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 examine the essential function of this class of proteins: binding of neurotransmitter triggering opening of an intrinsic ion channel. Toward understanding this binding-triggering process, we propose to (i) identify residues in a and non-a subunits that stabilize ACh when bound to open and closed states of the channel, (ii) determine how the AChR maintains uniform channel gating kinetics, (iv) determine mechanistic consequences of mutations underlying congenital myasthenic syndromes (CMS), (iv) determine whether conserved residues bounding putative secondary structures in the major extracellular domain contribute to AChR activation and (v) determine structural features of the major extracellular domain of the AChR. The approach combines site-directed mutagenesis and expression in mammalian cells, single channel recording and kinetic analysis, measurements of ligand binding and protein biochemistry. Completion of the proposed studies will advance our understanding of synaptic transmission and drug action at motor endplates, facilitate treatment of neuromuscular disorders such as CMS, while the general findings will provide insight into structure function relationships for other members of the neurotransmitter receptor superfamily.

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 #
5R37NS031744-13
Application #
6785387
Study Section
Special Emphasis Panel (ZRG1-MDCN-3 (01))
Program Officer
Stewart, Randall R
Project Start
1992-09-01
Project End
2005-08-31
Budget Start
2004-09-01
Budget End
2005-08-31
Support Year
13
Fiscal Year
2004
Total Cost
$558,618
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
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
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
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
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
Mukhtasimova, Nuriya; Sine, Steven M (2013) Nicotinic receptor transduction zone: invariant arginine couples to multiple electron-rich residues. Biophys J 104:355-67
daCosta, Corrie J B; Sine, Steven M (2013) Stoichiometry for drug potentiation of a pentameric ion channel. Proc Natl Acad Sci U S A 110:6595-600

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