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Vol. 273, Issue 3, 1719-1726, January 16, 1998
From the Chimeric proteins comprising connexins 26, 32, and 43 and aequorin, a chemiluminescent calcium indicator, were made by
fusing the amino terminus of aequorin to the carboxyl terminus of
connexins. The retention of function by the chimeric partners was
investigated. Connexin 32-aequorin and connexin 43-aequorin retained
chemiluminescent activity whereas that of connexin 26-aequorin was
negligible. Immunofluorescent staining of COS-7 cells expressing the
chimerae showed they were targeted to the plasma membrane. Gap junction intercellular channel formation by the chimerae alone and in
combination with wild-type connexins was investigated. Stable HeLa
cells expressing connexin 43-aequorin were functional, as demonstrated
by Lucifer yellow transfer. Pairs of Xenopus oocytes
expressing connexin 43-aequorin were electrophysiologically coupled,
but those expressing chimeric connexin 26 or 32 showed no detectable
levels of coupling. The formation of heteromeric channels constructed
of chimeric connexin 32 or connexin 43 and the respective wild-type
connexins was inferred from the novel voltage gating properties of the
junctional conductance. The results show that the preservation of
function by each partner of the chimeric protein is dictated mainly by the nature of the connexin, especially the length of the cytoplasmic carboxyl-terminal domain. The aequorin partner of the connexin 43 chimera reported calcium levels in COS-7 cells in at least two
different calcium environments.
Assembly of Chimeric Connexin-Aequorin Proteins into Functional
Gap Junction Channels
REPORTING INTRACELLULAR AND PLASMA MEMBRANE CALCIUM
ENVIRONMENTS
,,,,
Department of Medical Biochemistry,
University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN,
United Kingdom and § Departamento de Investigacion, Servicio
de Neurologìa Experimental, Hospital "Ramon y Cajal,"
Carretera de Colmenar Km 9.1, 28034 Madrid, Spain
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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