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

Patrik Schadzek; Doris Hermes; Yannick Stahl; Nadine Dilger; Anaclet Ngezahayo

doi:10.3390/ijms19092742

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Original language	English
Article number	2742
Journal	International Journal of Molecular Sciences
Volume	19
Issue number	9
Publication status	Published - 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.

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

ASJC Scopus subject areas

Chemical Engineering(all)
Catalysis
Biochemistry, Genetics and Molecular Biology(all)
Molecular Biology
Chemistry(all)
Spectroscopy
Computer Science(all)
Computer Science Applications
Chemistry(all)
Physical and Theoretical Chemistry
Chemistry(all)
Organic Chemistry
Chemistry(all)
Inorganic Chemistry

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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, Vol. 19, No. 9, 2742, 13.09.2018.

Research output: Contribution to journal › Article › Research › peer review

Schadzek, P, Hermes, D, Stahl, Y, Dilger, N & Ngezahayo, A 2018, 'Concatenation of Human Connexin26 (hCx26) and Human Connexin46 (hCx46) for the Analysis of Heteromeric Gap Junction Hemichannels and Heterotypic Gap Junction Channels', International Journal of Molecular Sciences, vol. 19, no. 9, 2742. https://doi.org/10.3390/ijms19092742, https://doi.org/10.15488/4250

Schadzek, P., Hermes, D., Stahl, Y., Dilger, N., & Ngezahayo, A. (2018). Concatenation of Human Connexin26 (hCx26) and Human Connexin46 (hCx46) for the Analysis of Heteromeric Gap Junction Hemichannels and Heterotypic Gap Junction Channels. International Journal of Molecular Sciences, 19(9), Article 2742. https://doi.org/10.3390/ijms19092742, https://doi.org/10.15488/4250

Schadzek P, Hermes D, Stahl Y, Dilger N, Ngezahayo A. Concatenation of Human Connexin26 (hCx26) and Human Connexin46 (hCx46) for the Analysis of Heteromeric Gap Junction Hemichannels and Heterotypic Gap Junction Channels. International Journal of Molecular Sciences. 2018 Sept 13;19(9):2742. doi: 10.3390/ijms19092742, 10.15488/4250

Schadzek, Patrik ; Hermes, Doris ; Stahl, Yannick et al. / Concatenation of Human Connexin26 (hCx26) and Human Connexin46 (hCx46) for the Analysis of Heteromeric Gap Junction Hemichannels and Heterotypic Gap Junction Channels. In: International Journal of Molecular Sciences. 2018 ; Vol. 19, No. 9.

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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.",

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language = "English",

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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.

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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

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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

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ER -

Research@Leibniz University

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

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