The Experts below are selected from a list of 543 Experts worldwide ranked by ideXlab platform
G. E. Sosinsky - One of the best experts on this subject based on the ideXlab platform.
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Correlation analysis of gap junction lattice images
2015Co-Authors: G. E. Sosinsky, T. S. Baker, T D. L. D. Caspar, D. A. GoodenoughllAbstract:ABSTRACT Fourier averages of Connexon images computed from low-irradiation electron micrographs of isolated negatively stained gap junction domains exhibited differences in stain distribution and Connexon orientation. To analyze these polymorphic structures, correlation averaging methods were applied to images from negatively stained and frozen-hydrated specimens. For the negatively stained specimens, separate averages over two subsets of Connexons with differing degrees of stain accumulation in the axial channel were obtained. Two populations of Connexons with opposite skew orientations were distinguishable within a single junctional domain of a frozen-hydrated specimen Correlation maps calculated using the left- and right-skewed references showed that the selected Connexons tend to locally cluster. Using correlation methods to analyze packing disorder in a typical Connexon lattice, we estimated the root-mean-square variation in the nearest neighbor pair separation to be- 1 1 % of the lattice constant. Displacements of the Connexons relative to each other increased with increasing pair separation in the lattice, rather like a liquid, although long-range orientation order was conserved as in a crystal. These results support the hypothesis that the hexagonal ordering of the Connexons results from short-range repulsive forces
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pannexin channels are not gap junction hemichannels
Channels, 2011Co-Authors: G. E. Sosinsky, Silvia Penuela, Dale W Laird, Heather S Duffy, Christian C Naus, Daniela Boassa, Rolf Dermietzel, Brian A Macvicar, Eliana Scemes, David C SprayAbstract:Pannexins, a class of membrane channels, bear significant sequence homology with the invertebrate gap junction proteins, innexins, and more distant similarities in their membrane topologies and pharmacological sensitivities with the gap junction proteins, connexins. However, the functional role for the pannexin oligomers or pannexons, is different from connexin oligomers, the Connexons. Many pannexin publications have used the term “hemichannels” to describe pannexin oligomers while others use the term “channels” instead. This has led to confusion within the literature about the function of pannexins that promotes the idea that pannexons serve as gap junction hemichannels and thus, have an assembly and functional state as gap junctional intercellular channels. Here, we present the case that unlike the connexin gap junction intercellular channels, so far, pannexin oligomers have repeatedly been shown to be channels that are functional in single membranes, but not as intercellular channels in appositional m...
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Mutation of a Conserved Threonine in the Third Transmembrane Helix of α- and β-Connexins Creates a Dominant-negative Closed Gap Junction Channel
The Journal of biological chemistry, 2005Co-Authors: Derek L. Beahm, Bruce J Nicholson, Galen M. Hand, A. Oshima, Guido M. Gaietta, Amy Smock, Shoshanna N. Zucker, Masoud M. Toloue, Anjana Chandrasekhar, G. E. SosinskyAbstract:Abstract 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 α- and β-connexin subgroups but not the γ-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.
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conformational changes in surface structures of isolated connexin 26 gap junctions
The EMBO Journal, 2002Co-Authors: Andreas Engel, Galen M. Hand, Daniel J Muller, G. E. SosinskyAbstract: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 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 specific 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.
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three dimensional structure of the gap junction Connexon
Biophysical Journal, 1997Co-Authors: Guy A. Perkins, Daniel A. Goodenough, G. E. SosinskyAbstract:The gap junction membrane channel is composed of macular aggregations of intercellular channels permitting the direct intercellular transfer of ions and small molecules. Each intercellular channel is formed by the apposition of two hexameric transmembrane channels (Connexons), one from each cell. The interlocking of the two channels occurs extracellularly in a narrow 3.5-nm "gap" separating the junctional membranes. The channel-channel interaction is known to be selective between members of the family of proteins, called connexins, which oligomerize into the Connexons. In addition to selectivity, the molecular interfaces involved in the extracellular interactions between Connexons must be very congruent, since the intercellular channel must provide high resistances to the leakage of small ions between the channel lumen and the extracellular space. By using a recently developed biochemical procedure for obtaining ordered arrays of Connexons from gap junctions split in the extracellular gap, (Ghoshroy, S., D. A. Goodenough, and G. E. Sosinsky. 1995. Preparation, characterization, and structure of half gap junctional layers split with urea and EGTA. J. Membr. Biol. 146:15–28) a three-dimensional reconstruction of a Connexon has been obtained by electron crystallographic methods. This reconstruction emphasizes the structural asymmetry between the extracellular and cytoplasmic domains and assigns lobed structural features to the extracellular domains of the Connexon. The implication of our hemichannel structure is discussed in relation to the in vivo state of unpaired Connexons, which have been shown to exist in the plasma membrane.
Simon Scheuring - One of the best experts on this subject based on the ideXlab platform.
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high speed atomic force microscopy cooperative adhesion and dynamic equilibrium of junctional microdomain membrane proteins
Journal of Molecular Biology, 2012Co-Authors: Adai Colom, Ignacio Casuso, Thomas Boudier, Simon ScheuringAbstract:Junctional microdomains, paradigm for membrane protein segregation in functional assemblies, in eye lens fiber cell membranes are constituted of lens-specific aquaporin-0 tetramers (AQP0(4)) and connexin (Cx) hexamers, termed Connexons. Both proteins have double function to assure nutrition and mediate adhesion of lens cells. Here we use high-speed atomic force microscopy to examine microdomain protein dynamics at the single-molecule level. We found that the adhesion function of head-to-head associated AQP0(4) and Cx is cooperative. This finding provides first experimental evidence for the mechanistic importance for junctional microdomain formation. From the observation of lateral association-dissociation events of AQP0(4), we determine that the enthalpic energy gain of a single AQP0(4)-AQP0(4) interaction in the membrane plane is -2.7 k(B)T, sufficient to drive formation of microdomains. Connexon association is stronger as dynamics are rarely observed, explaining their rim localization in junctional microdomains.
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binding kinetics of inter Connexon interaction
Biophysical Journal, 2011Co-Authors: Felix Rico, Atsunori Oshima, Yoshinori Fujiyoshi, Peter Hinterdorfer, Simon ScheuringAbstract:Gap junctions are pairs of hexameric half-channels called Connexons, which coaxially dock to connect two adjacent cells, mediating both adhesion and channeling between cells in many types of tissue. Connexons are formed of six connexin proteins (Cx). Gap junctions form by interdigitating the two extracellular loops of each connexin. While gap junction structure and function has been widely characterized using different techniques, the binding affinity of the inter-Connexon interaction remains unknown. The goal of this work was to determine the binding affinity of gap junctions using dynamic force spectroscopy atomic force microscopy (AFM). Among the residues that mediate inter-Connexon interaction, an exposed stretch of conserved amino acids ‘NTVD’ within the extracellular loop 2 (E2) has been identified. For dynamic force spectroscopy, we covalently linked mimetic peptides ‘NTVD‘ that mimic loop E2 of Cx26 to the AFM tip, while Cx26 two-dimensional (2D) crystals were immobilized on a mica substrate. We report the first characterization of the binding strength of the gap junction interaction. Force curves at various retraction speeds were acquired to determine the dissociation kinetics of the peptide-Cx26 interaction, while adhesion probability measurements at different contact times revealed the binding kinetics. The relatively fast intrinsic dissociation rate (koff) inferred a rather dynamic inter-Connexon interaction, while the slow association rate (kon) probably reflects the restricted mobility and degrees of freedom of the Connexons in the densely packed organization observed in native gap junction plaques and the reduced flexibility and dimensions of the extracellular loops. Our results suggest that gap junction formation may occur before plaque formation.
Robert G Gourdie - One of the best experts on this subject based on the ideXlab platform.
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connexin 43 Connexon to gap junction transition is regulated by zonula occludens 1
Molecular Biology of the Cell, 2011Co-Authors: Matthew J Rhett, Jane L Jourdan, Robert G GourdieAbstract:Cx43 gap junctions (GJs) are integral to the function of the mammalian heart. It is shown that ZO-1 dynamically regulates the transition between Cx43 Connexons and GJ intercellular channels, determ...
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connexin 43 Connexon to gap junction transition is regulated by zonula occludens 1
Molecular Biology of the Cell, 2011Co-Authors: Matthew J Rhett, Jane L Jourdan, Robert G GourdieAbstract:Connexin 43 (Cx43) is a gap junction (GJ) protein widely expressed in mammalian tissues that mediates cell-to-cell coupling. Intercellular channels comprising GJ aggregates form from docking of paired Connexons, with one each contributed by apposing cells. Zonula occludens-1 (ZO-1) binds the carboxy terminus of Cx43, and we have previously shown that inhibition of the Cx43/ZO-1 interaction increases GJ size by 48 h. Here we demonstrated that increases in GJ aggregation occur within 2 h (∼Cx43 half-life) following disruption of Cx43/ZO-1. Immunoprecipitation and Duolink protein-protein interaction assays indicated that inhibition targets ZO-1 binding with Cx43 in GJs as well as Connexons in an adjacent domain that we term the "perinexus." Consistent with GJ size increases being matched by decreases in Connexons, inhibition of Cx43/ZO-1 reduced the extent of perinexal interaction, increased the proportion of Connexons docked in GJs relative to undocked Connexons in the plasma membrane, and increased GJ intercellular communication while concomitantly decreasing hemichannel-mediated membrane permeance in contacting, but not noncontacting, cells. ZO-1 small interfering RNA and overexpression experiments verified that loss and gain of ZO-1 function govern the transition of Connexons into GJs. It is concluded that ZO-1 regulates the rate of undocked Connexon aggregation into GJs, enabling dynamic partitioning of Cx43 channel function between junctional and proximal nonjunctional domains of plasma membrane.
Kathrin A Stauffer - One of the best experts on this subject based on the ideXlab platform.
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the gap junction proteins β1 connexin connexin 32 and β2 connexin connexin 26 can form heteromeric hemichannels
Journal of Biological Chemistry, 1995Co-Authors: Kathrin A StaufferAbstract:Abstract Two different types of gap junction proteins, β1- and β2-connexin, were expressed in insect cells, either singly or together, using infection with recombinant baculovirus. Membrane fractions enriched in gap junction proteins were isolated, and Connexons (hemichannels) were solubilized with detergent. These solubilized Connexons were then run out on a gel filtration column which was capable of partially separating the two homomeric Connexons. It was found that Connexons from cells co-infected with both types of baculovirus ran together on this column, whereas Connexons from cells infected separately and mixed before solubilization did not, suggesting that in the co-infected cells the two types of connexin are assembled into heteromeric hemichannels.
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the gap junction proteins β1 connexin connexin 32 and β2 connexin connexin 26 can form heteromeric hemichannels
Journal of Biological Chemistry, 1995Co-Authors: Kathrin A StaufferAbstract:Two different types of gap junction proteins, beta 1- and beta 2-connexin, were expressed in insect cells, either singly or together, using infection with recombinant baculovirus. Membrane fractions enriched in gap junction proteins were isolated, and Connexons (hemichannels) were solubilized with detergent. These solubilized Connexons were then run out on a gel filtration column which was capable of partially separating the two homomeric Connexons. It was found that Connexons from cells co-infected with both types of baculovirus ran together on this column, whereas Connexons from cells infected separately and mixed before solubilization did not, suggesting that in the co-infected cells the two types of connexin are assembled into heteromeric hemichannels.
Luc Leybaert - One of the best experts on this subject based on the ideXlab platform.
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connexin 43 is an emerging therapeutic target in ischemia reperfusion injury cardioprotection and neuroprotection
Pharmacology & Therapeutics, 2015Co-Authors: Rainer Schulz, Philipp Maximilian Gorge, Aniko Gorbe, Peter Ferdinandy, Paul D Lampe, Luc LeybaertAbstract:Connexins are widely distributed proteins in the body that are crucially important for heart and brain functions. Six connexin subunits form a Connexon or hemichannel in the plasma membrane. Interactions between two hemichannels in a head-to-head arrangement result in the formation of a gap junction channel. Gap junctions are necessary to coordinate cell function by passing electrical current flow between heart and nerve cells or by allowing exchange of chemical signals and energy substrates. Apart from its localization at the sarcolemma of cardiomyocytes and brain cells, connexins are also found in the mitochondria where they are involved in the regulation of mitochondrial matrix ion fluxes and respiration. Connexin expression is affected by age and gender as well as several pathophysiological alterations such as hypertension, hypertrophy, diabetes, hypercholesterolemia, ischemia, post-myocardial infarction remodeling or heart failure, and post-translationally connexins are modified by phosphorylation/de-phosphorylation and nitros(yl)ation which can modulate channel activity. Using knockout/knockin technology as well as pharmacological approaches, one of the connexins, namely connexin 43, has been identified to be important for cardiac and brain ischemia/reperfusion injuries as well as protection from it. Therefore, the current review will focus on the importance of connexin 43 for irreversible injury of heart and brain tissues following ischemia/reperfusion and will highlight the importance of connexin 43 as an emerging therapeutic target in cardio- and neuroprotection.