Metabolic Inhibition Activates a Non-selective Current Through Connexin Hemichannels in Isolated Ventricular Myocytes

Kondo, Richard P.; Wang, Shengyong; John, Scott A.; Weiss, James N.; Goldhaber, Joshua I.

doi:10.1006/jmcc.2000.1220

Cited by 179 publications

(171 citation statements)

References 33 publications

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“…Another proof that indeed Cx43 hemichannels are monitored is blocking the activity by lanthanum ions. Several studies (35)(36)(37) have shown the blockade of hemichannel activity by lanthanum. In our setup, the addition of 26 M La 3ϩ clearly blocked the channel activity of Cx43-GFP (Fig.…”

Section: Production and Purification Of Cx43 And Cx43-gfp-mousementioning

confidence: 99%

Green Fluorescent Protein Changes the Conductance of Connexin 43 (Cx43) Hemichannels Reconstituted in Planar Lipid Bilayers

Carnarius

Kreir

Krick

et al. 2012

Journal of Biological Chemistry

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Background: Connexin 43 hemichannels participate in many cellular processes. To elucidate their location and function within living cells, they were labeled with GFP. Results: Recombinantly expressed Cx43 and Cx43-GFP form conducting hemichannels in reconstituted planar membranes. Their conductance states and voltage dependence differ. Conclusion: Fusion of GFP to Cx43 significantly affects the electrophysiological behavior of Cx43 hemichannels. Significance: GFP can significantly alter channel activities.

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Section: Production and Purification Of Cx43 And Cx43-gfp-mousementioning

confidence: 99%

Green Fluorescent Protein Changes the Conductance of Connexin 43 (Cx43) Hemichannels Reconstituted in Planar Lipid Bilayers

Carnarius

Kreir

Krick

et al. 2012

Journal of Biological Chemistry

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“…If DTT increases uptake of EtdBr by opening Cx43-EGFP hemichannels, this effect should be inhibited by hemichannel blockers such as La 3ϩ (4,9,17) and the gap 26 peptide, which has the same sequence as a region of the first extracellular loop of Cx43 (6). We measured basal uptake for 10 min and added DTT, which increased uptake.…”

Section: Dtt-induced Increase In Etdbr Uptake Is Sensitive To Hemichamentioning

confidence: 99%

Opening of connexin 43 hemichannels is increased by lowering intracellular redox potential

Retamal

Schalper

Shoji

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

154

153

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Nonjunctional membrane in many cells contains connexin gap junction hemichannels (or connexons) that can open to allow permeation of small molecules. Opening of Cx43 hemichannels is infrequent in normal extracellular Ca 2؉ and enhanced by low Ca 2؉ , positive membrane potentials, and dephosphorylation of critical residues. Here we report that lowering intracellular redox potential increases Cx43 hemichannel open probability under otherwise normal conditions. We studied dye uptake and single-channel activity in HeLa cells transfected with wild-type Cx43, Cx43 with enhanced GFP attached to its C terminus (Cx43-EGFP), and Cx43 with enhanced GFP attached to its N terminus (EGFP-Cx43). Dithiothreitol [(DTT) 10 mM], a membrane permeant-reducing agent, increased the rate of dye uptake by cells expressing Cx43 and Cx43-EGFP, but not by parental cells or cells expressing EGFP-Cx43. Induced dye uptake was blocked by La 3؉ , by a peptide gap junction and hemichannel blocker (gap 26), and by flufenamic acid. DTT increased Cx43-EGFP hemichannel opening at positive voltages. Bath application of reduced glutathione, a membrane impermeantreducing agent, did not increase dye uptake, but glutathione in the recording pipette increased hemichannel opening at positive voltages, suggesting that it acted intracellularly. DTT caused little change in levels of surface Cx43 or Cx43-EGFP, or in intracellular pH. These findings suggest that lowering intracellular redox potential increases the opening of Cx43 and Cx43-EGFP hemichannels, possibly by action on cytoplasmic cysteine residues in the connexin C terminus.

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“…The use of an extracellular dye with molecular mass above the exclusion limit of hemichannels has been useful to rule out membrane damage as a possible nonspecific pathway of dye uptake (Li et al 1996;Kondo et al 2000;Contreras et al 2002;De Vuyst et al 2006). In addition, molecular shape could significantly influence the relative permeability measurement (Goldberg et al 2004).…”

Section: Dye Uptake Assaysmentioning

confidence: 99%

“…Whole-cell voltage clamp studies of unitary events have also provided information on connexin hemichannel permeability selectivity, opening and closing kinetics, presence of subconductance states, and open probability (latency), among other properties (Valiunas and Weingart 2000;Kondo et al 2000;Valiunas 2002Gómez-Hernandez et al 2003;Contreras et al 2003;Retamal et al 2007b). In addition, these analyses are useful in distinguishing between connexin and pannexin hemichannels.…”

Section: Electrophysiological Characterization Of Hemichannelsmentioning

confidence: 99%

Currently Used Methods for Identification and Characterization of Hemichannels

Schalper

Palacios-Prado

Orellana

et al. 2008

Cell Communication & Adhesion

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Connexins and pannexins are vertebrate transmembrane proteins that form hexameric conduits termed hemichannels. Functional hemichannels allow the diffusional transport of ions and small molecules across the plasma membrane and serve as paracrine and autocrine communication pathways. During the last decade, interest in the hemichannel field increased substantially. Today, there is evidence for the existence of connexin hemichannels in vertebrate cells and bulk of information supports their function in diverse physiological and pathological responses. Controversy regarding the molecular identity of the hemichannel type mediating many responses arose recently with the identification of pannexin-based hemichannels. Here, the authors describe the most frequently used methods for studying hemichannels in living mammalian cells and focus on those with which they have more experience. Although the available in vitro evidence is substantial, further studies and possibly new experimental approaches are required to understand the role and properties of connexin and pannexin hemichannels in vivo.

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Metabolic Inhibition Activates a Non-selective Current Through Connexin Hemichannels in Isolated Ventricular Myocytes

Cited by 179 publications

References 33 publications

Green Fluorescent Protein Changes the Conductance of Connexin 43 (Cx43) Hemichannels Reconstituted in Planar Lipid Bilayers

Green Fluorescent Protein Changes the Conductance of Connexin 43 (Cx43) Hemichannels Reconstituted in Planar Lipid Bilayers

Opening of connexin 43 hemichannels is increased by lowering intracellular redox potential

Currently Used Methods for Identification and Characterization of Hemichannels

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