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The Marshall W. Nirenberg Papers

Title:
Laboratory project: "Studies of Action Potential and Receptor Ionophores" pdf (248,355 Bytes) transcript of pdf
Laboratory project: "Studies of Action Potential and Receptor Ionophores"
Description:
Summary of work for this project as indicated on the report: "The objective of this project is to develop biochemical methods for studies of action potential and receptor ionophores leading eventually to isolation of these macromolecules and characterization at both the molecular and cellular levels. Our current efforts are directed toward developing reagents, mainly neurotoxins, which act on the action potential Na+ ionophore, preparing radioactively labelled (sic) derivatives, and using these reagents to characterize the ionophore at the cellular level and to solubilize and eventually isolate it. The nicotinic acetylcholine receptor ionophore is also under study at the cellular level."
Item is a photocopy.
Number of Image Pages:
3 (248,355 Bytes)
Date:
1976-09 (September 1976)
Creator:
Catterall, W. A.
Ray, Radharaman
Huang, L. M.
National Heart and Lung Institute. Laboratory of Biochemical Genetics
Rights:
This item is in the public domain. It may be used without permission.
Exhibit Categories:
From Neuroblastoma to Homeobox Genes, 1976-1992
Neuroblastoma Research, 1967-1976
Relation:
Metadata Record Annual Report of the Laboratory of Biochemical Genetics, October 1, 1975 - September 30, 1976 (September 1976)
Box Number:
31 of 31
Unique Identifier:
JJBBPG
Accession Number:
2001-020
Document Type:
Reports
Excerpts
Language:
English
Format:
application/pdf
image/tif
Physical Condition:
Good
Transcript:
Project Number: Z01 HL 00004-03 LBG
Period Covered: July 1, 1975 through June 30, 1976
Title of Project: Studies of Action Potential and Receptor Ionophores
Names, Laboratory and Institute Affiliations, and Titles of Principal Investigations and All Other Professional Personnel Engaged on the Project:
PI: W.A. Catterall, Staff Fellow, LBG NHLI
R. Ray, MARC Fellow/NIGMS, LBG NHLI
L. M. Huang, NIH Postdoctoral Fellow, LBG NHLI and BP NINCDS
Cooperating Units (if any): Laboratory of Biophysics, NINCDS
Lab/Branch: Laboratory of Biochemical Genetics
Section: Section on Molecular Biology
Institute and Location: NHLI, NIH, Bethesda, Maryland 20014
Total Man Years: 2.7
Professional: 1.7
Other: 1
Summary of Work:
The objective of this project is to develop biochemical methods for studies of action potential and receptor ionophores leading eventually to isolation of these macromolecules and characterization at both the molecular and cellular levels. Our current efforts are directed toward developing reagents, mainly neurotoxins, which act on the action potential Na+ ionophore, preparing radioactively labeled derivatives, and using these reagents to characterize the ionophore at the cellular level and to solubilize and eventually isolate it. The nicotinic acetylcholine receptor ionophore is also under study at the cellular level.
Project Description:
Objectives: The objectives of this project are (1) to develop biochemical methods for study of action potential and receptor ionophores, (2) to use these methods to study the mechanism of action of these macromolecules at the cellular and membrane levels, and (3) to solubilize, purify, and characterize these ionophores at the molecular level.
Methods Employed: Biochemical assays which measure changes in passive Na+ influx were used to study the acetylcholine receptor ionophore and the action potential Na+ ionophore.
Major Findings: Previous results led to the conclusion that (1) the neurotoxic alkaloids veratridine, batrachotoxin, and aconitine activate the action potential Na+ ionophore by interaction with a single class of sites; (2) scorpion venom activates the ionophore by interaction with a different class of sites; (3) the sites of action of these 2 classes of toxin are allosterically coupled in a highly cooperative manner; and (4) the inhibitors tetrodotoxin and saxitoxin act at a separate site directly involved in ion transport by the ionophore.
The active component of scorpion venom has been purified using its ability to activate the action potential Na+ ionophore as a specific assay. The toxin is a polypeptide having a molecular weight of 6700, an isoelectric point of 9.8, and lacking methionine and histidine. The purified toxin retains the ability to act cooperatively with each of the 3 alkaloids. It acts reversibly at a single class of sites with an apparent dissociation constant of 1 to 2 nM. The action of the toxin is highly membrane potential dependent. Depolarization of the cells causes a 30 fold increase in apparent dissociation constant. These results suggest that scorpion toxin binds to a voltage sensitive component of the Na+ ionophore that acts cooperatively in regulating its ion transport activity.
We have prepared an 125I-labeled derivative of scorpion toxin which retains biologic activity. Using this derivative we have detected a small class of saturable binding sites in electrically excitable neuroblastoma cells but not in neuroblastoma cells defective in electrical activity. Binding of scorpion toxin to these sites is voltage dependent as is the effect of the toxin on ion transport activity. Preliminary estimates of the number of sites are in the range of 3 to 6 fmole/mg cell protein or less than 1 site per mu-m2 of cell membrane. This labeled toxin derivative appears to provide an important new tool in studies of the Na+ ionophore.
Significance to Biomedical Research: The results provide new insights into the mechanism of action and regulation of membrane macromolecules involved in information transfer and processing in the nervous system and in maintenance of normal beating in heart.
Proposed Course: Planned investigations include (1) completing the analysis of scorpion toxin binding to excitable membranes of neuroblastoma cells, nerve axons, and heart muscle; (2) preparing labeled derivatives of saxitoxin and comparing binding with scorpion toxin; (3) studying the voltage dependent aspects of scorpion toxin binding in detail and relating them to the electrophysiologic properties of the ionophore; and (4) attempting to solubilize and purify the binding sites for scorpion toxin and saxitoxin and thus isolate the action potential Na+ ionophore.
Publications:
1. Catterall, W. A.: Activation of the action potential Na+ ionophore of cultured neuroblastoma cells by veratridine and batrachotoxin. J. Biol. Chem. 250: 4053-4059, 1975.
2. Catterall, W. A.: Cooperative activation of the action potential Na+ ionophore by neurotoxins. Proc. Natl. Acad. Sci. USA 72: 1782-1786, 1975.
3. Catterall, W. A. and Ray, R.: Interactions of neurotoxins with the action potential Na+ ionophore. J. Supramolecular Structure, in press.
4. Catterall, W. A.: Purification of a topic protein from scorpion venom which activates the action potential Na+ ionophore. J. Biol. Chem., in press.
5. Catterall, W. A., Ray, R. and Morrow, Cynthia S.: Membrane potential dependent binding of scorpion toxin to the action potential Na+ ionophore. Proc. Natl. Acad. Sci. USA, in press.
Metadata Last Modified Date:
2010-10-04
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