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 Modal affinities of endplate acetylcholine receptors caused by loop C mutations

2015 ◽  
Vol 146 (5) ◽  
pp. 375-386 ◽  
Author(s):  
Ridhima Vij ◽  
Prasad Purohit ◽  
Anthony Auerbach
Keyword(s):  
Binding Site ◽  
Acetylcholine Receptors ◽  
Time Course ◽  
Single Channel ◽  
Equilibrium Constants ◽  
Open Probability ◽  
In The Wild ◽  
Single Channel Currents ◽  
Almost All ◽  
Channel Currents

The time course of the endplate current is determined by the rate and equilibrium constants for acetylcholine receptor (AChR) activation. We measured these constants in single-channel currents from AChRs with mutations at the neurotransmitter-binding sites, in loop C. The main findings are: (a) Almost all perturbations of loop C generate heterogeneity in the channel open probability (“modes”). (b) Modes are generated by different affinities for ACh that can be either higher or lower than in the wild-type receptors. (c) The modes are stable, in so far as each receptor maintains its affinity for at least several minutes. (d) Different agonists show different degrees of modal activity. With the loop C mutation αP197A, there are four modes with ACh but only two with partial agonists. (e) The affinity variations arise exclusively from the αδ-binding site. (f) Substituting four γ-subunit residues into the δ subunit (three in loop E and one in the β5–β5′ linker) reduces modal activity. (g) At each neurotransmitter-binding site, affinity is determined by a core of five aromatic residues. Modes are eliminated by an alanine mutation at δW57 but not at the other aromatics. (h) Modes are eliminated by a phenylalanine substitution at all core aromatics except αY93. The results suggest that, at the αδ agonist site, loop C and the complementary subunit surface can each adopt alternative conformations and interact with each other to influence the position of δW57 with respect to the aromatic core and, hence, affinity.

scholarly journals Divalent Cation Interactions with Light-Dependent K Channels

1999 ◽  
Vol 114 (5) ◽  
pp. 653-672 ◽  
Author(s):  
Enrico Nasi ◽  
Maria del Pilar Gomez
Keyword(s):  
Binding Site ◽  
Single Channel ◽  
Divalent Cations ◽  
Light Response ◽  
Relaxation Methods ◽  
Physiological Conditions ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Single Binding Site

The light-dependent K conductance of hyperpolarizing Pecten photoreceptors exhibits a pronounced outward rectification that is eliminated by removal of extracellular divalent cations. The voltage-dependent block by Ca2+ and Mg2+ that underlies such nonlinearity was investigated. Both divalents reduce the photocurrent amplitude, the potency being significantly higher for Ca2+ than Mg2+ (K1/2 ≈ 16 and 61 mM, respectively, at Vm = −30 mV). Neither cation is measurably permeant. Manipulating the concentration of permeant K ions affects the blockade, suggesting that the mechanism entails occlusion of the permeation pathway. The voltage dependency of Ca2+ block is consistent with a single binding site located at an electrical distance of δ ≈ 0.6 from the outside. Resolution of light-dependent single-channel currents under physiological conditions indicates that blockade must be slow, which prompted the use of perturbation/relaxation methods to analyze its kinetics. Voltage steps during illumination produce a distinct relaxation in the photocurrent (τ = 5–20 ms) that disappears on removal of Ca2+ and Mg2+ and thus reflects enhancement or relief of blockade, depending on the polarity of the stimulus. The equilibration kinetics are significantly faster with Ca2+ than with Mg2+, suggesting that the process is dominated by the “on” rate, perhaps because of a step requiring dehydration of the blocking ion to access the binding site. Complementary strategies were adopted to investigate the interaction between blockade and channel gating: the photocurrent decay accelerates with hyperpolarization, but the effect requires extracellular divalents. Moreover, conditioning voltage steps terminated immediately before light stimulation failed to affect the photocurrent. These observations suggest that equilibration of block at different voltages requires an open pore. Inducing channels to close during a conditioning hyperpolarization resulted in a slight delay in the rising phase of a subsequent light response; this effect can be interpreted as closure of the channel with a divalent ion trapped inside.

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.

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.

Proton Modulation of α1β3δ GABAA Receptor Channel Gating and Desensitization

2004 ◽  
Vol 92 (3) ◽  
pp. 1577-1585 ◽  
Author(s):  
Hua-Jun Feng ◽  
Robert L. Macdonald
Keyword(s):  
Steady State ◽  
Single Channel ◽  
Dependent Manner ◽  
Human Embryonic Kidney Cells ◽  
Receptor Isoforms ◽  
Long Duration ◽  
A Subunit ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Receptor Channel

αβγ GABAA receptor currents are phasic and desensitizing, whereas αβδ GABAA receptor currents are tonic and have no fast desensitization. αβγ receptors are subsynaptic and mediate phasic inhibition, whereas αβδ receptors are extra- or perisynaptic and mediate tonic inhibition. Given the different roles of these GABAA receptor isoforms and the fact that GABAA receptors are allosterically regulated by extracellular pH in a subunit-dependent manner, we compared the effects of changing pH on rat δ or γ2L subunit–containing GABAA receptor currents. Human embryonic kidney cells (HEK293T) were transfected with cDNAs encoding rat α1, β3, γ2L, or δ GABAA receptor subunits in several binary and ternary combinations, and whole cell and single channel patch-clamp recordings were obtained. Lowering pH substantially enhanced α1β3 receptor currents. This effect was significantly more pronounced for ternary α1β3δ receptors, whereas ternary α1β3γ2L receptors were relatively insensitive to lowered pH. Lowering pH did not affect the extent of desensitization of α1β3 and α1β3γ2L receptor currents, but significantly increased the extent of desensitization of α1β3δ receptor currents. Lowering pH prolonged deactivation of α1β3 and α1β3δ receptor currents and enhanced the “steady-state” currents of α1β3δ receptors evoked by long-duration (28 s) GABA applications. Lowering pH significantly increased mean open duration of α1β3δ steady-state single channel currents due to introduction of a longer-duration open state, suggesting that low pH enhances α1β3δ receptor steady-state currents by modifying GABAA receptor gating properties.

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 Surface charge potentiates conduction through the cardiac ryanodine receptor channel.

1994 ◽  
Vol 103 (5) ◽  
pp. 853-867 ◽  
Author(s):  
Q Tu ◽  
P Velez ◽  
M Cortes-Gutierrez ◽  
M Fill
Keyword(s):  
Ionic Strength ◽  
Surface Charge ◽  
Single Channel ◽  
Reverse Direction ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Selective Channel ◽  
Negative Surface ◽  
Single Channel Currents ◽  
Channel Currents

Single channel currents through cardiac sarcoplasmic reticulum (SR) Ca2+ release channels were measured in very low levels of current carrier (e.g., 1 mM Ba2+). The hypothesis that surface charge contributes to these anomalously large single channel currents was tested by changing ionic strength and surface charge density. Channel identity and sidedness was pharmacologically determined. At low ionic strength (20 mM Cs+), Cs+ conduction in the lumen-->myoplasm (L-->M) direction was significantly greater than in the reverse direction (301.7 +/- 92.5 vs 59.8 +/- 38 pS, P < 0.001; mean +/- SD, t test). The Cs+ concentration at which conduction reached half saturation was asymmetric (32 vs 222 mM) and voltage independent. At high ionic strength (400 mM Cs+), conduction in both direction saturated at 550 +/- 32 pS. Further, neutralization of carboxyl groups on the lumenal side of the channel significantly reduced conduction (333.0 +/- 22.5 vs 216.2 +/- 24.4 pS, P < 0.002). These results indicate that negative surface charge exists near the lumenal mouth of the channel but outside the electric field of the membrane. In vivo, this surface charge may potentiate conduction by increasing the local Ca2+ concentration and thus act as a preselection filter for this poorly selective channel.

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