Large-conductance Ca2+-activated K+(BK) channels are reported to be essential for NADPH oxidase-dependent microbial killing and innate immunity in leukocytes. Using human peripheral blood and mouse bone marrow neutrophils, pharmacological targeting, and BK channel gene-deficient (BK−/−) mice, we stimulated NADPH oxidase activity with 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 H2O2production, our patch-clamp experiments failed to show PMA-activated BK channel currents 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 elevated intracellular Ca2+and membrane depolarization. We did not observe BK channel currents, even during extreme depolarization to +140 mV and after elevation of intracellular Ca2+by N-formyl-l-methionyl-l-leucyl-phenylalanine. As a control, we examined BK channel currents in cerebral and tibial artery smooth muscle cells, which showed characteristic BK channel current pharmacology. Iberiotoxin did not block killing of Staphylococcus aureus or Candida albicans. Moreover, we addressed the role of BK channels in a systemic S. aureus and Yersinia enterocolitica mouse infection model. After 3 and 5 days of infection, we found no differences in the number of bacteria in 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.
Erythrocyte NADPH oxidase activity modulated by Rac GTPases, PKC, and plasma cytokines contributes to oxidative stress in sickle cell disease
