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

Publikation: Beitrag in FachzeitschriftArtikelForschungPeer-Review

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Externe Organisationen

  • Georg-August-Universität Göttingen
  • Stiftung Tierärztliche Hochschule Hannover
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OriginalspracheEnglisch
Aufsatznummer2742
FachzeitschriftInternational Journal of Molecular Sciences
Jahrgang19
Ausgabenummer9
PublikationsstatusVeröffentlicht - 13 Sept. 2018

Abstract

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.

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Concatenation of Human Connexin26 (hCx26) and Human Connexin46 (hCx46) for the Analysis of Heteromeric Gap Junction Hemichannels and Heterotypic Gap Junction Channels. / Schadzek, Patrik; Hermes, Doris; Stahl, Yannick et al.
in: International Journal of Molecular Sciences, Jahrgang 19, Nr. 9, 2742, 13.09.2018.

Publikation: Beitrag in FachzeitschriftArtikelForschungPeer-Review

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title = "Concatenation of Human Connexin26 (hCx26) and Human Connexin46 (hCx46) for the Analysis of Heteromeric Gap Junction Hemichannels and Heterotypic Gap Junction Channels",
abstract = "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.",
keywords = "Channel stoichiometry, Concatenated connexins, Dual whole-cell patch-clamp, Dye transfer, Gap junction, Heteromeric connexons, Human connexin26, Human connexin46, Inside-out patch-clamp configuration, Oligomerization",
author = "Patrik Schadzek and Doris Hermes and Yannick Stahl and Nadine Dilger and Anaclet Ngezahayo",
note = "Funding Information: This research was partly funded by TransRegio TR37. The publication of this article was funded by the Open Access Fund of the Leibniz Universit{\"a}t Hannover. Acknowledgments: Nadine Dilger was partly supported by the DFG project Elektrodenoptimierung f{\"u}r Neuroprothesen NG 4/10-1. We thank Viviana Berthoud for the hCx46 clone.",
year = "2018",
month = sep,
day = "13",
doi = "10.3390/ijms19092742",
language = "English",
volume = "19",
journal = "International Journal of Molecular Sciences",
issn = "1661-6596",
publisher = "Multidisciplinary Digital Publishing Institute",
number = "9",

}

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TY - JOUR

T1 - Concatenation of Human Connexin26 (hCx26) and Human Connexin46 (hCx46) for the Analysis of Heteromeric Gap Junction Hemichannels and Heterotypic Gap Junction Channels

AU - Schadzek, Patrik

AU - Hermes, Doris

AU - Stahl, Yannick

AU - Dilger, Nadine

AU - Ngezahayo, Anaclet

N1 - Funding Information: This research was partly funded by TransRegio TR37. The publication of this article was funded by the Open Access Fund of the Leibniz Universität Hannover. Acknowledgments: Nadine Dilger was partly supported by the DFG project Elektrodenoptimierung für Neuroprothesen NG 4/10-1. We thank Viviana Berthoud for the hCx46 clone.

PY - 2018/9/13

Y1 - 2018/9/13

N2 - 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.

AB - 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.

KW - Channel stoichiometry

KW - Concatenated connexins

KW - Dual whole-cell patch-clamp

KW - Dye transfer

KW - Gap junction

KW - Heteromeric connexons

KW - Human connexin26

KW - Human connexin46

KW - Inside-out patch-clamp configuration

KW - Oligomerization

UR - http://www.scopus.com/inward/record.url?scp=85053463122&partnerID=8YFLogxK

U2 - 10.3390/ijms19092742

DO - 10.3390/ijms19092742

M3 - Article

C2 - 30217016

AN - SCOPUS:85053463122

VL - 19

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 9

M1 - 2742

ER -