scholarly journals Conductance and Permeability of the Residual State of Connexin43 Gap Junction Channels

2002 ◽  
Vol 119 (2) ◽  
pp. 171-186 ◽  
Author(s):  
Feliksas F. Bukauskas ◽  
Angele Bukauskiene ◽  
Vytas K. Verselis
Keyword(s):  
Gap Junction ◽  
Fluorescent Protein ◽  
Single Channel ◽  
Electrical Coupling ◽  
Dye Transfer ◽  
Channel Pore ◽  
Gap Junction Channels ◽  
Gating Mechanism ◽  
Gated Channel ◽  
Green Fluorescent

We used cell lines expressing wild-type connexin43 and connexin43 fused with the enhanced green fluorescent protein (Cx43-EGFP) to examine conductance and perm-selectivity of the residual state of Cx43 homotypic and Cx43/Cx43-EGFP heterotypic gap junction channels. Each hemichannel in Cx43 cell–cell channel possesses two gates: a fast gate that closes channels to the residual state and a slow gate that fully closes channels; the transjunctional voltage (Vj) closes the fast gate in the hemichannel that is on the relatively negative side. Here, we demonstrate macroscopically and at the single-channel level that the I-V relationship of the residual state rectifies, exhibiting higher conductance at higher Vjs that are negative on the side of gated hemichannel. The degree of rectification increases when Cl− is replaced by Asp− and decreases when K+ is replaced by TEA+. These data are consistent with an increased anionic selectivity of the residual state. The Vj-gated channel is not permeable to monovalent positively and negatively charged dyes, which are readily permeable through the fully open channel. These data indicate that a narrowing of the channel pore accompanies gating to the residual state. We suggest that the fast gate operates through a conformational change that introduces positive charge at the cytoplasmic vestibule of the gated hemichannel, thereby producing current rectification, increased anionic selectivity, and a narrowing of channel pore that is largely responsible for reducing channel conductance and restricting dye transfer. Consequently, the fast Vj-sensitive gating mechanism can serve as a selectivity filter, which allows electrical coupling but limits metabolic communication.

scholarly journals Trafficking, Assembly, and Function of a Connexin43-Green Fluorescent Protein Chimera in Live Mammalian Cells

1999 ◽  
Vol 10 (6) ◽  
pp. 2033-2050 ◽  
Author(s):  
Karen Jordan ◽  
Joell L. Solan ◽  
Michel Dominguez ◽  
Michael Sia ◽  
Art Hand ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Gap Junctions ◽  
Gap Junction ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Rat Kidney ◽  
Dye Transfer ◽  
Green Fluorescent

To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin–Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 μm and 0.5–1.5 μm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.

scholarly journals Gating Properties of Gap Junction Channels Assembled from Connexin43 and Connexin43 Fused with Green Fluorescent Protein

2001 ◽  
Vol 81 (1) ◽  
pp. 137-152 ◽  
Author(s):  
Feliksas F. Bukauskas ◽  
Angele Bukauskiene ◽  
Michael V.L. Bennett ◽  
Vytas K. Verselis
Keyword(s):  
Green Fluorescent Protein ◽  
Gap Junction ◽  
Fluorescent Protein ◽  
Gap Junction Channels ◽  
Green Fluorescent

scholarly journals Concatenation of Human Connexin26 (hCx26) and Human Connexin46 (hCx46) for the Analysis of Heteromeric Gap Junction Hemichannels and Heterotypic Gap Junction Channels

2018 ◽  
Vol 19 (9) ◽  
pp. 2742 ◽  
Author(s):  
Patrik Schadzek ◽  
Doris Hermes ◽  
Yannick Stahl ◽  
Nadine Dilger ◽  
Anaclet Ngezahayo
Keyword(s):  
Gap Junction ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
Single Channels ◽  
Dye Transfer ◽  
Channel Conductance ◽  
Gap Junction Channels ◽  
Plaque Area ◽  
Gap Junction Hemichannels ◽  
Inside Out

Gap junction channels and hemichannels formed by concatenated connexins were analyzed. Monomeric (hCx26, hCx46), homodimeric (hCx46-hCx46, hCx26-hCx26), and heterodimeric (hCx26-hCx46, hCx46-hCx26) constructs, coupled to GFP, were expressed in HeLa cells. Confocal microscopy showed that the tandems formed gap junction plaques with a reduced plaque area compared to monomeric hCx26 or hCx46. Dye transfer experiments showed that concatenation allows metabolic transfer. Expressed in Xenopus oocytes, the inside-out patch-clamp configuration showed single channels with a conductance of about 46 pS and 39 pS for hemichannels composed of hCx46 and hCx26 monomers, respectively, when chloride was replaced by gluconate on both membrane sides. The conductance was reduced for hCx46-hCx46 and hCx26-hCx26 homodimers, probably due to the concatenation. Heteromerized hemichannels, depending on the connexin-order, were characterized by substates at 26 pS and 16 pS for hCx46-hCx26 and 31 pS and 20 pS for hCx26-hCx46. Because of the linker between the connexins, the properties of the formed hemichannels and gap junction channels (e.g., single channel conductance) may not represent the properties of hetero-oligomerized channels. However, should the removal of the linker be successful, this method could be used to analyze the electrical and metabolic selectivity of such channels and the physiological consequences for a tissue.

The origin of annular junctions: a mechanism of gap junction internalization

2001 ◽  
Vol 114 (4) ◽  
pp. 763-773 ◽  
Author(s):  
K. Jordan ◽  
R. Chodock ◽  
A.R. Hand ◽  
D.W. Laird
Keyword(s):  
Cell Surface ◽  
Gap Junction ◽  
Fluorescent Protein ◽  
Rat Kidney ◽  
Time Lapse ◽  
Gap Junctional Intercellular Communication ◽  
Gap Junction Channels ◽  
Normal Rat ◽  
Connexin Proteins ◽  
Green Fluorescent

Gap junctional intercellular communication is established when connexin proteins oligomerize into connexon hemichannels, which then pair at the cell surface with connexons from neighboring cells to form functional gap junction channels. Gap junction channels routinely cluster into gap junction plaques, which can exhibit dynamic characteristics while under the frequent processes of formation and removal from the cell surface. We have three lines of evidence to suggest that one mechanism of gap junction removal occurs when one of two contacting cells internalizes the gap junction contribution from both cells. First, in coculture experiments, green fluorescent protein-tagged connexin43 (Cx43-GFP) expressed in normal rat kidney (NRK) cells can be internalized into contacting cells that do not express Cx43-GFP, and the incidences of identifying these internalized structures increase in the presence of lysosomal inhibitors. Secondly, time-lapse imaging of live NRK cells revealed that large areas of gap junction plaques containing Cx43-GFP were internalized as vesicular-like structures into one of two adjacent cells. Finally, when live NRK cells that express endogenous Cx43 were microinjected with anti-Cx43 antibodies, antibody-tagged gap junctions were visualized in cells that contacted the microinjected cell within 3–6.5 hours. Together our results strongly suggest that one mechanism of gap junction removal from the cell surface involves a unique process in which the entire gap junction or a fragment of it is internalized into one of the two contacting cells as an annular junction.

Transport of Connexins and Gap Junction Turnover in Living Cells Visualized Using Green Fluorescent Protein Tagged Connexins.

1999 ◽  
Vol 5 (S2) ◽  
pp. 1020-1021
Author(s):  
D.W. Laird ◽  
K. Jordan ◽  
P. Fistouris ◽  
J.L. Solan ◽  
P.D. Lampe ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Gap Junction ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Living Cells ◽  
Adjacent Cell ◽  
Gap Junction Channels ◽  
Amino Terminal ◽  
Type Iv ◽  
Green Fluorescent

Connexins oligomerize into hemichannels, traffic to the cell surface and dock with hemichannels from an adjacent cell to form intercellular gap junction channels. Pulsechase studies have revealed that connexins are subject to a short half-life ranging from 1- 5 hrs. To examine the mechanisms involved in connexin trafficking, gap junction assembly and internalization in living cells we fused green fluorescent protein (GFP) to the amino or carboxyl terminus of full length connexins (Cx). When cDNA encoding Cx43-GFP was transfected into communication-competent NRK cells, Cx43-negative MDCK cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into functional gap junctions (Figure 1). Most notably, the fusion of GFP to the amino terminal of Cx43 (GFP-Cx43) or Cx32 (GFP-Cx32) did not inhibit the co-translational insertion of these polytopic type IV connexins into the membrane and as a result gap junction plaques assembled at cellcell interfaces.

scholarly journals Mechanism of v-Src- and mitogen-activated protein kinase-induced reduction of gap junction communication

2003 ◽  
Vol 284 (2) ◽  
pp. C511-C520 ◽  
Author(s):  
G. Trevor Cottrell ◽  
Rui Lin ◽  
Bonnie J. Warn-Cramer ◽  
Alan F. Lau ◽  
Janis M. Burt
Keyword(s):  
Gap Junction ◽  
Single Channel ◽  
Electrical Coupling ◽  
Mitogen Activated Protein Kinase ◽  
Dye Coupling ◽  
Open Probability ◽  
Unitary Conductance ◽  
Gap Junction Communication ◽  
Mitogen Activated Protein ◽  
Cx 43

Connexin (Cx)43 gap junction channels are phosphorylated by numerous protein kinases, with the net effect typically being a reduction in gap junction communication (GJC). This reduction must result from a decrease in channel open probability, unitary conductance, or permselectivity, because previous results suggest that channel number is unaffected. Coexpression of v-Src with wild-type Cx43 (Cx43-wt) but not Cx43 with tyrosine to phenylalanine substitutions at 247 and 265 (Cx43-Y247,265F) resulted in reduced electrical and dye coupling but no change in single-channel amplitudes. EGF treatment of cells expressing Cx43-wt but not Cx43 with serine to alanine substitutions at 255, 279, and 282 (Cx43-S255,279,282A) resulted in reduced GJC, also with no change in single-channel amplitude. Dye coupling was reduced to a far greater extent than electrical coupling, suggesting that channel selectivity was also altered but with minimal effect on unitary conductance. The absence of Src- and MAPK-induced reductions in single-channel amplitude suggests that the decreases in GJC induced by these kinases result from reduced channel open probability and possibly altered selectivity.

Targeting motifs and functional parameters governing the assembly of connexins into gap junctions

2000 ◽  
Vol 349 (1) ◽  
pp. 281-287 ◽  
Author(s):  
Patricia E. M. MARTIN ◽  
James STEGGLES ◽  
Claire WILSON ◽  
Shoeb AHMAD ◽  
W. Howard EVANS
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gap Junctions ◽  
Gap Junction ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Lucifer Yellow ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Green Fluorescent

To study the assembly of gap junctions, connexin-green-fluorescent-protein (Cx-GFP) chimeras were expressed in COS-7 and HeLa cells. Cx26- and Cx32-GFP were targeted to gap junctions where they formed functional channels that transferred Lucifer Yellow. A series of Cx32-GFP chimeras, truncated from the C-terminal cytoplasmic tail, were studied to identify amino acid sequences governing targeting from intracellular assembly sites to the gap junction. Extensive truncation of Cx32 resulted in failure to integrate into membranes. Truncation of Cx32 to residue 207, corresponding to removal of most of the 78 amino acids on the cytoplasmic C-terminal tail, led to arrest in the endoplasmic reticulum and incomplete oligomerization. However, truncation to amino acid 219 did not impair Cx oligomerization and connexon hemichannels were targeted to the plasma membrane. It was concluded that a crucial gap-junction targeting sequence resides between amino acid residues 207 and 219 on the cytoplasmic C-terminal tail of Cx32. Studies of a Cx32E208K mutation identified this as one of the key amino acids dictating targeting to the gap junction, although oligomerization of this site-specific mutation into hexameric hemichannels was relatively unimpaired. The studies show that expression of these Cx-GFP constructs in mammalian cells allowed an analysis of amino acid residues involved in gap-junction assembly.

scholarly journals Electron microscopy and functional analysis of recombinant innexin gap junctions

2014 ◽  
Vol 70 (a1) ◽  
pp. C851-C851
Author(s):  
Atsunori Oshima ◽  
Tomohiro Matsuzawa ◽  
Kazuyoshi Murata ◽  
Kouki Nishikawa ◽  
Yoshinori Fujiyoshi
Keyword(s):  
Electron Microscopy ◽  
Gap Junctions ◽  
Gap Junction ◽  
Single Particle ◽  
Particle Analysis ◽  
Single Particle Analysis ◽  
Dye Transfer ◽  
Gap Junction Channels ◽  
Class Average ◽  
Detergent Micelles

Innexin is a molecular component of invertebrate gap junctions, which have an important role in neural and muscular electrical activity in invertebrates. Although the structure of vertebrate connexin26 was revealed by X-ray crystallography [1], the structure of innexin channels remains poorly understood. To study the structure of innexin gap junction channels, we expressed and purified Caenorhabditis elegans innexin-6 (INX-6) gap junction channels, and characterized their molecular dimensions and channel permeability using electron microscopy (EM) and a fluorescent dye transfer assay, respectively [2]. Negative-staining and thin-section EM of isolated INX-6 gap junction plaques revealed a loosely packed hexagonal lattice. We performed single particle analysis of purified INX-6 channels with negative-staining and cryo EM. Based on the negative-stain EM images, the class average of the junction form had a longitudinal height of 220 Å, a channel diameter of 110 Å in the absence of detergent micelles, and an extracellular gap space of 60 Å, whereas the class average of the hemichannels had diameters of up to 140 Å in the presence of detergent micelles. Cryo EM images revealed rotational peaks that could be related to the INX-6 subunits. Structural analysis of the reconstituted INX-6 channels with single particle analysis and electron tomography suggested that the oligomeric number of the INX-6 channel was distinct from that of the dodecameric connexin channel. Dye transfer experiments indicated that the INX-6-GFP-His channels were permeable to 3-kDa and 10-kDa dextran-conjugated tracers. These findings indicate that INX-6 channels have a characteristic oligomer component that differs from that in connexin gap junction channels.

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