Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity

Abstract

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.

SK3 channel and mitochondrial ROS mediate NADPH oxidase-independent NETosis induced by calcium influx

“…It has recently been shown that BK channel activity is absent in neutrophils and is not required for antimicrobial defense (10,28). 1-EBIO is an activator of both calcium-activated potassium channel of intermediate conductance (IK) and SK channels (29); however, 1-EBIO does not activate IK channels in human neutrophils (11).…”

“…It has been shown that the calcium-activated potassium channel of small conductance (SK channel), which is the major calciumactivated potassium channel known to be present on neutrophils (8)(9)(10)(11), mediates NOX-independent neutrophil "apoptosis" (11). In this pathway, SK channels activate mitochondrial ROS production.…”

Hydrogen sulfide preconditioning or neutrophil depletion attenuates ischemia-reperfusion-induced mitochondrial dysfunction in rat small intestine

“…It does not appear that the effects of NaHS-PC to prevent mucosal mitochondrial dysfunction can be attributed to a direct action on neutrophils or mast cells during I/R since recent reports indicate that BK Ca channel activity is absent in both human and mouse neutrophils whereas mast cell function is not inhibited by iberiotoxin, a highly selective BK Ca channel inhibitor (7,31). On the other hand, platelets express BK Ca channels, which, when activated, hyperpolarize platelet membranes, thereby limiting P-selectin expression and platelet adhesion to endothelial cells under static and flow conditions (22).…”

VSOP/Hv1 proton channels sustain calcium entry, neutrophil migration, and superoxide production by limiting cell depolarization and acidification

“…More recently, potassium ions were proposed to provide the compensating charge and, by increasing the ionic strength within the phagosome, to activate cationic proteases required for bacterial killing (Reeves et al, 2002). However, the claim that Ca 2+ -activated K + channels (BK Ca ) are required for bacterial killing by neutrophils (Ahluwalia et al, 2004) was disproved because neutrophils lack BK Ca currents and kill microbes efficiently in the presence of BK Ca inhibitors or after genetic ablation of the BK Ca channel (Femling et al, 2006;Essin et al, 2007Essin et al, , 2009). Modeling indicates that other potassium channels and swellingactivated chloride channels (Ahluwalia, 2008) contribute no more than 5% of the compensating charge, which is provided essentially by proton channels (Murphy and DeCoursey, 2006).…”