The 43-kD polypeptide of heart gap junctions: immunolocalization, topology, and functional domains.

Abstract

Abstract. Analysis by SDS-PAGE of gap junction fractions isolated from heart suggests that the junctions are comprised of a protein with an Mr 43,000. Antibodies against the electroeluted protein and a peptide representing the 20 amino terminal residues bind specifically on immunoblots to the 43-kD protein and to the major products arising from proteolysis during isolation. By immunocytochemistry, the protein is found in ventricle and atrium in patterns consistent with the known distribution of gap junctions. Both antibodies bind exclusively to gap junctions in fractions from heart examined by EM after gold labeling. Since only domains of the protein exposed at the cytoplasmic surface should be accessible to antibody, we conclude that the 43-kD protein is assembled in gap junctions with the amino terminus of the molecule exposed on the cytoplasmic side of the bilayer, that is, on the same side as the carboxy terminus as determined previously. By combining proteolysis experiments with data from immunoblotting, we can identify a third cytoplasmic region, a loop of some 4 kD between membrane protected domains. This loop carries an antibody binding site. The protein, if transmembrane, is therefore likely to cross the membrane four times. We have used the same antisera to ascertain if the 43-kD protein is involved in cell-cell communication. The antiserum against the amino terminus blocked dye coupling in 90% of cell pairs tested; the antiserum recognizing epitopes in the cytoplasmic loop and cytoplasmic tail blocked coupling in 75 % of cell pairs tested. Preimmune serum and control antibodies (one against MIP and another binding to a cardiac G protein) had no or little effect on dye transfer. Our experimental evidence thus indicates that, in spite of the differences in amino acid sequence, the gap junction proteins in heart and liver share a general organizational plan and that there may be several domains (including the amino terminus) of the molecule that are involved in the control of junctional permeability.

Connexin expression and gap junction communication compartments in the developing mouse limb

“…The limbs were clamped in wells filled with bathing buffer consisting of 133 mM NaC1, 3.6 mM KC1, 0.3 mM MgCl,, 16 mM glucose, and 3.0 mM HEPES, pH 7.3-7.4. Cells of the AER, mesenchyme, or nonridge ectoderm were microinjected with 5% Lucifer yellow CH dissolved in 0.15 M LiCl, pH to 7.4 with 10 mM HEPES as described (Yancey et al, 1989). Dye injected limbs were incubated at room temperature for 15-45 min prior to fixing in 1% formaldehyde (made fresh from paraformaldehyde) for 2 hr to overnight a t 4°C.…”

GJA1mutations, variants, and connexin 43 dysfunction as it relates to the oculodentodigital dysplasia phenotype

“…Beyer et al [1987] published hydropathicity plots of Cx43, delineating the hydrophobic membrane spanning domains in their model of connexin topology. Since then, various Cx43 topology predictions have been published [Yancey et al, 1989;Laird and Revel, 1990;Yeager and Gilula, 1992;Yeager, 1998;van der Heyden et al, 2004]. Web transmembrane prediction tools HMMTOP, TMHMM, SOSUI, Tmpred, and TopPred (ExPASy Proteomics tools: http://www.…”

Incompatibility of connexin 40 and 43 Hemichannels in gap junctions between mammalian cells is determined by intracellular domains.

“…The topology of a type connexin proteins was inferred from hydropathy plots of amino acid sequences and experimental results concerning rat Cx43 (Yancey et al, 1989). Mouse Cx4O domains (Hennemann et al, 1992) were exchanged for the corresponding domains of mouse Cx43 (Beyer and Steinberg, 1991;Hennemann et al, 1992) by site-directed mutagenesis (see Figure 1).…”