Assembly of chimeric connexin-aequorin proteins into functional gap junction channels. Reporting intracellular and plasma membrane calcium environments.
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. Paris 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.
Martin
P E
PE
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom.
George
C H
CH
Castro
C
C
Kendall
J M
JM
Capel
J
J
Campbell
A K
AK
Revilla
A
A
Barrio
L C
LC
Evans
W H
WH
D000331:Aequorin / Q000235:genetics*; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002450:Cell Communication; D002462:Cell Membrane / Q000378:metabolism; D018031:Connexin 43 / Q000235:genetics*; D017630:Connexins / Q000235:genetics*; D017629:Gap Junctions / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D015640:Ion Channel Gating; D008163:Luminescent Measurements; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014162:Transfection; D000097002:Gap Junction beta-1 Protein
D000331:Aequorin; D002118:Calcium; D018031:Connexin 43; D017630:Connexins; D011993:Recombinant Fusion Proteins; D000097002:Gap Junction beta-1 Protein