MOLECULAR PHYSIOLOGY OF CLONED ION CHANNELS

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date4/1/913/31/92

Funding

  • National Heart, Lung, and Blood Institute

ASJC

  • Cellular and Molecular Neuroscience
  • Biophysics
  • Structural Biology
  • Physiology
  • Pharmacology

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