The overall goal of this research is to understand how transmitter-gated membrane channels (receptors) function and how their function is modified by pharmacological agents. These receptors allow cells in the brain to communicate, and their proper function is essential to normal brain function. They are also the targets for many drugs, both clinically used and recreational. This research focuses on a major class of human brain nicotinic receptors, the one composed of a4 and ?2 subunits (a4?2 receptors). The functional receptor is composed of five subunits: two pairs of a4-?2 subunits form the two acetylcholine-binding sites (ACh, the neurotransmitter which activates the receptor) while the 5th subunit does not contribute to an ACh-binding site. The a4?2 receptor has two forms, one in which the 5th subunit is an a4 subunit and the other in which it is a ?2 subunit. The properties of the two forms differ physiologically and pharmacologically - for example when the 5th subunit is a4 the receptor has a low sensitivity to ACh, whereas when it is ?2 the sensitivity to ACh is high. Accordingly, the proposed research will use techniques to define the subunit stoichiometry and arrangement so that the properties of a defined type of receptor will be studied. The first two aims of this proposal are to determine the mechanism (Aim 1) and site of action (Aim 2) for drugs which potentiate the activation of a4?2 receptors. Such drugs have been used or proposed for treatment of a number of disorders, including Alzheimer's disease. However, the site and mechanism of action of potentiating drugs are not known. Studies of function will emphasize analysis of receptor kinetics to provide novel information, while molecular studies will manipulate amino acids at specific subunit interfaces.
The final aim will examine the structures in the a4 and ?2 subunits which influence the ability of these subunits to assemble as the 5th subunit in the a4?2 receptor. Little is known about the structural basis of the rules for assembly of this receptor. Since the properties of the assembled receptor depend on its subunit composition, understanding the basis for selecting the 5th subunit is important for understanding how the overall properties of receptors in a neuron are affected by the palette of subunits expressed in the cell. The knowledge to be gained in the research will provide insights into how receptor function is modulated by chemicals, including clinically used drugs, and will increase our understanding of the mechanisms for the control of receptor function. Further, this work will provide new information on the physiological properties of the two forms of the a4?2 receptor and examine the mechanism for determining the choice of the 5th subunit. To accomplish these aims, receptors of known subunit composition and arrangement, and with mutations at specific interfaces, will be studied.

Public Health Relevance

Receptors for neurotransmitters allow cells in the brain to communicate, and their proper function is essential to normal brain function. They are also the targets for many drugs, both clinically used and recreational. The goal of this research is to understand how these receptors function, how cells assemble receptors and how frequently used drugs act on receptors.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS022356-28
Application #
8460532
Study Section
Neurotransporters, Receptors, and Calcium Signaling Study Section (NTRC)
Program Officer
Silberberg, Shai D
Project Start
1984-12-01
Project End
2014-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
28
Fiscal Year
2013
Total Cost
$314,445
Indirect Cost
$107,573
Name
Washington University
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Bracamontes, John R; Akk, Gustav; Steinbach, Joe Henry (2016) Introduced Amino Terminal Epitopes Can Reduce Surface Expression of Neuronal Nicotinic Receptors. PLoS One 11:e0151071
Jin, Xiaochun; Steinbach, Joe Henry (2015) Potentiation of Neuronal Nicotinic Receptors by 17β-Estradiol: Roles of the Carboxy-Terminal and the Amino-Terminal Extracellular Domains. PLoS One 10:e0144631
Jin, Xiaochun; Bermudez, Isabel; Steinbach, Joe Henry (2014) The nicotinic *5 subunit can replace either an acetylcholine-binding or nonbinding subunit in the *4*2* neuronal nicotinic receptor. Mol Pharmacol 85:11-7
Haller, Gabe; Li, Ping; Esch, Caroline et al. (2014) Functional characterization improves associations between rare non-synonymous variants in CHRNB4 and smoking behavior. PLoS One 9:e96753
Akk, Gustav; Eaton, Megan; Li, Ping et al. (2013) Energetic contributions to channel gating of residues in the muscle nicotinic receptor β1 subunit. PLoS One 8:e78539
Haller, Gabe; Druley, Todd; Vallania, Francesco L et al. (2012) Rare missense variants in CHRNB4 are associated with reduced risk of nicotine dependence. Hum Mol Genet 21:647-55
Tammimäki, Anne; Herder, Penelope; Li, Ping et al. (2012) Impact of human D398N single nucleotide polymorphism on intracellular calcium response mediated by α3β4α5 nicotinic acetylcholine receptors. Neuropharmacology 63:1002-11
Hinrichs, Anthony L; Murphy, Sharon E; Wang, Jen C et al. (2011) Common polymorphisms in FMO1 are associated with nicotine dependence. Pharmacogenet Genomics 21:397-402
Steinbach, Joe Henry; Akk, Gustav (2011) Use of concatemers of ligand-gated ion channel subunits to study mechanisms of steroid potentiation. Anesthesiology 115:1328-37
Jin, Xiaochun; Steinbach, Joe Henry (2011) A portable site: a binding element for 17β-estradiol can be placed on any subunit of a nicotinic α4β2 receptor. J Neurosci 31:5045-54

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