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

MARTIN, P. E.; STEGGLES, J.; WILSON, C.; AHMAD, S.; EVANS, W. H.

doi:10.1042/bj3500943

Cited by 4 publications

(7 citation statements)

References 37 publications

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“…Regulation of connexin export to the plasma membrane and gap junction assembly. Live cell and other imaging techniques show multiple regulatory aspects of Cx26, Cx32, and Cx43 transport to the plasma membrane, including microtubule-based vesicle transport (Martin et al, 2000;Johnson et al, 2002;Lauf et al, 2002;Shaw et al, 2007). Imaging studies have also shown that Cx43 can move from the plasma membrane into the periphery of a larger plaque; thus plaques grow by adding channels to the outside, and the oldest proteins are found in the center of the plaque where they get selectively turned over (Gaietta et al, 2002).…”

Section: F Connexin Posttranslational Modificationsmentioning

confidence: 99%

Connexins in Cardiovascular and Neurovascular Health and Disease: Pharmacological Implications

Leybaert¹,

Lampe²,

Dhein³

et al. 2017

Pharmacological Reviews

205

192

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Connexins are ubiquitous channel forming proteins that assemble as plasma membrane hemichannels and as intercellular gap junction channels that directly connect cells. In the heart, gap junction channels electrically connect myocytes and specialized conductive tissues to coordinate the atrial and ventricular contraction/relaxation cycles and pump function. In blood vessels, these channels facilitate long-distance endothelial cell communication, synchronize smooth muscle cell contraction, and support endothelial-smooth muscle cell communication. In the central nervous system they form cellular syncytia and coordinate neural function. Gap junction channels are normally open and hemichannels are normally closed, but pathologic conditions may restrict gap junction communication and promote hemichannel opening, thereby disturbing a delicate cellular communication balance. Until recently, most connexin-targeting agents exhibited little specificity and several off-target effects. Recent work with peptide-based approaches has demonstrated improved specificity and opened avenues for a more rational approach toward independently modulating the function of gap junctions and hemichannels. We here review the role of connexins and their channels in cardiovascular and neurovascular health and disease, focusing on crucial regulatory aspects and identification of potential targets to modify their function. We conclude that peptide-based investigations have raised several new opportunities for interfering with connexins and their channels that may soon allow preservation of gap junction communication, inhibition of hemichannel opening, and mitigation of inflammatory signaling.

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Section: F Connexin Posttranslational Modificationsmentioning

confidence: 99%

Connexins in Cardiovascular and Neurovascular Health and Disease: Pharmacological Implications

Leybaert¹,

Lampe²,

Dhein³

et al. 2017

Pharmacological Reviews

205

192

View full text Add to dashboard Cite

show abstract

“…We have shown previously that the trafficking determinants of connexins do not reside on the cytoplasmic carboxyl tail that varies in length between different connexins, except for a short sequence proximal to TM4 (Martin et al, 2000b;Ahmad et al, 2001). Thus, trafficking to gap junctions of Cx26 containing the carboxyl tail of Cx43 or Cx32 and fused to aequorin was unaffected by BFA treatment, but was disrupted by nocodazole treatment (George et al, 1999).…”

Section: Effects Of Disruption Of the Secretory Pathway On Gap Junction Communicationmentioning

confidence: 95%

“…To create constructs Cx26, 32 and 43-GFP, the open reading frame of the relevant Cx cDNA was amplified from plasmids containing the cDNA using primers containing BglII (for Cx26 and Cx43) or HindIII (for Cx32) restriction enzyme sites to ensure inframe fusion with the amino terminus of enhanced-GFP (eGFP) in the vector pe-GFP-N1 (Clontech) (Martin et al, 2000b;Paemeleire et al, 2000). With Cx26-YFP and Cx32-CFP chimerae, the relevant Cx was isolated from the GFP chimeric construct by restriction enzyme digestion such that the Cx cDNA was fused in frame to the relevant FP vector (BglII for Cx26 and HindIII for Cx32) followed by direct ligation of Cx26 into the BglII site of pe-YFP-N1 and of Cx32 into the HindIII site of vector pe-CFP-N1 (Clontech) and subsequent transformation into E. coli (DH5α) (Gibco-BRL).…”

Section: Construction Of Chimeric Cx-fp Cdnamentioning

confidence: 99%

“…HeLa Ohio cells (ECACC, UK) were selected to express Cx26-GFP or Cx43-GFP on the basis of their sensitivity to Geneticin (G418sulphate 4 mg/ml) and selection by FACS as previously described (Martin et al, 2000b;Paemeleire et al, 2000). Cell populations selected for the GFP chimerae were maintained in Dulbecco's modified Eagles medium, supplemented with 10% fetal calf serum, penicillin/streptomycin (100 µg/ml), amphotericin (100 µg/ml) and Lglutamine (2 mM) and geneticin).…”

Section: Expression Of Cx-fp Proteins In Hela and Cos-7 Cellsmentioning

confidence: 99%

“…The present work further analyses the trafficking properties of connexins by use of Cx26, Cx32 or 43-fluorescent protein chimerae and point mutations in TM1 and TM4 that distinguish between Cx26 and Cx32. Cx26, 32 and Cx43-GFP chimerae were shown previously to be of the predicted molecular weight when expressed in COS-7 or HeLa cells (Martin et al, 2000b). These chimeric connexins were targeted to gap junctions that transferred Lucifer yellow and electrical current and they also propagated intercellular Ca 2+ waves (Paemeleire et al, 2000).…”

Section: Introductionmentioning

confidence: 97%

See 2 more Smart Citations

Multiple pathways in the trafficking and assembly of connexin 26, 32 and 43 into gap junction intercellular communication channels

Martin

Blundell

Ahmad

et al. 2001

Journal of Cell Science

150

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The assembly of gap junctions was investigated in mammalian cells expressing connexin (Cx) 26, 32 and 43 fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Targeting of Cx32-CFP and 43-GFP to gap junctions and gap junctional communication was inhibited in cells treated with Brefeldin A, a drug that disassembles the Golgi. However gap junctions constructed of Cx26-GFP were only minimally affected by Brefeldin A. Nocodazole, a microtubule disruptor, had little effect on the assembly of Cx43-GFP gap junctions, but perturbed assembly of Cx26-GFP gap junctions. Co-expression of Cx26-YFP and Cx32-CFP in cells treated with Brefeldin A resulted in assembly of gap junctions constructed of Cx26-YFP. Two amino acids that distinguish Cx26 from Cx32 in transmembrane domains were mutated in Cx32 to investigate underlying mechanisms determining trafficking routes to gap junctions. One mutation, Cx32I28L, conferred on it partial Cx26-like trafficking properties as well the post-translational membrane insertion characteristics of Cx26, suggesting that a key determinant regulating trafficking was present in the first transmembrane domain. The results provide a protein trafficking basis for specifying and regulating connexin composition of gap junctions and thus selectivity of intercellular signaling, with Cx32 and 43 trafficking through the secretory pathway and Cx26 also following an alternative pathway.

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Multicolor and Electron Microscopic Imaging of Connexin Trafficking

Gaietta¹,

Deerinck²,

Adams

et al. 2002

Science

873

680

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Recombinant proteins containing tetracysteine tags can be successively labeled in living cells with different colors of biarsenical fluorophores so that older and younger protein molecules can be sharply distinguished by both fluorescence and electron microscopy. Here we used this approach to show that newly synthesized connexin43 was transported predominantly in 100- to 150-nanometer vesicles to the plasma membrane and incorporated at the periphery of existing gap junctions, whereas older connexins were removed from the center of the plaques into pleiomorphic vesicles of widely varying sizes. Selective imaging by correlated optical and electron microscopy of protein molecules of known ages will clarify fundamental processes of protein trafficking in situ.

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

Cited by 4 publications

References 37 publications

Connexins in Cardiovascular and Neurovascular Health and Disease: Pharmacological Implications

Connexins in Cardiovascular and Neurovascular Health and Disease: Pharmacological Implications

Multiple pathways in the trafficking and assembly of connexin 26, 32 and 43 into gap junction intercellular communication channels

Multicolor and Electron Microscopic Imaging of Connexin Trafficking

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