The nicotinic acetylcholine receptor channel (nAChRC) is the best understood ion channel, it transduces the chemical signal at the neuromuscular junction and is the target of pathogenic antibodies in myasthenia gravis. The goal of this proposal is to better understand how the channel protein structure produces its functional properties, specifically, how electrical charge on the surface of the protein influences ion transport. A series of receptor channels with single charged amino acid mutations will be created by site-directed mutagenesis using a uracil-enriched template cDNA. Channel protein expression in Xenopus oocytes and single channel recording under conditions of varied ionic strength and permeant ion concentration will be used to determine the conductance of the channel. The conductance and the shape of the current- voltage relations of the mutant receptor channels will be compared to the wild type receptor. These data will be interpreted in terms of a computational model of the electric potential profile of the nAChRC. From this analysis the relative electrical distances of the point charges to the pore mouth will be calculated. Examination of the sidedness of the effect of changes to the pore mouth will be calculated. Examination of the sidedness of the effect of changes in ionic strength and the rectification of the current-voltage relation will allow the localization of the charge change to the cytoplasmic or extracellular side of the pore complementary to what is currently known about the transmembrane topology from protein chemistry and antibody analysis. The candidate, through work on this project, hopes to refine his skills in ion channel expression and single channel recording, and acquire new skills in DNA cloning and site-directed mutagenesis. He will ultimately apply these techniques to the study of voltage-dependent ion channels from cardiac tissue, specifically the sodium channel from ventricular muscle. He hopes to study questions related to the molecular nature of the local anesthetic antiarrhythmic drug binding site in this channel.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08HL002421-03
Application #
3082814
Study Section
Research Manpower Review Committee (MR)
Project Start
1990-04-01
Project End
1995-03-31
Budget Start
1992-04-01
Budget End
1993-03-31
Support Year
3
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
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Nuss, H B; Tomaselli, G F; Marban, E (1995) Cardiac sodium channels (hH1) are intrinsically more sensitive to block by lidocaine than are skeletal muscle (mu 1) channels. J Gen Physiol 106:1193-209
Ashen, M D; O'Rourke, B; Kluge, K A et al. (1995) Inward rectifier K+ channel from human heart and brain: cloning and stable expression in a human cell line. Am J Physiol 268:H506-11
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Tomaselli, G F; McLaughlin, J T; Jurman, M E et al. (1991) Mutations affecting agonist sensitivity of the nicotinic acetylcholine receptor. Biophys J 60:721-7