The occurrence of intercellular channels formed by pannexin1 has been challenged for more than a decade. Here, we provide an electrophysiological characterization of exogenous human pannexin1 (hPanx1) cell-cell channels expressed in HeLa cells knocked out for connexin45. The observed hPanx1 cell-cell channels show two phenotypes: O-state and S-state. The former displayed low transjunctional voltage (Vj) sensitivity and singlechannel conductance of ∼175 pS, with a substate of ∼35 pS; the latter showed a peculiar dynamic asymmetry in Vj dependence and single-channel conductance identical to the substate conductance of the O-state. S-state hPanx1 cell-cell channels were also identified between TC620 cells, a human oligodendroglioma cell line that endogenously expresses hPanx1. In these cells, dye and electrical coupling increased with temperature and were strongly reduced after hPanx1 expression was knocked down by small interfering RNA or inhibited with Panx1 mimetic inhibitory peptide. Moreover, cell-cell coupling was augmented when hPanx1 levels were increased with a doxycycline-inducible expression system. Application of octanol, a connexin gap junction (GJ) channel inhibitor, was not sufficient to block electrical coupling between HeLa KO Cx45-hPanx1 or TC620 cell pairs. In silico studies suggest that several arginine residues inside the channel pore may be neutralized by hydrophobic interactions, allowing the passage of DAPI, consistent with dye coupling observed between TC620 cells. These findings demonstrate that endogenously expressed hPanx1 forms intercellular cell-cell channels and their unique properties resemble those described in innexin-based GJ channels. Since Panx1 is ubiquitously expressed, finding conditions to recognize Panx1 cell-cell channels in different cell types might require special attention.
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|Publication status||Published - 2022 May 3|
Bibliographical noteFunding Information:
11201113 (to Y.D.), 1221498 (to F.D.G.-N.), and 1191329 (to J.C.S. and Y.D.); Programa de Atracción e Inserción de Capital Humano Avanzado, Agencia Nacional de Investigación y Desarrollo + REC grant/award 77200056 (to V. Márquez), as well as grant ICM677 ANID, Project P09 from the Centro Inter-disciplinario de Neurociencias de Valparáıso (to J.C.S. and F.D.G.-N.); and by Basic Science Research Program through the National Research Foundation of Korea funded by Ministry of Education grant 2018R1A6A1A03023718 (to J.L.) and 2020R1I1A1A01061656 (to E.J.C.).
ACKNOWLEDGMENTS. We thank Teresa Vergara, Paola Fernández, and Juan Guiza for excellent technical assistance. This research was partially supported by Fondo Nacional de Desarrollo Científico y Tecnológico grants 3180272 (to N.P.-P.),
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