Laboratory project: "Studies of Action Potential and Receptor Ionophores"
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."
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1976-09 (September 1976)
Catterall, W. A.
Huang, L. M.
National Heart and Lung Institute. Laboratory of Biochemical Genetics
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From Neuroblastoma to Homeobox Genes, 1976-1992
Neuroblastoma Research, 1967-1976
Annual Report of the Laboratory of Biochemical Genetics, October 1, 1975 - September 30, 1976 (September 1976)
31 of 31
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
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.
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
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.
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.