Gap junction structures. I. Correlated electron microscopy and x-ray diffraction

X-ray crystallographic methods and electron microscope image analysis have been used to correlate the structure and the chemical composition of gap junction plaques isolated intact from mouse liver. The requirement that the interpretations of X-ray, electron microscope, and chemical measurements be consistent reduces the uncertainties inherent in the separate observations and leads to a unified picture of the gap junction structures. Gap junctions are built up of units called connexons that are hexagonally arrayed in the pair of connected cell membranes. X-ray diffraction and electron microscope measurements show that the lattice constant of this array varies from about 80 to 90 A. Analysis of electron micrographs of negatively stained gap junctions shows that there is significant short range disorder in the junction lattice. even though the long range order of the array is remarkably regular. Analysis of the disorder provides information about the nature of the intermolecular forces that hold the array together.     

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

Micrographs of mouse liver gap junctions, isolated with detergents, and negatively stained with uranyl acetate, have been recorded by low-irradiation methods. Our Fourier-averaged 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 approximately 34 A from the connexon center, 8 degrees 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 junction structures. VI. Variation and conservation in connexon conformation and packing.

Correlation of structural changes in isolated gap junctions with the mechanism of channel gating is complicated by the effects of isolation procedures and the lack of a direct functional assay. The effect of variations in the isolation procedure are examined by comparison of the structures of gap junctions isolated by different protocols. X-ray diffraction data from over two hundren specimens are compared to provide a basis for identification of invariant aspects of the connexon structure and variable properties related either to functional switching or experimental modifications. We discuss the relationship between subunit tilt, lattice symmetry and packing, and membrane curvature and demonstrate that membrane curvature may be a natural consequence of the structure of the connexons and the patterns of interactions between them.     

Gap junction structures after experimental alteration of junctional channel conductance

Gap junctions are known to present a variety of different morphologies in electron micrographs and x-ray diffraction patterns. This variation in structure is not only seen between gap junctions in different tissues and organisms, but also within a given tissue. In an attempt to understand the physiological meaning of some aspects of this variability, gap junction structure was studied following experimental manipulation of junctional channel conductance. Both physiological and morphological experiments were performed on gap junctions joining stage 20-23 chick embryo lens epithelial cells. Channel conductance was experimentally altered by using five different experimental manipulations, and assayed for conductance changes by observing the intercellular diffusion of Lucifer Yellow CH. All structural measurements were made on electron micrographs of freeze-fracture replicas after quick-freezing of specimens from the living state; for comparison, aldehyde-fixed specimens were measured as well. Analysis of the data generated as a result of this study revealed no common statistically significant changes in the intrajunctional packing of connexons in the membrane plane as a result of experimental alteration of junctional channel conductance, although some of the experimental manipulations used to alter junctional conductance did produce significant structural changes. Aldehyde fixation caused a dramatic condensation of connexon packing, a result not observed with any of the five experimental uncoupling conditions over the 40-min time course of the experiments.     

Gap junction structures: Analysis of the x-ray diffraction data

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 to the two-dimensional electron density projection onto the plane of the membrane. Differences in the structure of the connexons as seen in the meridional profile 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 the X-ray data from different specimens. The combination of these results with electron microscope and chemical data provides low resolution three- dimensional representations of the structures of gap junctions.     

Gap junction structures. VII. Analysis of connexon images obtained with cationic and anionic negative stains

Micrographs of isolated gap junction specimens, negatively stained with one molybdate, three tungstate and three uranyl stains, were recorded at low and high irradiation. Fourier-averaged images of the negatively stained gap junctions have been self-consistently scaled to identify conserved and variable features. Intrinsic features in the hexagonally averaged images have been distinguished from residual noise by statistical comparisons among similarly prepared specimens. The cationic uranyl stains can penetrate the axial connexon channel, whereas the anionic stains are largely excluded; these observations indicate that the channel is negatively charged. Variability in the extent of the axial stain penetration, and enhancement of this staining by radiation damage and heating may be accounted for by a leaky, labile channel gate. The peripheral stain concentrations marking the perimeter of the skewed, six-lobed connexon image and the stain-excluding region at the 3-fold axis of the lattice, which are seen only under conditions of low irradiation with both anionic and cationic stains, are identified as intrinsic features of the isolated gap junction structure. The stain concentrations located approximately 30 A from the connexon center appear to be symmetrically related on opposite sides of the junction by non-crystallographic 2-fold axes oriented approximately 8 degrees to the lattice axes at the plane of the gap. The radiation-sensitive hexagonal features seen in the negatively stained images may correspond to substructure on the cytoplasmic surfaces of the paired gap junction membranes.     

Gap junction channel gating

Over the last two decades, the view of gap junction (GJ) channel gating has changed from one with GJs having a single transjunctional voltage-sensitive (V(j)-sensitive) gating mechanism to one with each hemichannel of a formed GJ channel, as well as unapposed hemichannels, containing two, molecularly distinct gating mechanisms. These mechanisms are termed fast gating and slow or 'loop' gating. It appears that the fast gating mechanism is solely sensitive to V(j) and induces fast gating transitions between the open state and a particular substate, termed the residual conductance state. The slow gating mechanism is also sensitive to V(j), but there is evidence that this gate may mediate gating by transmembrane voltage (V(m)), intracellular Ca(2+) and pH, chemical uncouplers and GJ channel opening during de novo channel formation. A distinguishing feature of the slow gate is that the gating transitions appear to be slow, consisting of a series of transient substates en route to opening and closing. Published reports suggest that both sensorial and gating elements of the fast gating mechanism are formed by transmembrane and cytoplamic components of connexins among which the N terminus is most essential and which determines gating polarity. We propose that the gating element of the slow gating mechanism is located closer to the central region of the channel pore and serves as a 'common' gate linked to several sensing elements that are responsive to different factors and located in different regions of the channel.     

Gap junction regulation by calmodulin

Intracellular Ca²⁺ activated calmodulin (CaM) inhibits gap junction channels in the low nanomolar to high micromolar range of [Ca²⁺]_i. This regulation plays an essential role in numerous cellular processes that include hearing, lens transparency, and synchronized contractions of the heart. Previous studies have indicated that gap junction mediated cell-to-cell communication was inhibited by CaM antagonists. More recent evidence indicates a direct role of CaM in regulating several members of the connexin family. Since the intracellular loop and carboxyl termini of connexins are largely “invisible” in electron microscopy and X-ray crystallographic structures due to disorder in these domains, peptide models encompassing the putative CaM binding sites of several intracellular domains of connexins have been used to identify the Ca²⁺-dependent CaM binding sites of these proteins. This approach has been used to determine the CaM binding affinities of peptides derived from a number of different connexin-subfamilies.     

Gap junction structures: V. Structural chemistry inferred from X-ray diffraction measurements on sucrose accessibility and trypsin susceptibility

X-ray diffraction patterns have been recorded from partially oriented specimens of gap junctions isolated from mouse liver and suspended in sucrose solutions of different concentration and thus of different electron density. Analysis of these diffraction patterns has shown that sucrose is excluded from the 6-fold rotation axis of the junction lattice for a length of about 100 Å. This indicates that the aqueous channel of the junctions is in the closed, high resistance state in these preparations. Mapping of the sucrose-accessible space in the junction indicates that the cross-sectional area of the channel entrance on the cytoplasmic side of the membrane could be up to five times larger than the area of the transmembrane channel. Sucrose does not penetrate more than 20 Å into the membrane along the channel. Apparently the aqueous channel, 8 to 10 Å in radius for most of its length, is narrowed or blocked by a small feature about 50 Å from the center of the gap. Very close interactions exist between the gap junction protein and the lipid polar head groups on the cytoplasmic surface of the membrane. In this region, the protein intercalates between the polar head groups. These results suggest that the gap junction protein may have a functional two-domain structure. One domain, with a molecular weight of about 15,000, spans one bilayer and half of the gap and is contained largely within a radius of 25 Å from the 6-fold axis. The second domain is smaller and occupies the cytoplasmic surface of the gap junction membrane. Trypsin digestion removes about 4000 M_rmr from the cytoplasmic surface domain of the junction protein. Most of the material susceptible to trypsin digestion is located more than 28 å from the 6-fold axis.     

Gap junction protein connexin-43 interacts directly with microtubules.

Gap junctions are specialized cell-cell junctions that mediate intercellular communication. They are composed of connexin proteins, which form transmembrane channels for small molecules [1, 2]. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated in the regulation of Cx43 channel gating by growth factors [3-5]. The Cx43 tail contains various protein interaction sites, but little is known about binding partners. To identify Cx43-interacting proteins, we performed pull-down experiments using the C-terminal tail of Cx43 fused to glutathione-S-transferase. We find that the Cx43 tail binds directly to tubulin and, like full-length Cx43, sediments with microtubules. Tubulin binding to Cx43 is specific in that it is not observed with three other connexins. We established that a 35-amino acid juxtamembrane region in the Cx43 tail, which contains a presumptive tubulin binding motif, is necessary and sufficient for microtubule binding. Immunofluorescence and immunoelectron microscopy studies reveal that microtubules extend to Cx43-based gap junctions in contacted cells. However, intact microtubules are dispensable for the regulation of Cx43 gap-junctional communication. Our findings suggest that, in addition to its well-established role as a channel-forming protein, Cx43 can anchor microtubule distal ends to gap junctions and thereby might influence the properties of microtubules in contacted cells.     

Gap junctional communication during neuromuscular junction formation

We have tested whether gap junctions form between nerve and muscle during their initial contact, before establishing the chemical synapse. Embryonic Xenopus stage 18-20 myotomes and neural tubes were permeabilized with DMSO to load appropriate reagents, dissociated, and cocultured. When myotomes, loaded with Lucifer yellow, were cocultured with unlabeled neural tube cells, 23% of the neurons contained dye after 24 hr. Affinity-purified gap junction antibodies loaded into myocytes or neurons reduced neuronal labeling significantly to 5%. [3H]uridine nucleotide transfer was observed in both directions between myocytes and neurons. Again gap junction antibodies substantially reduced recipient label. In all cases preimmune IgGs did not reduce transfer. When acetylcholine receptor clustering was examined in cultures containing gap junction antibodies, no difference in the number of neuronally induced AChR clusters was observed. This suggests that the cluster-inducing signal between nerve and muscle does not pass through gap junctions.     

Gap junction channels: distinct voltage-sensitive and -insensitive conductance states.

All mammalian gap junction channels are sensitive to the voltage difference imposed across the junctional membrane, and parameters of voltage sensitivity have been shown to vary according to the gap junction protein that is expressed. For connexin43, the major gap junction protein in the cardiovascular system, in the uterus, and between glial cells in brain, voltage clamp studies have shown that transjunctional voltages (Vj) exceeding +/- 50 mV reduce junctional conductance (gj). However, substantial gj remains at even very large Vj values; this residual voltage-insensitive conductance has been termed gmin. We have explored the mechanism underlying gmin using several cell types in which connexin43 is endogenously expressed as well as in communication-deficient hepatoma cells transfected with cDNA encoding human connexin43. For pairs of transfectants exhibiting series resistance-corrected maximal gj (gmax) values ranging from < 2 to > 90 nS, the ratio gmin/gmax was found to be relatively constant (about 0.4-0.5), indicating that the channels responsible for the voltage-sensitive and -insensitive components of gj are not independent. Single channel studies further revealed that different channel sizes comprise the voltage-sensitive and -insensitive components, and that the open times of the larger, more voltage-sensitive conductance events declined to values near zero at large voltages, despite the high gmin. We conclude that the voltage-insensitive component of gj is ascribable to a voltage-insensitive substate of connexin43 channels rather than to the presence of multiple types of channels in the junctional membrane. These studies thus demonstrate that for certain gap junction channels, closure in response to specific stimuli may be graded, rather than all-or-none.     

Gap junction intercellular communication during lymphocyte transendothelial migration

Migration of lymphocytes across the endothelium of central or peripheral tissues, a process occurring following activation or differentiation, involves cell to cell interactions featuring adhesion and heterotypic signalling 'cross-talk'. Since lymphocytes and endothelial cells express connexins, the subunit proteins of gap junction intercellular channels, we investigated whether these channels feature in heterotypic signalling during transendothelial migration of lymphocytes. We show, using FACS analysis, that calcein, a gap junction permeant fluorescent dye, was transferred from endothelial cell layers to lymphocytes. The gap junction involvement in intercellular dye transfer was reinforced by studies showing that the process was inhibited by connexin mimetic peptides, a new class of reagents shown to block gap junction communication. Further evidence for the involvement of lymphocyte gap junctions in intercellular communication during transendothelial migration was obtained by two-photon laser scanning microscopy. Although gap junctional communication was inhibited by connexin mimetic peptides, they had little influence on the transmigration process.     

Gap junction channel gating modulated through protein phosphorylation

As a ubiquitous post-translation modification process, protein phosphorylation has proven to be a key mechanism in regulating the function of several membrane proteins, including transporters and channels. Connexins, pannexins, and innexins are protein families that form gap junction channels essential for intercellular communication. Connexins have been intensely studied, and most of their isoforms are known to be phosphorylated by protein kinases that lead to modifications in tyrosine, serine, and threonine residues, which have been reported to affect, in one way or another, intercellular communication. Despite the abundant reports on changes in intercellular communication due to the activation or inactivation of numerous kinases, the molecular mechanisms by which phosphorylation alters channel gating properties have not been elucidated completely. Hence, this chapter will cover some of the current, relevant research that attempt to explain how phosphorylation triggers and/or modulates gap junction channel gating.     

Gap junction ultrastructure in three states of conductance

Summary Junctional conductance between the epidermal cells of the beetle Tenebrio molitor is raised after exposure to the hormone 20-hydroxyecdysone and lowered reversibly by exposure to chlorpromazine. Gap Junctional particle size, density and arrangement associated with these conductance changes were studied. We found no significant difference in particle density in gap junctions of control (2456±471 particles/m², mean ±S.D.) and hormone-treated epidermis (2490±315); however, a significant increase in packing density occurred in chlorpromazine-uncoupled epidermis (3133±665). The particles are randomly arranged in all three states of conductance. Particle size measurements show that the E-face gap junctional particles are heterogeneous with a mean diameter ±S.D. of 15.2±2.0 nm. No significant difference in particle size between controls and experimentals was detected. Although glutaraldehyde irreversibly uncoupled these cells, the absence of glutaraldehyde fixation but presence of glycerol induced marked alterations in the appearance of the gap junctions such that quantification was no longer possible. From this particle-packing data and our previous thin-section data, we estimate that there are 90000 gap junctional particles per cell (within junctional plaques). The conductance of a single gap junctional channel (assuming one population) changes from 94 pS to 213 pS after hormone treatment.