Gap junction structures. IV. Asymmetric features revealed by low-irradiation microscopy.

Abstract

Micrographs of mouse liver gap junctions, isolated with detergents, and negatively stained with uranyl acetate, have been recorded by low-irradiation methods. Our Fourieraveraged micrographs of the hexagonal junction lattice show skewed, hexameric connexons with less stain at the threefold axis than at the six indentations between the lobes of the connexon image. These substructural features, not clearly observed previously, are acutely sensitive to irradiation. After an electron dose less than that normally used in microscopy, the image is converted to the familiar doughnut shape, with a darkly stained center and a smooth hexagonal outline, oriented with mirror symmetry in the lattice. Differences in appearance among 25 reconstructed images from our low-irradiation micrographs illustrate variation in staining of the connexon channel and the space between connexons. Consistently observed stain concentration at six symmetrically related sites ~34 tk from the connexon center, 8 ° to the right or left of the (1, 1) lattice vector may reveal an intrinsic asymmetric feature of the junction structure. The unexpected skewing of the six-lobed connexon image suggests that the pair of hexagonal membrane arrays that form the junction may not be structurally identical. Because the projected image of the connexon pair itself appears mirror symmetric, each pair may consist of two identical connexon hexamers related by local (noncrystallographic) twofold axes in the junctional plane at the middle of the gap. All connexons may be chemically identical, but their packing in the hexagonal arrays on the two sides of the junction appears to be nonequivalent.Gap junctions in liver (13, 28) and many other tissues (5, 10) are built of connexon units (6) hexagonally arrayed with crystaUike regularity in the pair of connected cell membranes. Makowski et al. (21) proposed a model for the junction structure based on data from x-ray diffraction, electron microscopy and chemical analysis in which the connexon units were pictured as symmetric hexamers of identical connexin molecules that are equivalently paired across the gap with dihedral symmetry in the two-sided hexagonal plane group (p622; see reference 16). This model predicts that, as normally viewed in electron micrographs of untilted, negatively stained specimens, the junction lattice should appear mirror-symmetric in projection (with p6m plane group symmetry).Conventional high-irradiation micrographs of negatively stained specimens show doughnut-shaped connexons that appear nearly circularly symmetric in the Fourier-averaged images, and, therefore, the hexagonal lattice array, appear to have mirror symmetry (6). Higher resolution, hexagonally averaged reconstructions calculated by Zampighi and Unwin (33) from minimal irradiation micrographs of two forms of gap junctions, negatively stained with uranyl acetate, showed hexagonal shaped connexons arrayed with approximate mirror symmetry in the projected lattice images. However, the hexagonally shaped connexon images occured in ...

Distribution of transport proteins over animal cell membranes

“…The structural basis for this pathway is probably the gap junction (Loewenstein, 1981) which appears as an aggregate of ~ 8 nm intramembranous particles in freeze-fracture images (Peracchia, 1980). It is thought that each particle consists of six subunits, and that when two such hexamers in adjacent membranes connect, they form the "connexon'" which constitutes a cell-tocell channel (Henderson, Eibl & Weber, 1979;Unwin & Zampighi, 1980;Baker et al, 1983). For a detailed discussion of structure-function relationships of this transport protein, the reader is referred to the informative and comprehensive review by Loewenstein (1981).…”

Detergent sensitivity and splitting of isolated liver gap junctions

“…Recent advances in the understanding of the structure (Peracchia, /980;Hirokawa & Heuser, 1982;Baker, Caspar, Hollingshead & Goodenough, 1983) and protein composition (Henderson, Eibl & Weber, 1979;Hertzberg & Gilula, 1979;Finbow, Yancey, Johnson & Revel, 1980;Hertzberg, /980;Kensler & Goodenough, 1980;Ehrhart, 1981;Nicholson et al, 1981;Takemoto & Hansen, /981;Hertzberg, Anderson, Friedlander & Gilula, 1982;Hertzberg & Gilula, /982;Manjunath, Goings & Page, 1982a, b;Nicholson et al, 1983) of gap junctions, as well as the hypothesis that each of their channels is formed by the coming together and interlocking of "protochannels" present in the lipid bilayers of the adjacent plasma membranes (Loewenstein, 1981), have made it desirable to examine the properties of split gap junctions. Although studies with the freeze fracture technique suggest that gap junctions in situ can be split by exposure to hypertonic saline (Goodenough & Gilula, 1974;Hirokawa & Heuser, 1982) or Ca 2+ free solutions (Hirokawa & Heuser, 1982), the splitting of gap junctions that have been isolated and purified by biochemical techniques has not been reported.…”

Conservation of a cytoplasmic carboxy-terminal domain of connexin 43, a gap junctional protein, in mammal heart and brain

“…These structures, now termed "gap junctions," are formed between two cells by the close association of plasma membrane regions containing hexagonal arrays of particles (Revel & Karnovsky, 1967): the connexons. Ultrastructural, biophysical and physiological investigations suggest that the connexons of a membrane, tightly connected, in the extracellular space, to the apposed-membrane connexons are channelmaking hexameric structures providing a direct route for the passage of ions and small molecules between the cytoplasms of adjacent cells (McNutt & Weinstein, 1970;Unwin & Zampighi, 1980;Loewenstein, 1981;Baker et al, 1983;Sosinsky et al, 1988). Thus the gap junctions are implicated in metabolic cooperation and electrical coupling between cells (Pitts & Finbow, 1986;Warner, 1988).…”