Abstract

An interdisciplinary approach, involving molecular biology and electrophysiology, for cloning and studying structure and expression of genes coding for ion channels and neurotransmitter receptors expressed in mammalian brain and skeletal and cardiac muscle is proposed. Major emphasis will be placed on developing a new general method for cloning such genes for those cases in which the conventional approach through protein purification is not readily applicable. The method is based on electrophysiological assays for channels or receptors expressed on the oocyte surface by microinjection of mRNA into Xenopus oocytes, combined with cDNA cloning, and hybrid selection or hybrid arrest procedures. The first goals will be the isolation of genes coding for voltage sensitive calcium channels and for serotonin receptors. The chromosomal genes for the voltage sensitive sodium channels of brain, heart, and muscle will be isolated by standard methods from a presently available partial cDNA clone for the brain gene. Special emphasis will be placed on determining the molecular basis of the diversity of gene products for all the channels of interest, and to the significance of this diversity in signal processing. In all cases, high precision characterization of the electrophysiological properties of the channels expressed in abundance in the oocyte will be carried out by single channel patch clamping methods.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM010991-30
Application #
3268203
Study Section
Neurology C Study Section (NEUC)
Project Start
1977-06-01
Project End
1991-05-31
Budget Start
1989-06-01
Budget End
1990-05-31
Support Year
30
Fiscal Year
1989
Total Cost
Indirect Cost

  Institution

Name
California Institute of Technology
Department
Type
Biomed Engr/Col Engr/Engr Sta
DUNS #
078731668
City
Pasadena
State
CA
Country
United States
Zip Code
91125

  Publications

Ho, B Y; Karschin, A; Raymond, J R et al. (1992) Expression in animal cells of the 5-HT1A receptor by a vaccinia virus vector system. FEBS Lett 301:303-6
Karschin, A; Ho, B Y; Labarca, C et al. (1991) Heterologously expressed serotonin 1A receptors couple to muscarinic K+ channels in heart. Proc Natl Acad Sci U S A 88:5694-8
Yang, X C; Karschin, A; Labarca, C et al. (1991) Expression of ion channels and receptors in Xenopus oocytes using vaccinia virus. FASEB J 5:2209-16
Karschin, A; Aiyar, J; Gouin, A et al. (1991) K+ channel expression in primary cell cultures mediated by vaccinia virus. FEBS Lett 278:229-33
Walter, A E; Hoger, J H; Labarca, C et al. (1991) Low molecular weight mRNA encodes a protein that controls serotonin 5-HT1c and acetylcholine M1 receptor sensitivity in Xenopus oocytes. J Gen Physiol 98:399-417
Hoger, J H; Rudy, B; Lester, H A et al. (1991) Characterization of maintained voltage-dependent K(+)-channels induced in Xenopus oocytes by rat brain mRNA. Brain Res Mol Brain Res 10:1-11
Guastella, J; Nelson, N; Nelson, H et al. (1990) Cloning and expression of a rat brain GABA transporter. Science 249:1303-6
Snutch, T P; Leonard, J P; Gilbert, M M et al. (1990) Rat brain expresses a heterogeneous family of calcium channels. Proc Natl Acad Sci U S A 87:3391-5
Krafte, D S; Goldin, A L; Auld, V J et al. (1990) Inactivation of cloned Na channels expressed in Xenopus oocytes. J Gen Physiol 96:689-706
Leonard, R J; Karschin, A; Jayashree-Aiyar, S et al. (1989) Expression of Drosophila Shaker potassium channels in mammalian cells infected with recombinant vaccinia virus. Proc Natl Acad Sci U S A 86:7629-33

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