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Activation of endogenously expressed ion channels by active complement in the retinal pigment epithelium

  • Signaling and cell physiology
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Abstract

Defective regulation of the alternative pathway of the complement system is believed to contribute to damage of retinal pigment epithelial (RPE) cells in age-related macular degeneration. Thus we investigated the effect of complement activation on the RPE cell membrane by analyzing changes in membrane conductance via patch-clamp techniques and Ca2+ imaging. Exposure of human ARPE-19 cells to complement-sufficient normal human serum (NHS) (25 %) resulted in a biphasic increase in intracellular free Ca2+ ([Ca2+]i); an initial peak followed by sustained Ca2+ increase. C5- or C7-depleted sera did not fully reproduce the signal generated by NHS. The initial peak of the Ca2+ response was reduced by sarcoplasmic Ca2+-ATPase inhibitor thapsigargin, L-type channel blockers (R)-(+)-BayK8644 and isradipine, transient-receptor-potential (TRP) channel blocker ruthenium-red and ryanodine receptor blocker dantrolene. The sustained phase was carried by CaV1.3 L-type channels via tyrosine-phosphorylation. Changes in [Ca2+]I were accompanied by an abrupt hyperpolarization, resulting from a transient increase in membrane conductance, which was absent under extracellular Ca2+- or K+-free conditions and blocked by (R)-(+)-BayK8644 or paxilline, a maxiK channel inhibitor. Single-channel recordings confirmed the contribution of maxiK channels. Primary porcine RPE cells responded to NHS in a comparable manner. Pre-incubation with NHS reduced H2O2-induced cell death. In summary, in a concerted manner, C3a, C5a and sC5b-9 increased [Ca2+]i by ryanodine-receptor-dependent activation of L-type channels in addition to maxi-K channels and TRP channels absent from any insertion of a lytic pore.

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Acknowledgments

The authors thank Andrea Dannullis, Elfriede Eckert and Renate Föckler for expert technical assistance. Financial support: OS: Novartis Professorship; BR: in part by the National Institutes of Health (NIH) (R01EY019320), a Department for Veterans Affairs merit award RX000444 and an unrestricted grant to MUSC from Research to Prevent Blindness (RPB), New York, NY; CS: Deutsche Forschungsgemeinschaft (DFG) SK46/2-1, SK46/2-2.

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Authors and Affiliations

  1. Max-Planck Institute of Psychiatry, Munich, Germany

    Andreas Genewsky

  2. Experimental Ophthalmology, Eye Clinic, University Medical Center Regensburg, Regensburg, Germany

    Andreas Genewsky, Ingmar Jost, Julia Stindl & Olaf Strauß

  3. Department of Infection Biology, Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany

    Christine Skerka & Peter F. Zipfel

  4. Friedrich Schiller University, Jena, Germany

    Peter F. Zipfel

  5. Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, 29425, USA

    Bärbel Rohrer

  6. Research Service, Ralph H. Johnson VA Medical Center, Charleston, SC, 29401, USA

    Bärbel Rohrer

  7. Experimental Ophthalmology, Department of Ophthalmology, Charite University Medicine Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353, Berlin, Germany

    Catharina Busch, Christian Huber & Olaf Strauß

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Genewsky, A., Jost, I., Busch, C. et al. Activation of endogenously expressed ion channels by active complement in the retinal pigment epithelium. Pflugers Arch - Eur J Physiol 467, 2179–2191 (2015). https://doi.org/10.1007/s00424-014-1656-2

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  • DOI: https://doi.org/10.1007/s00424-014-1656-2

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