Conformational changes in surface structures of isolated connexin 26 gap junctions
2002; Springer Nature; Volume: 21; Issue: 14 Linguagem: Inglês
10.1093/emboj/cdf365
ISSN1460-2075
Autores Tópico(s)Nicotinic Acetylcholine Receptors Study
ResumoArticle15 July 2002free access Conformational changes in surface structures of isolated connexin 26 gap junctions Daniel J. Müller Corresponding Author Daniel J. Müller Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany BIOTEC, Technical University Dresden, Dresden, Germany Search for more papers by this author Galen M. Hand Galen M. Hand National Center for Microscopy and Imaging Research, Department of Neurosciences, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Andreas Engel Andreas Engel M.E.Müller Institute for Structural Biology, Biozentrum, Basel, Switzerland Search for more papers by this author Gina E. Sosinsky Corresponding Author Gina E. Sosinsky National Center for Microscopy and Imaging Research, Department of Neurosciences, University of California, San Diego, La Jolla, CA, USA San Diego Supercomputer Center, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Daniel J. Müller Corresponding Author Daniel J. Müller Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany BIOTEC, Technical University Dresden, Dresden, Germany Search for more papers by this author Galen M. Hand Galen M. Hand National Center for Microscopy and Imaging Research, Department of Neurosciences, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Andreas Engel Andreas Engel M.E.Müller Institute for Structural Biology, Biozentrum, Basel, Switzerland Search for more papers by this author Gina E. Sosinsky Corresponding Author Gina E. Sosinsky National Center for Microscopy and Imaging Research, Department of Neurosciences, University of California, San Diego, La Jolla, CA, USA San Diego Supercomputer Center, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Author Information Daniel J. Müller 1,2, Galen M. Hand3, Andreas Engel4 and Gina E. Sosinsky 3,5 1Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany 2BIOTEC, Technical University Dresden, Dresden, Germany 3National Center for Microscopy and Imaging Research, Department of Neurosciences, University of California, San Diego, La Jolla, CA, USA 4M.E.Müller Institute for Structural Biology, Biozentrum, Basel, Switzerland 5San Diego Supercomputer Center, University of California, San Diego, La Jolla, CA, USA *Corresponding authors. E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2002)21:3598-3607https://doi.org/10.1093/emboj/cdf365 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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 flexibility, 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 Ca2+ 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 specific among the divalent cations tested. Ca2+ 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. Introduction Gap junctions are the sites of direct cell-to-cell communication in vertebrates, facilitating the exchange of molecules and signals. These intercellular channels are pairs of hexameric half-channels, called connexons or hemichannels, which are connected coaxially to bridge the extracellular space between two adjacent plasma membranes. Thousands of docked connexon pairs are tightly packed into junctional plaques that are planar, double-layered structures with distinct boundaries. Connexons are assembled from one or more connexins, which are a family of highly conserved polypeptides. At present, 21 members of this family have been identified in mammals and more have been identified in non-mammalian vertebrates that are orthologs of these mammalian isoforms (Willecke et al., 2002). Connexins exhibit a conserved core comprising the N-terminus, four transmembrane domains and two extracellular loops, as well as a variable portion consisting of the cytoplasmic loop and C-terminus. The three-dimensional (3D) structure of recombinant gap junction channels derived from cryo-electron microscopy at a resolution of 7 Å has recently provided direct evidence for α-helical folding of four transmembrane domains within each connexin subunit (Unger et al., 1999). Complementary to electron microscopy (EM), atomic force microscopy (AFM; Binnig et al., 1986) provided the first insight into the connexon extracellular surface structure (Hoh et al., 1991, 1993). Sample preparation and imaging procedures of AFM have been improving steadily, thereby allowing surface structures of native proteins to be observed at a vertical resolution of 0.1 nm and a lateral resolution of <1 nm (Engel et al., 1997; Czajkowsky and Shao, 1998). Most attractive for biologists, AFM reveals the biological object in buffer solution, at ambient temperatures and with an outstanding signal-to-noise ratio, allowing observation of single proteins at work in their native environment (Engel et al., 1999; Engel and Müller, 2000). Early experiments measuring cell–cell coupling in several cell systems have shown that Ca2+ ions decrease or suppress electrical coupling mediated by gap junctions (Loewenstein et al., 1967; Oliveira-Castro and Loewenstein, 1971; DeMello, 1975). These observations and other experiments formed the basis of Loewenstein's 'calcium hypothesis', which states that cytoplasmic calcium ion levels regulate the gating of gap junction channels (Loewenstein, 1966). Large fluxes of cytoplasmic calcium ion concentrations have long been postulated as a cellular apoptotic mechanism that uncouples the gap junctions in dying cells, thereby isolating dying cells from healthy neighbors. Following up on Loewenstein's hypothesis, Unwin and colleagues published two EM structures of isolated rat liver gap junctions at ∼2.5 nm resolution, one prepared in the presence of calcium ions, the other prepared in the presence of calcium ion chelators (Unwin and Zampighi, 1980; Unwin and Ennis, 1984). Their interpretation of the differences between the Ca2+ and Ca2+-free structures lead them to postulate that Ca2+ ions cause a rotation of the subunits that closes the pore in a manner analogous to the closing of a camera iris. However, while calcium-mediated uncoupling has been studied since the 1960s, we still do not understand this effect, and neither can we resolve the puzzle of cells producing calcium waves that are transmitted from cell to cell (Rottingen and Iversen, 2000). Nevertheless, recent studies show that cells containing high concentrations (∼1–2 mM) of calcium ions in the external medium may close non-junctional gap junction hemichannels [connexin (Cx) 43 (Li et al., 1996); Cx46 (Pfahnl and Dahl, 1999); and Cx26 (Kamermans et al., 2001)]. Here we report the first high-resolution topographs of both connexon surfaces and their conformational changes. The cytoplasmic C-terminal domains form a hexameric pore exhibiting a high degree of structural flexibility. Upon increasing the force applied to the AFM stylus by a few tens of picoNewtons (1 pN = 10−12 N), these domains reversibly collapse onto the membrane surface. Concomitant with these considerable structural rearrangements, the cytoplasmic channel entrance appears to close. We demonstrate that in single connexon layers, the hexameric extracellular pore appears more rigid, with its channel entrance narrowing significantly in the presence of Ca2+ ions. A different structural change is observed with intact gap junctions upon calcium ion incubation. Our results demonstrate that the Ca2+-induced closure of gap junction hemichannels is the result of a change in conformation in the pore opening at the extracellular surface. Furthermore, the results presented here indicate that there is a different gating mechanism for closure at the extracellular surface from the one that occurs at the cytoplasmic surface. Therefore, two distinct structural entities within the connexon are likely to act as physical gates at the two ends of the pore. Results Imaging and dissection of gap junction plaques Gap junction plaques adsorbed to freshly cleaved mica and recorded in buffer solution by AFM (Figure 1A, marked as GJ) exhibited a thickness of 17.4 ± 0.7 nm (n = 47). In contrast, the lipid membranes (Figure 1A, marked as LM) surrounding the protein membranes had a height of 4.5 ± 0.5 nm (n = 58), while single layered connexon membranes (Figure 1A, marked as CX) showed a height of 8.0 ± 0.6 nm (n = 24). A summary of these thickness measurements is shown in Table I. Figure 1.Gap junction plaque imaged in buffer solution using AFM. (A) Overview of a gap junction plaque (marked as GJ) surrounded by a lipid membrane (marked as LM) and fragments of single-layered connexon membranes (marked as CX). (B) The same gap junction plaque but partly dissected. The gap junction membrane was dissected by enhancing the applied force from ∼50 pN (imaging force) to ∼500 pN. After removal, the gap junction plaque was re-imaged at ∼50 pN. (C and D) Extracellular surface of the connexon membrane at elevated magnifications. The hexagonal arrangement of the connexons is clearly visible (D). Topographs were recorded in buffer solution (5 mM Tris, 1 mM EGTA, 1 mM PMSF) with a force of ∼50 pN applied to the AFM stylus and a line frequency of 4.4 Hz, and were displayed as relief tilted by 5°. Topographs exhibited a vertical full gray level scale of 25 nm (A–C) and 2.5 nm (D), and were displayed as relief tilted by 5%. Download figure Download PowerPoint Table 1. Summary of pore and thickness sizes measured in AFM images Morphological feature Size ± SD (nm) 50 pN imaging force Size ± SD (nm) 70 pN imaging force Gap junction bilayer height 4.7 ± 0.6 NA Non-junctional bilayer height 4.5 ± 0.5 NA Gap junction height 17.4 ± 0.7 NA Single connexon layer height 8.0 ± 0.6 NA Cytoplasmic domain height 1.7 ± 0.2 0.2 ± 0.2 Extracellular domain height 1.6 ± 0.2 NA Outer diameter of cytoplasmic pore 5.6 ± 0.3 5.8 ± 0.3 Inner diameter of cytoplasmic pore 2.8 ± 0.3 4.7 ± 0.3 NA, not applicable. As visible from the overview image, only small regions of the extracellular surface were exposed to the AFM stylus (Figure 1A, marked as CX). To image a larger area of this interface, the upper connexon layer was removed carefully using the AFM stylus as a 'nanotweezer' (Figure 1B and C) (Hoh et al., 1991; Schabert et al., 1995; Fotiadis et al., 1998, 2000, 2002). Following this, the extracellular surface of the remaining single-layered connexon membrane was imaged at higher magnification and the hexagonal assembly of individual connexons into microcrystalline patches became visible (Figure 1D). Because the forces used for AFM image recording were not destructive to the specimen, the same gap junction or connexon layer can be imaged repeatedly. This was a prerequisite to observe reproducible structural changes of the membrane surface. Additionally, comparing membrane surfaces imaged using the same AFM stylus minimized possible errors arising from tip artifacts (Schwarz et al., 1994). Cytoplasmic domains of the gap junction form a hexagonal pore Imaged at a higher resolution and with the minimum force applied to the AFM stylus (50 ± 20 pN) to obtain stable images, the cytoplasmic gap junction surface (Figure 1A, GJ) exhibited donut-shaped structures assembled into a hexagonal lattice, with a unit cell distance of 7.7 ± 0.5 nm (Figure 2A, top). Such imaging conditions were obtained after carefully balancing electrostatic repulsion and van der Waals attraction between stylus and protein (Müller et al., 1999a). While individual connexons are distinct in the unprocessed topograph, their consistent structural features are revealed by their average (Figure 2B). Each gap junction exhibited six protruding cytoplasmic domains, forming a donut-like structure. The donuts had an outer diameter 5.6 ± 0.3 nm (n = 68) at full width half maximum (FWHM), and their pores had an inner diameter of 2.8 ± 0.3 nm (n = 68) with a depth of 1.0 ± 0.3 nm (n = 30). In the average structure, the cytoplasmic domains protruded by 1.7 ± 0.2 nm (n = 30) from the lipid bilayer (Figure 2B, +). A summary of these pore dimensions is shown in Table I. Figure 2.Two conformations of the cytoplasmic gap junction surface. (A) AFM topograph demonstrating the variability of cytoplasmic gap junction domains. Individual gap junction domains appear disordered (circle). The initial applied force of 50 pN (top to center of image) was enhanced at the center of the topograph (blue arrow) to 70 pN (center to bottom of image). A conformational change is distinct: pore forming gap junction hexamers collapse onto the membrane surface, thereby transforming into pores with larger channel diameters. (B) Average of the extended conformation of gap junction exhibiting a lateral resolution of ∼2 nm. The cytoplasmic domains form a pore (asterisks) and protrude by 1.7 ± 0.2 nm (n = 30) above the lipid bilayer (cross). (C) Average of gap junction domains collapsed onto the membrane surface. Here the cytoplasmic domains protrude only by 0.2 ± 0.2 nm (n = 30) above the lipid bilayer (+). Topograph was recorded in Ca2+-free buffer solution (5 mM Tris, 1 mM EGTA and 1 mM PMSF) at a line frequency of 5 Hz. All topographs were displayed as relief tilted by 5°. Topographs exhibit a vertical full gray level scale of 3 nm (A), of 2 nm (B) and of 1 nm (C). Download figure Download PowerPoint Cytoplasmic gap junction surface domains exhibit high structural flexibility When imaged with a slightly enhanced force applied to the stylus (70 ± 20 pN), the cytoplasmic domains were observed to collapse onto the membrane surface (Figure 2A, center to bottom). This structural change was fully reversible and could be repeated several times without detectable structural destruction. The collapse of the cytoplasmic domains formed a supra-structure on the membrane surface (Figure 2C) that reduced the gap junction thickness by 1.5 nm. Consequently, these surface structures protruded only 0.2 ± 0.2 nm (n = 30) above the lipid bilayer (Figure 2C, cross). The cytoplasmic domains of this conformation surrounded an enlarged channel entrance exhibiting an inner diameter of 4.7 ± 0.3 nm (n = 27) at FWHM and formed a pore (asterisks) with an outer diameter of 5.8 ± 0.3 nm (n = 27). The depth of the pore was ∼1.6 ± 0.2 nm (n = 27), while the center-to-center distance of the unit cell (7.7 ± 0.5 nm) remained unchanged. Extracellular connexon surface structure When imaging the extracellular connexon surface in the Ca2+-free buffer used for isolation at high magnification (pixel size 15 Å) structures (Sosinsky et al., 1988; Sosinsky, 1992; Perkins et al., 1997). The higher resolution structure of a truncation mutant of Cx43 (263 total aa; Unger et al., 1999) containing a longer C-terminus than Cx26 (226 total aa), shows some cytoplasmic surface structure. However, since these cytoplasmic segments were crystallographically disordered in the EM preparations investigated, the averaged surface structures were short and unconnected. EM images of thin sections of tannic acid-fixed rat liver gap junctions contained wispy protein extensions at the cytoplasmic surface (Sosinsky et al., 1988). This observation is in apparent contrast to our finding that the C-terminal domains can co-exist in a structurally ordered conformation. It may be concluded that the cytoplasmic domains observed in our experiments may not be sufficiently crystallographically ordered in preparations that are studied by EM. These results suggest that the specimen conditions are crucial in maintaining the native conformation of the cytoplasmic gap junction domains. The AFM topographs presented here provide a surface view of the structure, and show that the native cytoplasmic gap junction domains are indeed flexible but that these domains can rearrange into an ordered structure. The unperturbed structure of the domains exte
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