The muscle acetylcholine receptor (AChR) is an ion channel that mediates transmission at the nerve-muscle synapse. After binding two transmitter molecules, the AChR switches rapidly (and with high probability) from a closed-channel (C) to an open-channel (O) conformation. With prolonged exposure to agonist, AChRs also adopt inactivated (desensitized) conformations. We seek to understand the dynamics of the molecular events that constitute the binding, gating and desensitization reactions. Results to date suggest that the allosteric gating conformational change is asynchronous, with residues in the extracellular domain of the protein moving in advance of those in the membrane domain during the C-to-O isomerization. Could it be visualized, we hypothesize that this conformational change would appear as a staggering sequence of back and forth motions of a few rigid body domains rather than as a smooth transition between the C and O conformations. Perturbations to the protein (for example mutations that cause the disease slow-channel congenital myasthenic syndromes) alter gating, and, hence, synaptic function, by changing the propagation of this Brownian conformational 'wave'. We will use single-molecule electrophysiology and kinetic (phi-value) analysis to probe the properties of the brief intermediates that constitute the transition state of the gating reaction.
Our specific aims are to i) extend the map of phi-values, ii) measure the degree of synchrony between the five AChR subunits, iii) explore the discreteness of the rigid body gating domains, iv) quantify the temperature dependence of the channel-opening speed limit, and v) extend our theoretical analyses of the transition state. We also propose to use similar approaches to study the intermediate states of the transmitter binding and desensitization reactions. The results will illuminate the dynamic machinery of the AChR, and will provide fundamental insight into the mechanisms by which drugs, toxins, cellular perturbations and disease-causing mutations modify ion channel function. They will also serve as the basis for rational protein engineering.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37NS023513-25
Application #
8442323
Study Section
Special Emphasis Panel (NSS)
Program Officer
Silberberg, Shai D
Project Start
1987-07-01
Project End
2014-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
25
Fiscal Year
2013
Total Cost
$580,582
Indirect Cost
$211,135
Name
State University of New York at Buffalo
Department
Physiology
Type
Schools of Medicine
DUNS #
038633251
City
Buffalo
State
NY
Country
United States
Zip Code
14260
Auerbach, Anthony (2015) Agonist activation of a nicotinic acetylcholine receptor. Neuropharmacology 96:150-6
Purohit, Prasad; Bruhova, Iva; Gupta, Shaweta et al. (2014) Catch-and-hold activation of muscle acetylcholine receptors having transmitter binding site mutations. Biophys J 107:88-99
Gupta, Shaweta; Purohit, Prasad; Auerbach, Anthony (2013) Function of interfacial prolines at the transmitter-binding sites of the neuromuscular acetylcholine receptor. J Biol Chem 288:12667-79
Jadey, Snehal; Purohit, Prasad; Auerbach, Anthony (2013) Action of nicotine and analogs on acetylcholine receptors having mutations of transmitter-binding site residue ýýG153. J Gen Physiol 141:95-104
Auerbach, Anthony (2013) The energy and work of a ligand-gated ion channel. J Mol Biol 425:1461-75
Nayak, Tapan K; Auerbach, Anthony (2013) Asymmetric transmitter binding sites of fetal muscle acetylcholine receptors shape their synaptic response. Proc Natl Acad Sci U S A 110:13654-9
Auerbach, Anthony (2012) Thinking in cycles: MWC is a good model for acetylcholine receptor-channels. J Physiol 590:93-8
Nayak, Tapan K; Purohit, Prasad G; Auerbach, Anthony (2012) The intrinsic energy of the gating isomerization of a neuromuscular acetylcholine receptor channel. J Gen Physiol 139:349-58
Zheng, Wenjun; Auerbach, Anthony (2011) Decrypting the sequence of structural events during the gating transition of pentameric ligand-gated ion channels based on an interpolated elastic network model. PLoS Comput Biol 7:e1001046
Gupta, Shaweta; Auerbach, Anthony (2011) Temperature dependence of acetylcholine receptor channels activated by different agonists. Biophys J 100:895-903

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