The broad, long-term aim of our research is to understand how neurotransmitters activate receptor-coupled ion channels of the Cys-loop superfamily. Focusing on the acetylcholine receptor (AChR) at the motor synapse, we address the central question of how ACh binding triggers opening of an ion channel intrinsic to the AChR. Toward understanding the binding-triggering process, we propose to (i) determine how residues at the junction of binding and pore domains link agonist binding to channel gating, (ii) delineate a comprehensive reaction mechanism underlying AChR activation, (iii) identify structures at subunit interfaces that mediate inter-subunit global coupling, and (iv) develop a water-soluble AChR ligand binding domain for studies of structure and ligand recognition. The approach combines site-directed mutagenesis, naturally-occurring mutations, single channel kinetic analysis, structural modeling, computational methods and protein biochemical and structural methods. Completion of the proposal will advance understanding of synaptic transmission and drug action at motor endplates, facilitate treatment of inherited disorders of synaptic receptors, while the general principles will provide insight into how structure gives rise to mechanism for other members of the Cys-loop receptor superfamily.

Public Health Relevance

Throughout the nervous system, moment-to-moment communication relies on post-synaptic receptors to detect nerve-released neurotransmitter and change the membrane potential. The change in membrane potential elicits a response in the receptive cell, such as muscle contraction, neuro-secretion or a change in excitability and thus altered communication with other cells. In many neurological diseases and drug addiction the interaction between neurotransmitter and post- synaptic receptor is altered, which if understood, could provide bases for rational therapy. Furthermore, many clinically useful drugs act on post-synaptic receptors, but often have unwanted side effects. Understanding both structures and molecular mechanisms of post-synaptic receptors would enable design of more specific drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS031744-20
Application #
8022821
Study Section
Biophysics of Neural Systems Study Section (BPNS)
Program Officer
Silberberg, Shai D
Project Start
1992-09-01
Project End
2013-02-28
Budget Start
2011-03-01
Budget End
2012-02-29
Support Year
20
Fiscal Year
2011
Total Cost
$602,531
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905
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
daCosta, Corrie J B; Free, Chris R; Sine, Steven M (2015) Stoichiometry for α-bungarotoxin block of α7 acetylcholine receptors. Nat Commun 6:8057
Engel, Andrew G; Shen, Xin-Ming; Selcen, Duygu et al. (2015) Congenital myasthenic syndromes: pathogenesis, diagnosis, and treatment. Lancet Neurol 14:420-34
Sine, Steven M; Huang, Sun; Li, Shu-Xing et al. (2013) Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors. Biochem J 454:311-21
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
Huang, Sun; Li, Shu-Xing; Bren, Nina et al. (2013) Complex between α-bungarotoxin and an α7 nicotinic receptor ligand-binding domain chimaera. Biochem J 454:303-10
Andersen, Natalia; Corradi, Jeremías; Sine, Steven M et al. (2013) Stoichiometry for activation of neuronal α7 nicotinic receptors. Proc Natl Acad Sci U S A 110:20819-24
Shen, Xin-Ming; Brengman, Joan M; Sine, Steven M et al. (2012) Myasthenic syndrome AChRα C-loop mutant disrupts initiation of channel gating. J Clin Invest 122:2613-21

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