Biochemical and Biophysical Research Communications
Permeability and gating properties of human connexins 26 and 30 expressed in HeLa cells
Section snippets
Materials and methods
Reagents. Alexa Fluor 488, Alexa Fluor 594, and calcein were purchased from Molecular Probes (Eugene, OR). Neurobiotin and 7-amino-4-methylcoumarin-3-acetic acid (AMCA)–streptavidin were from Vector Laboratories (Burlingame, CA). The polyclonal anti-rat Cx26 antibody employed was from Zymed Laboratories (San Francisco, CA). The secondary antibody was a tetramethylrhodamine isothiocyanate (TRITC)-conjugated affinity purified goat anti-rabbit IgG from Jackson ImmunoResearch Laboratories (West
Subcellular localization of connexins in transfected HeLa cells
Immunofluorescence performed with an anti-Cx26 antibody produced no signal in parental (communication incompetent), untransfected HeLa cells, but showed positive staining localized at cell–cell contact areas in a clone of HeLa cells stably transfected with mCx26 (not shown).
To compare the properties of murine (mCx26) and human Cx26 (hCx26), the coding region of wild-type (wt) hCx26 cDNA was subcloned into pIRES-EGFP and transiently expressed in parental HeLa cells [19]. The cellular
Discussion and conclusions
Connexin functional studies have been carried out in expression system by reconstituting gap-junction communication either by microinjecting cRNAs in Xenopus laevis oocytes [15], [16], [30] or by transfecting mammalian cells devoid of endogenous connexins with relevant cDNAs [9], [10], [11], [12], [13], [14], [15], [16].
The construction of expression vectors that allow the identification of transfected cells by means of a co-expressed fluorescent reporter (e.g., the enhanced green fluorescent
Acknowledgments
This work was supported by Grants from Telethon (Grant GGP02043), MIUR-PRIN 2002, and MIUR-FIRB 2001 (F.M. and P.D’A.), Regione Friuli Venezia-Giulia (P.D’A.) and INFM TSSEBB2 (F.M.). We thank Sabina Morassutto for assistance, Roberto Bruzzone (Institut Pasteur, Paris, France) for hCx26 and Cx30 cDNAs, and Klaus Willecke (University of Bonn, Germany) and Marc Mesnil (University of Poitieres, France) for the generous gift of the communication-incompetent and mCx26 expressing HeLa clones,
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