Structure and functional studies of N-terminal Cx43 mutants linked to oculodentodigital dysplasia

Abstract

Mutations in the gene encoding connexin-43 (Cx43) cause the human development disorder known as oculodentodigital dysplasia (ODDD). In this study, ODDD-linked Cx43 N-terminal mutants formed nonfunctional gap junction-like plaques and exhibited dominant-negative effects on the coupling conductance of coexpressed endogenous Cx43 in reference cell models. Nuclear magnetic resonance (NMR) protein structure determination of an N-terminal 23-amino acid polypeptide of wild-type Cx43 revealed that it folded in to a kinked α-helical structure. This finding predicted that W4 might be critically important in intramolecular and intermolecular interactions. Thus we engineered and characterized a W4A mutant and found that this mutant formed a regular, nonkinked α-helix but did not form functional gap junctions. Furthermore, a G2V variant peptide of Cx43 showed a kinked helix that now included V2 interactions with W4, resulting in the G2V mutant forming nonfunctional gap junctions. Also predicted from the NMR structures, a G2S mutant was found to relieve these interactions and allowed the protein to form functional gap junctions. Collectively, these studies suggest that the nature of the mutation conveys loss of Cx43 function by distinctly different mechanisms that are rooted in the structure of the N-terminal region.

FIGURE 1:

Distribution of N-terminal Cx43 mutants in NRK cells. Cells were transfected with vectors encoding GFP-tagged Cx43 or the G2V, D3N, L7V, L11P, Y17S, or S18P mutant before immunolabeling with anti-Cx43 (red) antibodies. Hoechst 33342 was used to stain the nuclei Arrows indicate gap junction plaque-like structures at sites of cell–cell contact. Bars, 10 μm.

FIGURE 2:

Intracellular distribution of Cx43^Δ2-7-GFP in HeLa and NRK cells. HeLa (A) or NRK (B) cells were transfected with a vector that encoded a GFP-tagged Cx43 mutant (Cx43^Δ2-7-GFP) and immunolabeled with anti-Cx43 (red) or anti-PDI (red) antibodies. Hoechst 33342 was used to stain the nuclei. Bars, 10 μm.

FIGURE 3:

N-terminal–linked Cx43 mutants do not make functional intercellular channels and exhibit dominant-negative properties on coexpressed endogenous Cx43. HeLa (A) or NRK (B) cells expressing Cx43 or mutants were pressure microinjected with 5% Lucifer yellow, and the incidence of dye transfer to neighboring cells was assessed (n > 20 for each group). (C) N2A cells expressing wild-type or Cx43 mutants were patch-clamped to measure electrical coupling conductance. In all cases, the mutants failed to form functional gap junction channels (A, C) and exhibited dominant-negative properties on coexpressed Cx43 (B). WT, untransfected wild-type cells; control in C, untransfected N2A cells.

FIGURE 4:

Ribbon representations of Cx43 N-terminal and mutant peptide structures as revealed by NMR analysis. The superpositions of the 10 best structures for the native 23–amino acid Cx43 N-terminal peptide (WT) and peptides containing single–amino acid substitutions (W4A, G2V) are shown (left), along with a ribbon structure of the most representative structure (right). In each case, the side chains of key residues (described in the text) are shown along with the one-letter amino acid name and sequence number. All peptides are shown in a similar orientation based on the superposition of residues D12–Y17.

FIGURE 5:

Localization of the W4A and G2S Cx43 mutants in NRK cells. NRK cells were transfected with vectors encoding W4A-GFP (A) or G2S-GFP (B) and immunolabeled with anti-Cx43 antibodies (red) before Hoechst 33342 staining (blue). Arrows indicate that the mutants were localized to gap junction–like structures at cell–cell interfaces. Bars, 10 μm.

FIGURE 6:

The G2S, but not the G2V or W4A, mutant forms functional gap junctions. (A) NRK and HeLa cells were untreated (WT) or engineered to express W4A-GFP or G2S-GFP before microinjection with 5% Lucifer yellow and assessment of dye transfer. (B) Gap junctional intercellular communication–deficient N2A cells were engineered to express Cx43-GFP, W4A-GFP, G2V-GFP, or G2S-GFP. Patch-clamp analysis was used to assess gap junction coupling conductance. Whereas the W4A and G2V mutants did not form functional gap junction channels, the G2S mutant exhibited functional conductance that even exceeded that of Cx43. The numbers above each column depict the number of cell pairs recorded.