Functional Characterization of V-J-gating and Single Channel Conductance of Sheep Cx46 and Cx50 Gap Junctions

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Biophysical Journal

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Connexins form intercellular communication channels, known as gap junctions (GJs), found throughout vertebrate species (21 isoforms in human). GJs formed by different connexins harbor unique channel properties that have not been fully defined. High-resolution structures of native Cx46 and Cx50 GJs from sheep (sCx46 and sCx50) were recently resolved by single particle CryoEM. The CryoEM based structures of sCx46 and sCx50 were proposed to adopt a more stable open-state conformation, as compared to hCx26, based on structural differences and results of comparative molecular dynamics (MD) studies. These MD-studies identified the NT domains, especially the 9 th position, as key determinants in the differences of energetic barrier to K + permeation in sCx46 and sCx50 GJs. To better align functional studies with the CryoEM structure models, we studied functional properties of GJs formed by sCx46, sCx50, NT domain swapped chimeras (sCx46-50NT and sCx50-46NT), and point variants at the 9 th residue (sCx46-R9N and sCx50-N9R) in GJ-deficient N2A cells. All of them formed functional GJs except sCx46-50NT. Similar Vj-gating properties were observed for sCx46, sCx50, and sCx46-R9N. However, significantly different single channel conductances were observed for these connexin variants. MD-simulations performed on sCx46-R9N and sCx50-N9R show altered free-energy barriers to ion-permeation that are consistent with experimentally obtained single channel conductance. Together, these studies show the NT-domain encodes key functional differences in the rate of ion permeation and V j-gating properties of sCx46 and sCx50 GJs. Supported by NSERC and NIH.


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