The proposed research concerns the molecular basis of electrical excitability in nerve, muscle and related cells. The specific focus of our interest is the voltage-regulated sodium channel which mediates the early, regenerative inward currents of the propagated action potential. In our previous work we have isolated the tetrodotoxin (TTX) binding component of the sodium channel from the electric organ of Electrophorus electricus, and have provided information about its size, stability, peptide, amino-acid and carbohydrate compositions as well as preliminary observations of its appearance under the electron microscope, both in solubilized and reconstituted preparations. We have reincorporated this protein into artificial liposomes and demonstrated neurotoxin modulated ion transport. The protein exhibits sites for TTX and STX binding and blockade of transport, and sites for functional binding of alkaloid neurotoxins and local anesthetics, but apparently not for channel specific peptide neurotoxins. Our continuing research represents application of methods of protein biochemistry and membrane biophysics toward elucidation of the protein structure and mechanisms for ion-selective transport, voltage regulation and mechanisms of drug and anesthetic interaction with the channel.
The specific aims i nclude (1) determining the minimal components required to account for channel biophysics and pharmacology, and perfection of methods for their rapid isolation, (2) perfection of biochemical and biophysical assays of channel function, as regulated by voltage or pharmacological reagents, (3) to further describe the structure of the channel by methods of protein chemistry and electron microscopy, (4) to map the protein for sites which may be readily chemically modified, in portions of the molecule involved in conformational changes associated with gating, and (5) where suitable, to apply some of the methods, equipment and technology developed with the sodium channel to problems of other ion channels of excitable membranes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS017928-07
Application #
3397941
Study Section
Physiology Study Section (PHY)
Project Start
1981-12-01
Project End
1989-11-30
Budget Start
1987-12-01
Budget End
1988-11-30
Support Year
7
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Bendahhou, S; Cummins, T R; Agnew, W S (1997) Mechanism of modulation of the voltage-gated skeletal and cardiac muscle sodium channels by fatty acids. Am J Physiol 272:C592-600
Bendahhou, S; Agnew, W S (1996) Enhancement of the Shaker B delta6-46 current by fatty acids depends on the activation of the lipoxygenase metabolic pathway. Pflugers Arch 432:1091-3
Bendahhou, S; Cummins, T R; Potts, J F et al. (1995) Serine-1321-independent regulation of the mu 1 adult skeletal muscle Na+ channel by protein kinase C. Proc Natl Acad Sci U S A 92:12003-7
Cummins, T R; Zhou, J; Sigworth, F J et al. (1993) Functional consequences of a Na+ channel mutation causing hyperkalemic periodic paralysis. Neuron 10:667-78
Potts, J F; Regan, M R; Rochelle, J M et al. (1993) A glial-specific voltage-sensitive Na channel gene maps close to clustered genes for neuronal isoforms on mouse chromosome 2. Biochem Biophys Res Commun 197:100-4
Tong, J; Potts, J F; Rochelle, J M et al. (1993) A single B1 subunit mapped to mouse chromosome 7 may be a common component of Na channel isoforms from brain, skeletal muscle and heart. Biochem Biophys Res Commun 195:679-85
Emerick, M C; Shenkel, S; Agnew, W S (1993) Regulation of the eel electroplax Na channel and phosphorylation of residues on amino- and carboxyl-terminal domains by cAMP-dependent protein kinase. Biochemistry 32:9435-44
Ukomadu, C; Zhou, J; Sigworth, F J et al. (1992) muI Na+ channels expressed transiently in human embryonic kidney cells: biochemical and biophysical properties. Neuron 8:663-76
Hingorani, S R; Agnew, W S (1991) A rapid ion-exchange assay for detergent-solubilized inositol 1,4,5-trisphosphate receptors. Anal Biochem 194:204-13
Trimmer, J S (1991) Immunological identification and characterization of a delayed rectifier K+ channel polypeptide in rat brain. Proc Natl Acad Sci U S A 88:10764-8

Showing the most recent 10 out of 25 publications