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doi: 10.1242/10.1242/jcs.00604

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Specific amino-acid residues in the N-terminus and TM3 implicated in channel function and oligomerization compatibility of connexin43

Valérie Lagrée^1,*, Karin Brunschwig^1,, Patricia Lopez¹, Norton B. Gilula^1,, Gabriele Richard² and Matthias M. Falk^1,¶

¹ Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
² Department of Dermatology and Cutaneous Biology, and Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, PA, USA

View larger version (67K): [in a new window]   Fig. 3. Functional analysis of wildtype and Cx43 amino-acid substitution variants. Connexin-expressing BHK cells with assembled connexin clusters detectable by auto-fluorescence of their GFP-tags (GFP) were microinjected with a mixture of lucifer yellow (LY) and rhodamine dextran (RD), and their capacity to transfer LY to the neighboring cells was investigated. Although all Cx43 variants assembled into clusters, only Cx43 wt channels (row 2) and channels assembled from the R153W (P4) variant (row 7) transferred dye. BHK wild-type cells injected as controls (row 1) showed no dye coupling. Representative cells were imaged before and after microinjection (dye-filled injection capillaries visible on the LY images are marked with arrows). RD co-injected into the control did not pass to neighboring cells, indicating that cells were not leaking dye. Phase contrast images are shown on the left.

View larger version (62K): [in a new window]   Fig. 1. Sequence and structure of connexin polypeptides. (A) Amino-acid sequence alignment of human (h) and ß connexins and their mouse (m) homologues. Four positions (P1 to P4, boxed) at which the physico-chemical properties of amino-acid residues in all considered connexins drastically differed from all considered ß connexins were identified. P1 and P2 are located next to each other in the N-terminal region and P3 and P4 at the cytoplasmic end of TM3. The connexin (Cx) molecular weight nomenclature was used. (B) Topological model of Cx43. The four trans-membrane spanning helices (TM1 to TM4), the two extracellular loops (Loop 1, Loop 2), the intracellular loop and the N- and C-terminal domains are indicated. Asterisks indicate the four positions where -type specific amino-acid residues were replaced with corresponding residues of the ß-connexin, Cx32.

View larger version (75K): [in a new window]   Fig. 2. Expression and localization of wildtype and Cx43 amino-acid substitution variants. (A) BHK cells were transfected with cDNAs encoding GFP-tagged wt and mutated Cx43, respectively, and expressed proteins were detected by western blot analysis using polyclonal antibodies directed against the C-terminal domain of Cx43. Equal amounts of full-length fusion proteins were expressed with all constructs (labeled with an arrowhead). Small amounts of endogenous Cx43 in BHK cells (labeled with an asterisk) and some unspecific reaction products were also detected in all lanes. (B) Localization of GFP-tagged connexins was detected by GFP auto-fluorescence 24 hours post transfection. Connexin clusters were detected with all constructs at cell-cell appositions (marked with arrows) besides intracellular fluorescence. Phase-contrast images are shown on the left; the corresponding fluorescence images are shown on the right. Bar, 10 µm.

View larger version (60K): [in a new window]   Fig. 4. Dye transfer efficiencies of Cx43 variants (A), dominant-negative (B) and trans-dominant-negative dye transfer inhibition (C) of co-expressed Cx43 and Cx32, respectively. Cells that transferred dye to neighboring cells were counted, and the percentage of cells transferring dye are shown as bars. Injections at low (black bars) and high (gray bars) wild-type Cx43 or wt Cx32 expression levels are shown in B and C. Total numbers of injected cells are given in parentheses under the names of the expressed proteins.

View larger version (80K): [in a new window]   Fig. 5. Cx43 amino-acid substitution variants and Cx32 colocalize within connexin clusters at cell-cell appositions. Stably transfected BHK cells expressing Cx32 (BHK-Cx32) were co-transfected with wildtype or Cx43-GFP substitution variants, respectively. In all instances, both connexin isotypes localized to the same connexin clusters (labeled with arrows) as indicated by the yellow plaque color in the merged images (Merge). Cx32 was immunostained with Cx32-specific antibodies followed by TRITC-coupled secondary antibodies 24 hours after transfection and fixation (Anti-Cx32, red). Wild-type Cx43 and variants were detected by their GFP auto-fluorescence (GFP, green). Connexin clusters assembled between cells that expressed only Cx43 appeared green, clusters in cells that expressed only Cx32 appeared red (labeled with asterisks in the merged images of variants D12S, and R153W). Bar, 20 µm.

View larger version (52K): [in a new window]   Fig. 6A. Assessment of wildtype and Cx43 variant interaction and co-oligomerization. Transport-deficient DsRed-tagged Cx43 (A) or Cx32 (B) were co-expressed with GFP-tagged Cx43 variants. Interaction and co-assembly of DsRed-tagged wildtype and GFP-tagged variant connexin subunits into mixed connexons were evident by the successful trafficking of DsRed-tagged connexin subunits to the plasma membrane and their localization together with GFP-tagged connexins in connexin clusters that were detectable in the GFP and the DsRed channels (clusters are labeled with arrows). Clusters in merged images appear more or less yellow dependent on the ratios of GFP and DsRed-tagged subunits. Cx43 wildtype was co-expressed in control (rows 1 in A and B). Bar, 20 µm.

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