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

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.

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

Molecular Biology of the Cell ◽

10.1091/mbc.10.6.2033 ◽

1999 ◽

Vol 10 (6) ◽

pp. 2033-2050 ◽

Cited By ~ 143

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.

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

Journal of Cell Science ◽

10.1242/jcs.114.4.763 ◽

2001 ◽

Vol 114 (4) ◽

pp. 763-773 ◽

Cited By ~ 15

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.

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

The Journal of General Physiology ◽

10.1085/jgp.119.2.171 ◽

2002 ◽

Vol 119 (2) ◽

pp. 171-186 ◽

Cited By ~ 58

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.

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