative to desensitized state affinities, suggesting that the protein chain harboring εN182 and εD175 rearranges in the course of receptor desensitization. The overall results show that key residues at the ACh binding site differentially stabilize the agonist bound to closed, open and desensitized states, and provide a set point for gating of the channel.

Mapping the conformational wave of acetylcholine receptor channel gating

Nature ◽  
2000 ◽  
Vol 403 (6771) ◽  
pp. 773-776 ◽  
Author(s):  
Claudio Grosman ◽  
Ming Zhou ◽  
Anthony Auerbach
Keyword(s):  
Acetylcholine Receptor ◽  
Channel Gating ◽  
Acetylcholine Receptor Channel ◽  
Receptor Channel

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 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.

A stepwise mechanism for acetylcholine receptor channel gating

Nature ◽  
2007 ◽  
Vol 446 (7138) ◽  
pp. 930-933 ◽  
Author(s):  
Prasad Purohit ◽  
Ananya Mitra ◽  
Anthony Auerbach
Keyword(s):  
Acetylcholine Receptor ◽  
Channel Gating ◽  
Stepwise Mechanism ◽  
Acetylcholine Receptor Channel ◽  
Receptor Channel

Surfing the Conformational Wave of Acetylcholine Receptor Channel Gating

2001 ◽  
Vol 7 (S2) ◽  
pp. 24-25
Author(s):  
Gisela Cymes ◽  
Claudio Grosman ◽  
Anthony Auerbach
Keyword(s):  
Acetylcholine Receptor ◽  
Rate Constant ◽  
Single Molecule ◽  
Single Channel ◽  
Site Directed Mutagenesis ◽  
Kinetic Measurements ◽  
Ion Conducting ◽  
Single Channel Currents ◽  
Acetylcholine Receptor Channel ◽  
Receptor Channel

The muscle nicotinic acetylcholine receptor channel (AChR) is a cylindrical allosteric membrane protein (∼120 x 60 Å Fig. 1) that adopts alternative quaternary conformations (“open” and “closed”) with different functional properties (ion-conducting and ion-impermeable, respectively). We have characterized, residue-by-residue, the dynamics of the conformational change associated with gating using the framework of linear free energy relationships (LFER). The sequence of molecular events that underlies the closed-to-open gating transition was inferred from kinetic measurements of the receptor at the single molecule level.Specific regions of the AChR were perturbed using site-directed mutagenesis, changes in the membrane potential, or different agonists. Single-channel currents were recorded from cell-attached patches (Fig. 2). For the gain-of-function mutations, choline was used as the agonist because of its low efficacy. The opening rate constant was determined at a saturating concentration of agonist (for choline, 20 mM) in order to isolate gating from binding steps. to avoid bias introduced by fast channel blockade, the closing rate constant was measured at a low concentration (for choline, 200 μM). The diliganded channel opening (β) and closing (α) rate constants were estimated using the QuB suite of kinetic analysis programs. in general, a plot of the log rate constant vs. log equilibrium constant was linear.

scholarly journals The Intrinsic Electrostatic Potential and the Intermediate Ring of Charge in the Acetylcholine Receptor Channel

2000 ◽  
Vol 115 (2) ◽  
pp. 93-106 ◽  
Author(s):  
Gary G. Wilson ◽  
Juan M. Pascual ◽  
Natasja Brooijmans ◽  
Diana Murray ◽  
Arthur Karlin
Keyword(s):  
Acetylcholine Receptor ◽  
Electrostatic Potential ◽  
Rate Constants ◽  
Xenopus Laevis Oocytes ◽  
Mouse Muscle ◽  
Α Subunit ◽  
Intermediate Ring ◽  
Acetylcholine Receptor Channel ◽  
Positively Charged ◽  
Receptor Channel

A ring of aligned glutamate residues named the intermediate ring of charge surrounds the intracellular end of the acetylcholine receptor channel and dominates cation conduction (Imoto et al. 1988). Four of the five subunits in mouse-muscle acetylcholine receptor contribute a glutamate to the ring. These glutamates were mutated to glutamine or lysine, and combinations of mutant and native subunits, yielding net ring charges of −1 to −4, were expressed in Xenopus laevis oocytes. In all complexes, the α subunit contained a Cys substituted for αThr244, three residues away from the ring glutamate αGlu241. The rate constants for the reactions of αThr244Cys with the neutral 2-hydroxyethyl-methanethiosulfonate, the positively charged 2-ammonioethyl-methanethiosulfonate, and the doubly positively charged 2-ammonioethyl-2′-ammonioethanethiosulfonate were determined from the rates of irreversible inhibition of the responses to acetylcholine. The reagents were added in the presence and absence of acetylcholine and at various transmembrane potentials, and the rate constants were extrapolated to zero transmembrane potential. The intrinsic electrostatic potential in the channel in the vicinity of the ring of charge was estimated from the ratios of the rate constants of differently charged reagents. In the acetylcholine-induced open state, this potential was −230 mV with four glutamates in the ring and increased linearly towards 0 mV by +57 mV for each negative charge removed from the ring. Thus, the intrinsic electrostatic potential in the narrow, intracellular end of the open channel is almost entirely due to the intermediate ring of charge and is strongly correlated with alkali-metal-ion conductance through the channel. The intrinsic electrostatic potential in the closed state of the channel was more positive than in the open state at all values of the ring charge. These electrostatic properties were simulated by theoretical calculations based on a simplified model of the channel.

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