Gap junction channels mediate communication between adjacent cells. Using atomic force microscopy (AFM), we have imaged conformational changes of the cytoplasmic and extracellular surfaces of native connexin 26 gap junction plaques. The cytoplasmic domains of the gap junction surface, imaged at submolecular resolution, form a hexameric pore protruding from the membrane bilayer. Exhibiting an intrinsic¯exibility, these cytoplasmic domains, comprising the C-terminal connexin end, reversibly collapse by increasing the forces applied to the AFM stylus. The extracellular connexon surface was imaged after dissection of the gap junction with the AFM stylus. Upon injection of Ca 2+ into the buffer solution, the extracellular channel entrance reduced its diameter from 1.5 to 0.6 nm, a conformational change that is fully reversible and speci®c among the divalent cations tested. Ca 2+ had a profound effect on the cytoplasmic surface also, inducing the formation of microdomains. Consequently, the plaque height increased by 0.6 nm to 18 nm. This suggests that calcium ions induce conformational changes affecting the structure of both the hemichannels and the intact channels forming cell±cell contacts.

Single site mutations in connexins have provided insights about the influence specific amino acids have on gap junction synthesis, assembly, trafficking, and functionality. We have discovered a single point mutation that eliminates functionality without interfering with gap junction formation. The mutation occurs at a threonine residue located near the cytoplasmic end of the third transmembrane helix. This threonine is strictly conserved among members of the ␣-and ␤-connexin subgroups but not the ␥-subgroup. In HeLa cells, connexin43 and connexin26 mutants are synthesized, traffic to the plasma membrane, and make gap junctions with the same overall appearance as wild type. We have isolated connexin26T135A gap junctions both from HeLa cells and baculovirus-infected insect Sf9 cells. By using cryoelectron microscopy and correlation averaging, difference images revealed a small but significant size change within the pore region and a slight rearrangement of the subunits between mutant and wild-type connexons expressed in Sf9 cells. Purified, detergent-solubilized mutant connexons contain both hexameric and partially disassembled structures, although wild-type connexons are almost all hexameric, suggesting that the three-dimensional mutant connexon is unstable. Mammalian cells expressing gap junction plaques composed of either connexin43T154A or connexin26T135A showed an absence of dye coupling. When expressed in Xenopus oocytes, these mutants, as well as a cysteine substitution mutant of connexin50 (connexin50T157C), failed to produce electrical coupling in homotypic and heteromeric pairings with wild type in a dominant-negative effect. This mutant may be useful as a tool for knocking down or knocking out connexin function in vitro or in vivo.Intercellular communication is a fundamental feature of all multicellular organisms. Gap junctions are one means by which cells communicate with each other and arise as tissue cells grow and abut each other. The morphologically distinctive cell-cell junctional areas allow the exchange of ions, nutrients, and small metabolites between neighboring cells. Gap junction structures are found throughout vertebrates and invertebrates, although the primary sequences of constituent proteins are different from each other even though electron micrographs and physiological assays indicate similar quaternary structure and functionality. Gap junctions are composed of two oligomeric channel structures called connexons, with each cell supplying one connexon that docks with the other at their extracellular surfaces. The connexin family of proteins has a very conserved protein folding topology with highly conserved transmembrane and extracellular primary sequences, but contains variable regions of the cytoplasmic loop and C terminus that confer the individual physiological properties to each connexin. Each connexon is made up of a cyclic arrangement of six protein monomers, called connexins (abbreviated as Cx 5 plus the molecular mass of the protein as predicted by the amino acid sequence, e.g. ...

Gap junction channels regulate cell-cell communication by passing metabolites, ions, and signaling molecules. Gap junction channel closure in cells by acidification is well documented; however, it is unknown whether acidification affects connexins or modulating proteins or compounds that in turn act on connexins. Protonated aminosulfonates directly inhibit connexin channel activity in an isoform-specific manner as shown in previously published studies. High-resolution atomic force microscopy of force-dissected connexin26 gap junctions revealed that in HEPES buffer, the pore was closed at pH < 6.5 and opened reversibly by increasing the pH to 7.6. This pH effect was not observed in non-aminosulfonate buffers. Increasing the protonated HEPES concentration did not close the pore, indicating that a saturation of the binding sites occurs at 10 mM HEPES. Analysis of the extracellular surface topographs reveals that the pore diameter increases gradually with pH. The outer connexon diameter remains unchanged, and there is a ϳ6.5°rotation in connexon lobes. These observations suggest that the underlying mechanism closing the pore is different from an observed Ca 2؉ -induced closure.Gap junction channels (GJC) 3 are dynamic macromolecular complexes capable of opening and closing the channel pore in response to a number of stimuli such as divalent cations, signaling molecules, phosphorylation, pH, and modulators of specific isoforms (1). These regulated conduits for the passage of small molecules greatly influence homeostasis, development, ionic transmission, and other cellular processes. Whereas there exist strong cell biological, biochemical, and biophysical evidence for the effects of these modulators, there is not much information at the structural level as to the conformational changes that occur in closing the pore in response to these stimuli.Each connexin (Cx) channel is composed of two hexamers (connexons) that dock at their apposed extracellular surfaces. The cyclic arrangement of the subunits within the hexamers suggests that gating can occur by a rotation and translation of the transmembrane segments within all six monomers. It has been postulated that gating occurs as a "camera iris" shutter (2). An alternate hypothesis has been proposed in which intra-connexin associations occur to produce either a particle-receptor blockage at the cytoplasmic surface (3, 4) or as a physical gate near the extracellular surface ("loop gate") (5). Whether these proposed mechanisms correlate to the closure of fast and/or slow gates that have been characterized by electrophysiological methods (see Ref. 6) remain to be determined.Gating by intracellular acidification is one way that connexin channels open and close in response to stimuli. Experimentally determined decreases in intracellular pH are known to decrease junctional electrical coupling in cardiomyocytes and in Purkinje fibers (7-10) as well as in teleost and amphibian embryos (11). Stergiopoulos et al. (12) showed that many, but not all, connexins close in a pH-sensit...

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Gap junctions (GJ) are defined as contact regions between two adjacent cells containing tens to thousands of closely packed membrane channels. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Previously, we engineered a recombinant connexin43 (Cx43) by genetically appending a small tetracysteine peptide motif containing the sequence -Cys-Cys-Xaa-Xaa-Cys-Cys- to the carboxy terminus of Cx43 (Cx43-TC) (3). Cx43-TC was stably expressed in HeLa cells and was specifically labeled by exposing the cells to membrane-permeant non-fluorescent ligands, such as FlAsH (a fluorescein derivative) and ReAsH (a resorufin derivative). Direct correlation of live cell images with high resolution EM detection was possible because bound ReAsH not only becomes fluorescent, but can also be used to initiate the photoconversion of diaminobenzidine (DAB) that causes the localized polymerization of an insoluble osmiophilic precipitate then visible by EM. Cx43-TC GJ's could be labeled with ReAsH and photooxidized to give selectively stained channels. Here, how the development of these tetracysteine tags complexed with appropriate ligands are useful for experiments spanning resolution ranges from light microscopy to electron tomography to molecular purification and detection is described.