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Connexin-aequorin chimerae report cytoplasmic calcium environments along trafficking pathways leading to gap junction biogenesis in living COS-7 cells

George, Christopher H., Kendall, Jonathan M., Campbell, Anthony Keith and Evans, William Howard 1998. Connexin-aequorin chimerae report cytoplasmic calcium environments along trafficking pathways leading to gap junction biogenesis in living COS-7 cells. Journal of Biological Chemistry 273 (45) , pp. 29822-29829. 10.1074/jbc.273.45.29822

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Abstract

The cytoplasmic calcium environments along membrane trafficking pathways leading to gap junction intercellular communication channels at the plasma membrane were studied. Connexins, the constitutive proteins of gap junctions, were fused at their carboxyl terminus to the calcium-sensitive photoprotein aequorin. The cellular location of the chimeric proteins was determined by immunolocalization and subcellular fractionation. The generation of functional gap junctions by the connexin chimerae was monitored by the ability of the cells to exchange small dyes. Although aequorin fused to connexin-26 was nonfunctional, its ability to report Ca2+ and to form functional gap junctions was rescued by replacement of its cytoplasmic carboxyl tail with that of connexin-43. In COS-7 cells expressing these connexin-aequorin chimerae, calcium levels below the plasma membrane were higher (∼5 μm) than those in the cytoplasm (∼100 nm); gap junctions were able to transfer dyes under these conditions. Cytoplasmic levels of free calcium surrounding the ERGIC/Golgi reported by connexin-43 chimera (∼420 nm) were twice those measured by connexin-32 chimera (∼200 nm); both chimerae measured calcium levels substantially higher than those reported by a connexin-26 chimera (∼130 nm). Dispersion of the ERGIC and Golgi complex by brefeldin A led to a marked reduction in calcium levels. The results show that the various connexin chimerae were located in spatially different subcellular stores and that the ERGIC/Golgi regions of the cell maintain heterogeneous cytoplasmic domains of calcium. The implications of the subplasma-membrane Ca2+ levels on the gating of gap junctions are discussed.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Medicine
Pharmacy
Subjects: Q Science > QD Chemistry
R Medicine > RM Therapeutics. Pharmacology
Publisher: American Society for Biochemistry and Molecular Biology
ISSN: 0021-9258
Last Modified: 28 Jun 2019 02:45
URI: http://orca-mwe.cf.ac.uk/id/eprint/22518

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