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Biochem Biophys Res Commun
2006 Oct 20;3492:855-62. doi: 10.1016/j.bbrc.2006.08.121.
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Cloning, embryonic expression, and functional characterization of two novel connexins from Xenopus laevis.
de Boer TP
,
Kok B
,
Roël G
,
van Veen TA
,
Destrée OH
,
Rook MB
,
Vos MA
,
de Bakker JM
,
van der Heyden MA
.
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Vertebrate gap junctions are constituted of connexin (Cx) proteins. In Xenopus laevis, only seven different Cxs have been described so far. Here, we identify two new Cxs from X. laevis. Cx28.6 displays > 60% amino acid identity with human Cx25, Cx29 displays strong homology with mouse Cx26 and Cx30. Cx29 is expressed throughout embryonic development. Cx28.6 mRNA is only transiently found from stage 22 to 26 of development. While no Cx28.6 expression could be detected by whole mount in situ hybridization, expression of Cx29 was found in the developing endoderm, lateralmesoderm, liver anlage, pronephros, and proctodeum. Ectopic expression of Cx28.6 failed to produce functional gap-junctions. In contrast, ectopic expression of full-length Cx29 in HEK293 and COS-7 cells resulted in the formation of gap junction-like structures at the cell-cell interfaces. Ectopic expression of Cx29 in communication deficient N2A cell pairs led to functional electrical coupling.
Fig. 1.
(A) Comparison of the deduced amino acid sequence of Cx28.6 with that of a presumed X. tropicalis Cx, human Cx25, and two presumed Cxs from bovine and pufferfish. (B) Comparison of the deduced amino acid sequence of Cx29 with those of X. tropicalis Cx26, mouse Cx26 and Cx30, and chicken Cx31. Putative transmembrane regions (TM1 to TM4) were predicted with the web-based program TMHMM2.0 [25] and indicated with dashed lines above the sequence. Conserved cysteine-residues within the presumed extracellular loops are indicated by a “C” above the sequence. Identical amino acids between the Xenopus and other sequences are shaded black. Abbreviations: Bt, Bos taurus; Gg, Gallus gallus; Hs, Homo sapiens; Mm, Mus musculus; Tn, Tetraodon nigroviridis; Xl, Xenopus laevis; Xt, Xenopus tropicalis. Accession numbers and Ensembl gene identification (X. tropicalis Cx28.6 orthologue 18051) are depicted in Table 1.
Fig. 2.
Phylogenetic tree of 9 X. laevis Cxs. Halocynthia pyriformis (sea peach) Cx47 was used as an outgroup. The open reading frame nucleotide sequences were analyzed using ClustalW method and Neighbor-Joining algorithm. Support for each node (numbers) was determined by interior-branch test. α and β indicate the two main Cx subgroups.
Fig. 3.
Temporal expression of Cx28.6 and Cx29. RT-PCR analysis of X. laevis total RNA at different stages of development. Note that Cx28.6 is only weakly expressed at stages 22 and 26. Nieuwkoop and Faber stages are indicated at the top. PCR with histone H4 was used as loading control.
Fig. 4.
Dynamic expression of Cx29 during X. laevis development detected by whole-mount in situ hybridization. (A) Stage 10.25 embryo with dorsal side up. (B) Detail of stage 10.25 embryo shown in (A). (C–E) Stage 21 embryos with lateral view (C), dorsal view (D) and ventral view (E). Stage 25 (F), 26 (G), and 27 (H). I. Tailbud stage embryo shows specific expression in liver anlage (li) and pronephros (pn). pd, proctodeum; cg, cement gland; d, dorsal; v, ventral; a, anterior; p, posterior.
Fig. 5.
Localization of ectopically expressed Cx28.6-EGFP and Cx29-EGFP in HEK293 cells. Arrow indicates typical gap-junctional distribution of Cx29-EGFP-tagged fusion protein.
Fig. 6.
Western blot analysis of ectopically expressed Cx28.6 and Cx29. Cx28.6 (Cx28.6-EGFP) and Cx29 (Cx29-EGFP) fusion constructs were expressed in COS-7 cells. Cell lysates were analyzed for the presence of EGFP containing proteins by Western blotting using EGFP-antibody. Empty EGFP expression construct (EGFP) was used as control. Molecular weights (kDa) are indicated on the right.
Fig. 7.
Electrophysiological characterization of Cx29 gap junction channels in N2A cells. (A) Halothane mediated reversible inhibition of gap-junctional coupling. 1, full uncoupling following Halothane application. 2, full recovery following Halothane washout. (B) Recording of junctional currents showing mild time and voltage dependent inactivation in response to transjunctional potentials ranging between −100 and +100 mV, with 20 mV steps. Vertical scale 2 nA, horizontal scale 1 s. (C) Voltage dependence of gap junctional steady-state conductance. Mean normalized steady-state conductance ± SEM (N = 7) is plotted against transjunctional potential and fitted with a two-state Boltzmann equation. (D) Representative recording of single channel activity in a poorly coupled cell pair, transjunctional potential of −70 mV. Dashed lines represent conductance states of approximately 120, 60, and 0 pS (top to bottom).
gjb2 (gap junction protein beta 2) gene expression in Xenopus laevis, via in situ hybridization, NF stage 27 embryo, lateral view, anteriorleft.
gjb2 (gap junction protein beta 2) gene expression in Xenopus laevis, via in situ hybridization, NF stage 33 and 34 embryo, lateral view, anteriorleft.
