Abstract

This proposal will study the structure and function of the gap junction, and its role in intercellular communication. Electron image data will be obtained from frozen-hydrated specimens. Dynamic parts of the channel (connexon) involved in channel gating and in connexon-connexon interaction will be characterized by comparative electron microscopy and X-ray diffraction studies under a range of conditions which alter these flexible components. The domains indentified in the connexon structure will be correlated with the amino acid sequence determined from the recombinant DNA. Communication will also be studied using site- specific antisera generated using synthetic oligopeptides which correspond to defined domains within the amino acid sequence predicted from the liver gap junction 32kD cDNA. These antisera will be used for structural mapping of the topology of the gap junction protein by immunocytochemistry of isolated intact and urea-split gap junctions. The antisera will be tested for physiological activity in the AR4-2J pancreas cell line, and in Xenopus oocytes. Ovarian granulosa cDNA libraries will be made from pregnant mare serum gonadotrophin-stimulated rat ovaries prescreened by Northern analysis to demonstrate positive hybridization with the coding region of liver gap junction cDNA. This library will then be screened with the same probe in order to clone the granulosa gap junction cDNA. Synthetic mRNAs generated from liver, ovarian granulosa, and myocardial gap junction cNDAs will be intracellularly injected into Xenopus oocytes which have been manipulated into contact. In these """"""""reconstructed"""""""" systems, channel properties will be measured in voltage-clamped cells, and differences in liver, heart and granulosa channels measured in the context of an invariant oocyte cytoplasm. These differences in physiology will be explored further by attempting to construct liver/granulosa hybrid gap junctions: junctions with tissue-specific proteins apposed across the gap in asymmetrically-injected oocytes. Site-specific and tissue-specific antisera will be used to provide rigorous control of these experiments.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM018974-19
Application #
3484275
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1975-01-01
Project End
1992-12-31
Budget Start
1990-01-01
Budget End
1990-12-31
Support Year
19
Fiscal Year
1990
Total Cost
Indirect Cost

  Institution

Name
Harvard University
Department
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115

  Publications

Bruzzone, R; White, T W; Paul, D L (1994) Expression of chimeric connexins reveals new properties of the formation and gating behavior of gap junction channels. J Cell Sci 107 ( Pt 4):955-67
Bruzzone, R; White, T W; Scherer, S S et al. (1994) Null mutations of connexin32 in patients with X-linked Charcot-Marie-Tooth disease. Neuron 13:1253-60
Musil, L S; Goodenough, D A (1993) Multisubunit assembly of an integral plasma membrane channel protein, gap junction connexin43, occurs after exit from the ER. Cell 74:1065-77
Bruzzone, R; Haefliger, J A; Gimlich, R L et al. (1993) Connexin40, a component of gap junctions in vascular endothelium, is restricted in its ability to interact with other connexins. Mol Biol Cell 4:7-20
Flint, K K; Rosbash, M; Hall, J C (1993) Transfer of dye among salivary gland cells is not affected by genetic variations of the period clock gene in Drosophila melanogaster. J Membr Biol 136:333-42
White, T W; Bruzzone, R; Goodenough, D A et al. (1992) Mouse Cx50, a functional member of the connexin family of gap junction proteins, is the lens fiber protein MP70. Mol Biol Cell 3:711-20
Harris, A L; Walter, A; Paul, D et al. (1992) Ion channels in single bilayers induced by rat connexin32. Brain Res Mol Brain Res 15:269-80
Musil, L S; Goodenough, D A (1991) Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques. J Cell Biol 115:1357-74
Larson, D M; Haudenschild, C C; Beyer, E C (1990) Gap junction messenger RNA expression by vascular wall cells. Circ Res 66:1074-80
Musil, L S; Goodenough, D A (1990) Gap junctional intercellular communication and the regulation of connexin expression and function. Curr Opin Cell Biol 2:875-80

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