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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

1
Institut für Biophysik, Leibniz Universität Hannover, Herrenhäuser Straße 2, 30419 Hannover, Germany
2
Department of Clinical Neurophysiology, University of Göttingen, Robert-Koch Str. 40, D-37075 Göttingen, Germany
3
Zentrum für Systemische Neurowissenschaften Stiftung Tierärztliche Hochschule Hannover, Bünteweg 2, 30559 Hannover, Germany
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2018, 19(9), 2742; https://doi.org/10.3390/ijms19092742
Received: 20 August 2018 / Revised: 10 September 2018 / Accepted: 11 September 2018 / Published: 13 September 2018
(This article belongs to the Special Issue Interplay of Connexins and Pannexins in Tissue Function and Disease)
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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. View Full-Text
Keywords: oligomerization; concatenated connexins; gap junction; channel stoichiometry; heteromeric connexons; human connexin46; human connexin26; inside-out patch-clamp configuration; dual whole-cell patch-clamp; dye transfer oligomerization; concatenated connexins; gap junction; channel stoichiometry; heteromeric connexons; human connexin46; human connexin26; inside-out patch-clamp configuration; dual whole-cell patch-clamp; dye transfer
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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. Int. J. Mol. Sci. 2018, 19, 2742.

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