Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity.
Large-conductance Ca(2+)-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 O(2)(-) and H(2)O(2) production, 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 Zn(2+), which indicates activation of proton channel currents. BK channels are gated by elevated intracellular Ca(2+) and membrane depolarization. We did not observe BK channel currents, even during extreme depolarization to +140 mV and after elevation of intracellular Ca(2+) 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.
Essin
Kirill
K
Department of Nephrology and Medical Intensive Care, Charité-University Medicine Berlin, Humboldt University of Berlin, Germany.
Salanova
Birgit
B
Kettritz
Ralph
R
Sausbier
Matthias
M
Luft
Friedrich C
FC
Kraus
Dirk
D
Bohn
Erwin
E
Autenrieth
Ingo B
IB
Peschel
Andreas
A
Ruth
Peter
P
Gollasch
Maik
M
D000818:Animals; D001770:Blood Bactericidal Activity; D002118:Calcium / Q000378:metabolism; D002712:Chlorides / Q000494:pharmacology; D004195:Disease Models, Animal; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology; D006801:Humans; D007113:Immunity, Innate; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000494:pharmacology; D026902:Potassium Channel Blockers / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D016897:Respiratory Burst; D013203:Staphylococcal Infections / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology; D013211:Staphylococcus aureus; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015009:Yersinia Infections / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology; D015008:Yersinia enterocolitica; D017967:Zinc Compounds / Q000494:pharmacology
D002712:Chlorides; D020536:Enzyme Activators; D051036:Large-Conductance Calcium-Activated Potassium Channels; D010455:Peptides; D026902:Potassium Channel Blockers; D017382:Reactive Oxygen Species; D017967:Zinc Compounds; D009240:N-Formylmethionine Leucyl-Phenylalanine; C064719:iberiotoxin; C016837:zinc chloride; D019255:NADPH Oxidases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium