Abstract. Northern blot analysis of rat heart mRNA probed with a eDNA coding for the principal polypeptide of rat liver gap junctions demonstrated a 3.0-kb band. This band was observed only after hybridization and washing using low stringency conditions; high stringency conditions abolished the hybridization. A rat heart eDNA library was screened with the same eDNA probe under the permissive hybridization conditions, and a single positive clone identified and purified. The clone contained a 220-bp insert, which showed 55 % homology to the original eDNA probe near the 5' end. The 220-bp eDNA was used to rescreen a heart eDNA library under high stringency conditions, and three additional cDNAs that together spanned 2,768 bp were isolated. This composite eDNA contained a single 1,146-bp open reading frame coding for a predicted polypeptide of 382 amino acids with a molecular mass of 43,036 D. Northern analysis of various rat tissues using this heart eDNA as probe showed hybridization to 3.0-kb bands in RNA isolated from heart, ovary, uterus, kidney, and lens epithelium.Comparisons of the predicted amino acid sequences for the two gap junction proteins isolated from heart and liver showed two regions of high homology (58 and 42%), and other regions of little or no homology. A model is presented which indicates that the conserved sequences correspond to transmembrane and extracellular regions of the junctional molecules, while the nonconserved sequences correspond to cytoplasmic regions. Since it has been shown previously that the original eDNA isolated from liver recognizes mRNAs in stomach, kidney, and brain, and it is shown here that the eDNA isolated from heart recognizes mRNAs in ovary, uterus, lens epithelium, and kidney, a nomenclature is proposed which avoids categorization by organ of origin. In this nomenclature, the homologous proteins in gap junctions would be called connexins, each distinguished by its predicted molecular mass in kilodaltons. The gap junction protein isolated from liver would then be called connexin32; from heart, connexin43.AP junctions are composed of collections of membrane channels, called connexons, which join in mirror symmetry with connexons in the membrane of the adjacent cell. These pairs of connexons permit the intercytoplasmic exchange of small metabolites and ions between cells. Each connexon is composed ofa hexamer of an integral membrane protein, whose complete eDNA has been cloned from rat and human liver, with a predicted molecular mass of 32 kD (23, 32). The mRNA coding for this protein is not unique to the liver, but may be detected in other, but not all, organs within the same animal (32). In this paper, we show that a related mRNA is found in abundance in heart and other organs, and that mRNAs coding for both the liver and heart gap junction proteins are in some cases detected in the same organ.Thus, these gap junction mRNAs are not confined to the organs in which they were first observed, necessitating a nomenclature system which avoids mention of source. We propose ...

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Models for the spatial distribution of protein, lipid and water in gap junction structures have been constructed from the results of the analysis of X-ray diffraction data described here and the electron microscope and chemical data presented in the preceding paper (Caspar, D. L. D., D. A. Goodenough, L. Makowski, and W. C. Phillips. 1977. 74:605-628). The continuous intensity distribution on the meridian of the X-ray diffraction pattern was measured, and corrected for the effects of the partially ordered stacking and partial orientation of the junctions in the X-ray specimens. The electron density distribution in the direction perpendicular to the plane of the junction was calculated from the meridional intensity data. Determination of the interference function for the stacking of the junctions improved the accuracy of the electron density profile. The pair-correlation function, which provides information about the packing of junctions in the specimen, was calculated from the interference function. The intensities of the hexagonal lattice reflections on the equator of the X-ray pattern were used in coordination with the electron microscope data to calculate the two-dimensional electron density projection onto the plane of the membrane. Differences in the structure of the connexons as seen in the meridional profiles and equatorial projections were shown to be correlated to changes in lattice constant. The parts of the junction structure which are variable have been distinguished from the invariant parts by comparison of X-ray data from different specimens. The combination of these results with electron microscope and chemical data provides low-resolution threedimensional representations of the structures of gap junctions.The structural variations detailed in the preceding paper (5) establish that we are looking at not one structure of the gap junction, but at a family of structures. By observing closely related states of a molecular assembly, it is often possible to infer something about the way that transitions occur between states. These molecular rearrangements may be significant in the functional activity of the structure. Furthermore, polymorphism provides a constraint on the interpretation of the diffraction patterns. For example, the connexon structure is likely to be similar in arrays with different lattice constants, although the intensities in the X-ray patterns may be quite different.

The half-life of a gap-junction polypeptide band migrating at 21,000 Mr on SDS polyacrylamide gels isolated from mouse liver is measured to be 5 h . Two low-molecular weight bands, probably related to the 21,000 Mr material by proteolysis, have measured halflives of 4.6 and 5.2 h . Gap junctions are labeled in vivo using the ' 4 C-bicarbonate labeling procedure, followed by quantitative fluorography .Intercellular communication, which is mediated by gap junctions, can be regulated in most tissue systems by factors such as a rise in intracellular calcium and a decrease in intracellular pH (32,37,38) . The time-course of regulation, both the uncoupling and coupling of communication, varies over time-scales ranging from seconds (27) and minutes (5,21,22,38) at one extreme to tens of hours (9) at another. These differences in time-scale are partly the result of differences both in the experimental protocols and in the cells studied. Structurally, the regulation of intercellular communication could occur either at the level of the connexon (subunit) of the gap junction, involving the closing and opening of intraconnexon channels (29, 39), or by disassembly, followed by de novo assembly of new connexons .If the gap junction proteins have half-lives longer than the time necessary for cells to couple or uncouple, this would suggest that the uncoupling event is a reversible phenomenon at the level of the connexon itself. If the protein turnover is faster than the time required to uncouple and recouple cells, this would allow for disassembly and de novo connexon assembly to play a role in the regulation process .Data on the half-lives of gap junction proteins are limited .Gurd and Evans (17) used a double-label isotope technique to estimate the relative turnover time of a "sarcosine-resistant fraction" from rat liver plasma membranes, which contained isolated gap junctions . Turnover was reported to be extremely slow, compared to other cellular proteins . However, as is suggested by the enrichment for glycine in the amino acid analyses of these authors (6), the sarcosine-resistant fraction is heavily contaminated with collagen, an extracellular protein which turns over very slowly . In contrast, Yancey et al . (41) reported a 3-h time-point for peak incorporation of pulseinjected ["S]methionine into peptides at 10,000 Mr, derived from isolated rat liver gap junctions . A difficulty in measuring the half-lives of proteins using conventional pulse-chase techniques with single isotopes is centered on the multiple reutilization of the amino acids, thus giving longer apparent half-lives (11) . The [14 C]bicarbonate-