The long-term aim of our research is to understand how receptors for neurotransmitters operate in health and disease states. We have chosen the acetylcholine receptor (AChR) at the motor synapse to investigate the essential function of this class of proteins, that of transducing neurotransmitter binding into opening of an intrinsic ion channel. Toward understanding the binding-opening process, we propose to define the structure of the ligand binding site, identify residues that stabilize bound agonist, and identify structures that transmit free energy released by agonist binding to the channel gating apparatus. Our approach is to combine site-directed mutagenesis and expression in mammalian cells with a battery of functional measurements including ligand binding, single channel recording, and protein biochemistry.
AIM (I) will probe the transmitter binding site using the peptide ligand conotoxin M1. We will make mutations in both the AChR and conotoxin to identify pairs of residues that stabilize the toxin-AChR complex.
AIMS (II) and (III) will further define the structure of the binding site by identifying residues in non-alpha subunits of the AChR that confer selectivity of binding of the agonists acetylcholine and epibatidine.
AIM (IV) will identify residues that couple agonist binding to channel gating by making subunit chimeras and point mutations of conserved residues. Subunit association, ligand binding single channel currents will be measured to identify residues underlying inter-subunit communication.
AIM (V) will confirm pathogenicity and delineate structural and functional mechanisms of mutations in human AChR that underlie congenital myasthenic syndromes (CMS). Completion of this aim will contribute to treatment of patients with CMS, as well as provide insights into structure-function relationships of the AChR. Because of their position in the pathway of information flow, neurotransmitter receptors are logical targets for learning and memory processes, pathologic processes and therapeutic drugs. Completion of the aims outlined in this proposal will contribute to treatment and prevention of neuromuscular disorders such as CMS, while the general findings may lead to insights into the operation of members of the superfamily of neurotransmitter receptors.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS031744-09
Application #
6187931
Study Section
Physiology Study Section (PHY)
Program Officer
Talley, Edmund M
Project Start
1992-09-01
Project End
2001-08-31
Budget Start
2000-09-01
Budget End
2001-08-31
Support Year
9
Fiscal Year
2000
Total Cost
$392,498
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
City
Rochester
State
MN
Country
United States
Zip Code
55905
Shen, Xin-Ming; Brengman, Joan M; Shen, Shelley et al. (2018) Mutations causing congenital myasthenia reveal principal coupling pathway in the acetylcholine receptor ?-subunit. JCI Insight 3:
Bouzat, Cecilia; Sine, Steven M (2018) Nicotinic acetylcholine receptors at the single-channel level. Br J Pharmacol 175:1789-1804
Mukhtasimova, Nuriya; Sine, Steven M (2018) Full and partial agonists evoke distinct structural changes in opening the muscle acetylcholine receptor channel. J Gen Physiol 150:713-729
Mazzaferro, Simone; Bermudez, Isabel; Sine, Steven M (2017) ?4?2 Nicotinic Acetylcholine Receptors: RELATIONSHIPS BETWEEN SUBUNIT STOICHIOMETRY AND FUNCTION AT THE SINGLE CHANNEL LEVEL. J Biol Chem 292:2729-2740
Shen, Xin-Ming; Brengman, Joan; Neubauer, David et al. (2016) Investigation of Congenital Myasthenia Reveals Functional Asymmetry of Invariant Acetylcholine Receptor (AChR) Cys-loop Aspartates. J Biol Chem 291:3291-301
Mukhtasimova, Nuriya; daCosta, Corrie J B; Sine, Steven M (2016) Improved resolution of single channel dwell times reveals mechanisms of binding, priming, and gating in muscle AChR. J Gen Physiol 148:43-63
Engel, Andrew G; Shen, Xin-Ming; Selcen, Duygu et al. (2015) Congenital myasthenic syndromes: pathogenesis, diagnosis, and treatment. Lancet Neurol 14:420-34
daCosta, Corrie J B; Free, Chris R; Sine, Steven M (2015) Stoichiometry for ?-bungarotoxin block of ?7 acetylcholine receptors. Nat Commun 6:8057
Sine, Steven M; Huang, Sun; Li, Shu-Xing et al. (2013) Inter-residue coupling contributes to high-affinity subtype-selective binding of ?-bungarotoxin to nicotinic receptors. Biochem J 454:311-21
Huang, Sun; Li, Shu-Xing; Bren, Nina et al. (2013) Complex between ?-bungarotoxin and an ?7 nicotinic receptor ligand-binding domain chimaera. Biochem J 454:303-310

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