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1 Nephrology, Franz-Volhard Clinic at Max-Delbrueck-Center for Molecular Medicine, Wiltbergstr 50, Berlin, 13125, Germany
2 Franz Volhard Clinic, Humboldt University Berlin, Wiltbergstrasse 50, Berlin, 13125, Germany
3 Institut fur Medizinische Mikrobiologie une Krankenhaushygiene, Tubingen, United States; Institut fur Medizinische Mikrobiologie une Krankenhaushygiene, Tubingen
4 Universtät Tübingen
5 Dept. Nephrology and Intensive Care, Charité - Humboldt University, Augustenburger Platz 1, Berlin, 13353, Germany
* To whom correspondence should be addressed. E-mail: maik.gollasch{at}charite.de.
Large-conductance calcium-activated potassium (BK) channels are reported to be essential for NADPH oxidase-dependent microbial killing and innate immunity in leukocytes. We studied this issue using human peripheral blood and mouse bone marrow neutrophils, pharmacological targeting, and BK channel gene-deficient (BK-/-) mice. We stimulated NADPH oxidase activity using 12-O-tetradecanoylphorbol-13-acetate (PMA) and performed patch clamp recordings on isolated neutrophils. Although PMA stimulated NADPH oxidase activity as assessed by O2- and H2O2 production, our patch clamp experiments failed to show BK channel currents activated by PMA in neutrophils. In our studies, PMA induced slowly activating currents, which were insensitive to the BK channel inhibitor iberiotoxin. Instead, the currents were blocked by Zn2+, which indicates activation of proton channel currents. BK channels are gated by both elevated intracellular Ca2+ levels and membrane depolarization. We did not observe BK channel currents, even during extreme depolarization to +140mV and after elevation of intracellular [Ca2+] by N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP). As a control, we examined BK currents in cerebral and tibial artery smooth muscle cells, which showed characteristic BK channel current pharmacology. Iberiotoxin did not block killing of S.aureus or C.albicans. Moreover, we addressed the role of BK channels in a systemic S.aureus and Y.enterocolitica mouse infection model. After 3 and 5 days of infection, we found no differences in the bacterial numbers in the spleen and kidney between BK-/- and BK+/+ mice. In conclusion, our experiments failed to identify functional BK channels in neutrophils. We therefore conclude that BK channels are not essential for innate immunity.
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