Permeability and gating properties of human connexins 26 and 30 expressed in HeLa cells

https://doi.org/10.1016/S0006-291X(03)00868-4Get rights and content

Abstract

Human connexins 26 and 30 were expressed either through the bicistronic pIRES-EGFP expression vector or as EYFP-tagged chimeras. When transiently transfected in communication-incompetent HeLa cells, hCx26–pIRES transfectants were permeable to dyes up to 622 Da, but were significantly less permeable to 759 Da molecules. Under the same conditions, permeability of hCx26–EYFP fusion products was comparable to that of hCx26–pIRES, but with significant increase in diffusion at 759 Da, possibly as a consequence of having selected large fluorescent junctional plaques. Dye transfer was limited to 457 Da in hCx30–EYFP transfectants. When reconstructed from confocal serial sections, fluorescent plaques formed by hCx26–EYFP and hCx30–EYFP appeared irregular, often with long protrusions or deep invagination. Similar plaques were observed following immunostaining both in cells transfected with hCx26–pIRES and in HeLa cells stably transfected with mouse Cx26. Tissue conductance (Tgj) displayed significantly smaller values (28.8 ± 1.8 nS) for stably transfected mCx26 than transiently transfected hCx26 (43.5 ± 3.3 nS). These differences reflected in distinct functional dependence of normalized junctional conductance (Gj) on transjunctional voltage (Vj). The half-activation voltage for Gj was close to ±95 and ±58 mV in mCx26 and hCx26, respectively. The corresponding parameters for hCx30 transfectants were Tgj=45.2±3.5 nS and V0=±34 mV. These results highlight unexpected differences between mCx26 and hCx26 in this expression system, reinforce the concept that channel permeability may be related to Cx level expression, and indicate that fusion of hCx30 to GFP colour mutants produces channels that are suitable for permeability and gating studies.

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,

References (30)

  • J. Xia et al.

    Mutations in the gene encoding gap junction protein β-3 associated with autosomal dominant hearing impairment

    Nat. Genet.

    (1998)
  • J. Lamartine et al.

    Mutations in GJB6 cause hidrotic ectodermal dysplasia

    Nat. Genet.

    (2000)
  • P.E.M. Martin et al.

    Properties of connexin26 gap junctional proteins derived from mutations associated with non-syndromal hereditary deafness

    Hum. Mol. Gen.

    (1999)
  • E. Thonnissen et al.

    Human connexin26 (GJB2) deafness mutations affect the function of gap junction channels at different levels of protein expression

    Hum. Genet.

    (2002)
  • W.L. Di et al.

    Defective trafficking and cell death is characteristic of skin disease-associated connexin 31 mutations

    Hum Mol Genet.

    (2002)
  • Cited by (56)

    • Diversity in connexin biology

      2023, Journal of Biological Chemistry
    • Determinants of Cx43 Channel Gating and Permeation: The Amino Terminus

      2016, Biophysical Journal
      Citation Excerpt :

      Members of the Cx gene family have unique patterns of expression that frequently include coexpression of two or more isotypes in a cell type (38). Although Cx isotypes share significant sequence similarity, their differences, even among the most similar isotypes, underlie critical functional differences (39–44). Cx43 and Cx37 coexpress in several cell types in vivo including vascular endothelial and smooth muscle cells, where the functional consequences of their expression can be quite different (45,46).

    • Distinct permeation profiles of the connexin 30 and 43 hemichannels

      2014, FEBS Letters
      Citation Excerpt :

      In any case the difference between Cx30 and Cx43 hemichannel permeability does not seem to reflect the selectivity of cell–cell channels. Cx30 cell–cell channels are reported to be selective to cationic dyes with little or no permeability to anionic dyes [71–74], whereas Cx43 cell–cell channels seem to discriminate on size rather than charge [75]. Therefore the results indicate that caution should be taken when inferring properties of cell–cell channels directly to hemichannels.

    • The role of connexins in ear and skin physiology - Functional insights from disease-associated mutations

      2013, Biochimica et Biophysica Acta - Biomembranes
      Citation Excerpt :

      Cx26 gap junction channels are readily permeable to the negatively charged dyes, Lucifer yellow, calcein, and carboxyfluorescein, but Cx30 gap junction channels are largely impermeable to these molecules, even when comparisons are made between cells with very similar electrical coupling. In contrast to the anionic dyes, both connexin gap junctions are equally permeable to positively charged dyes like ethidium bromide or propidium iodide [76,79,80]. Cx26 was also found to mediate Ca++ wave propagation between cells more efficiently than Cx30 [36].

    View all citing articles on Scopus
    View full text