Characterization of gap junctions between cultured leptomeningeal cells

Spray, David C.; Moreno, Alonso P.; Kessler, JA; Dermietzel, Rolf

doi:10.1016/0006-8993(91)91373-9

Cited by 90 publications

(52 citation statements)

References 35 publications

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“…The looser interactions with residues Ser-364 -Ala-371 may play a more regulatory role with other Cx43 molecular partners. It is perhaps noteworthy that this region contains three phosphorylatable residues: protein kinase C phosphorylates Cx43 on Ser-368 and Ser-372 (7,63), leading to decreased junctional conductance (64 -66), whereas phosphorylation of Ser-364 by cAMP-dependent protein kinase increases gap junctional communication (67,68). Whether phosphorylation of this region alters binding affinity to intracellular ligands remains to be determined.…”

Section: Discussionmentioning

confidence: 99%

Structural Changes in the Carboxyl Terminus of the Gap Junction Protein Connexin43 Indicates Signaling between Binding Domains for c-Src and Zonula Occludens-1

Sorgen

Duffy

Sahoo

et al. 2004

Journal of Biological Chemistry

183

220

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Regulation of cell-cell communication by the gap junction protein connexin43 can be modulated by a variety of connexin-associating proteins. In particular, c-Src can disrupt the connexin43 (Cx43)-zonula occludens-1 (ZO-1) interaction, leading to down-regulation of gap junction intercellular communication. The binding sites for ZO-1 and c-Src correspond to widely separated Cx43 domains (ϳ100 residues apart); however, little is known about the structural modifications that may allow information to be transferred over this distance. Here, we have characterized the structure of the connexin43 carboxyl-terminal domain (Cx43CT) to assess its ability to interact with domains from ZO-1 and c-Src. NMR data indicate that the Cx43CT exists primarily as an elongated random coil, with two regions of ␣-helical structure. NMR titration experiments determined that the ZO-1 PDZ-2 domain affected the last 19 Cx43CT residues, a region larger than that reported to be required for Cx43CT-ZO-1 binding. The c-Src SH3 domain affected Cx43CT residues Lys-264 -Lys-287, Ser-306 -Glu-316, His-331-Phe-337, Leu-356 -Val-359, and Ala-367-Ser-372. Only region Lys-264 -Lys-287 contains the residues previously reported to act as an SH3 binding domain. The specificity of these interactions was verified by peptide competition experiments. Finally, we demonstrated that the SH3 domain could partially displace the Cx43CT-PDZ-2 complex. These studies represent the first structural characterization of a connexin domain when integrated in a multimolecular complex. Furthermore, we demonstrate that the structural characteristics of a disordered Cx43CT are advantageous for signaling between different binding partners that may be important in describing the mechanism of channel closure or internalization in response to pathophysiological stimuli.Gap junction channels serve to directly interconnect the cytoplasm of neighboring cells, allowing the passage of moderately small ions, metabolites, and signaling molecules. Mammalian gap junction channels are formed by as many as 21 different connexin proteins (1). Of these, connexin43 (Cx43) 1 is the most completely characterized in terms of channel gating properties (2-4), phosphorylation sites (5-7), mechanisms of pH sensitivity (8 -11), and overall molecular structure (12). Cx43 is the most abundant gap junction protein in various tissues, including heart and brain. Cx43 null mice have been extensively investigated, with important differences being found as compared with wild types with regard to numerous processes, including cardiac developmental abnormalities, electrical synchrony in the heart, spreading depression in brain, as well as global gene expression changes in heart and astrocytes (13)(14)(15)(16)(17)(18)(19)(20).Connexin molecules are tetraspan membrane proteins, with both amino and carboxyl termini within the cytoplasm. Although the structure of the membrane-spanning portions of Cx43 has been solved to a resolution of about 7.5 Å (in the membrane plane) using electron crystallography (12), a constr...

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Section: Discussionmentioning

confidence: 99%

Structural Changes in the Carboxyl Terminus of the Gap Junction Protein Connexin43 Indicates Signaling between Binding Domains for c-Src and Zonula Occludens-1

Sorgen

Duffy

Sahoo

et al. 2004

Journal of Biological Chemistry

183

220

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“…By contrast, in cells where the phosphorylated forms of Cx43 predominate (e.g., rat cardiocytes) TPA promotes intercellular communication with no detectable changes in the state of phosphorylation, as evaluated by Western blots (42). Nevertheless, there are exceptions: in rat leptomeningeal cells (8), and in a rat liver epithelial cell line (IRA 20) (54) the phosphorylated forms of Cx43 predominate and TPA induces uncoupling without detectable changes in the state of Cx43 phosphorylation detected by Western blots. In IRA 20 cells TPA did not change the levels of Cx43 or of its mRNA but did result in the loss of Cx43 immunoreactivity by indirect immunofluorescence (54), suggesting that the inhibition in intercellular gap junctional communication might involve a post-translational modification that perhaps cannot be detected in denaturing gels.…”

Section: Effect Of Cx43 Phosphorylation On Intercellular Gap Junctionmentioning

confidence: 99%

“…Frequently, a single cell can express more than one Cx, which can be localized in the same (6,7) or in different gap junction plaques (8). Moreover, functional gap junctions can be established in an exogenous expression system (4,5,(9)(10)(11)(12)(13)(14)(15)(16)(17) where the role of protein phosphorylation in channels formed by a particular Cx can be analyzed at the functional level.…”

Section: Introductionmentioning

confidence: 99%

Regulation of gap junctions by protein phosphorylation

Sáez

Martı́nez

Brañes

et al. 1998

Braz J Med Biol Res

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Gap junctions are constituted by intercellular channels and provide a pathway for transfer of ions and small molecules between adjacent cells of most tissues. The degree of intercellular coupling mediated by gap junctions depends on the number of gap junction channels and their activity may be a function of the state of phosphorylation of connexins, the structural subunit of gap junction channels. Protein phosphorylation has been proposed to control intercellular gap junctional communication at several steps from gene expression to protein degradation, including translational and post-translational modification of connexins (i.e., phosphorylation of the assembled channel acting as a gating mechanism) and assembly into and removal from the plasma membrane. Several connexins contain sites for phosphorylation for more than one protein kinase. These consensus sites vary between connexins and have been preferentially identified in the C-terminus. Changes in intercellular communication mediated by protein phosphorylation are believed to control various physiological tissue and cell functions as well as to be altered under pathological conditions.Correspondence

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“…These two connexins are found together in a number of tissues or organs such as mammary epithelium (Tomasetto et al, 1993), skin (Risek et al, 1994;Goliger and Paul, 1995), colchea (Kikuchi et al, 2000) and testes (Brehm et al, 2002). Indeed, in leptomeningeal cells (Spray et al, 1991), thyroid cells (Meda et al, 1993), epidermal cells (Risek et al, 1994), P19 embryonal carcinoma cells (Belliveau et al, 1997), hypothalamic neuronal cell lines (Charles et al, 1996), lung epithelial cells (Lee et al, 1997) and astrocytes (Nagy et al, 2001), Cx26 and Cx43 are present in the same cells. Both connexins localize to appositional membranes in co-expressing cells.…”

Section: Introductionmentioning

confidence: 99%

“…Yet, cell biological studies from a number of laboratories have suggested that they might not mix. For example, Spray et al (Spray et al, 1991) found that Cx26 and Cx43 localize to discrete positions at appositional membranes in cultured leptomeningeal cells and make identifiable channels of discrete sizes. Risek et al (Risek et al, 1994) identified very large gap junctions in epidermal cells that contained discrete domains of Cx26 or Cx43 immunoreactivity.…”

Section: Introductionmentioning

confidence: 99%

Connexin43 and connexin26 form gap junctions, but not heteromeric channels in co-expressing cells

Gemel

Valiūnas

Brink

et al. 2004

Journal of Cell Science

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solubilized connexons from co-expressing cells by centrifugation through sucrose gradients or by affinity purification using a Ni-NTA column showed no evidence of mixing of Cx26 and Cx43. These results contrast with our observations in cells co-expressing other connexins with Cx43 and suggest that Cx26 and Cx43 do not form heteromeric hemichannels. Moreover, the incorporation of Cx26 and Cx43 into oligomers and into the membrane were similarly affected by treatment of co-expressing cells with brefeldin A or nocodazole, suggesting that the lack of mixing is due to incompatibility of these connexins, not to differences in biosynthetic trafficking.

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Characterization of gap junctions between cultured leptomeningeal cells

Cited by 90 publications

References 35 publications

Structural Changes in the Carboxyl Terminus of the Gap Junction Protein Connexin43 Indicates Signaling between Binding Domains for c-Src and Zonula Occludens-1

Structural Changes in the Carboxyl Terminus of the Gap Junction Protein Connexin43 Indicates Signaling between Binding Domains for c-Src and Zonula Occludens-1

Regulation of gap junctions by protein phosphorylation

Connexin43 and connexin26 form gap junctions, but not heteromeric channels in co-expressing cells

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