SK channels mediate NADPH oxidase-independent reactive oxygen species production and apoptosis in granulocytes

Abstract

Neutrophils are immune cells that bind to, engulf, and destroy bacterial and fungal pathogens in infected tissue, and their clearance by apoptosis is essential for the resolution of inflammation. Killing involves both oxidative and nonoxidative processes, the oxidative pathway requiring electrogenic production of superoxide by the membrane-bound NADPH oxidase complex. A variety of stimuli, from bacterial chemotactic peptides to complement-or IgG-opsonized microbes, can induce the production of reactive oxygen species (ROS) by neutrophils, presumably by means of NADPH oxidase. We report here that 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of Ca 2؉ -activated potassium channels of small conductance (SK) and intermediate conductance (IK), causes production of superoxide and hydrogen peroxide by neutrophils and granulocyte-differentiated PLB-985 cells. This response can be partially inhibited by the SK blocker apamin, which inhibits a Ca 2؉ -activated K ؉ current in these cells. Analysis of RNA transcripts indicates that channels encoded by the SK3 gene carry this current. The effects of 1-EBIO and apamin are independent of the NADPH oxidase pathway, as demonstrated by using a PLB-985 cell line lacking the gp91phox subunit. Rather, 1-EBIO and apamin modulate mitochondrial ROS production. Consistent with the enhanced ROS production and K ؉ efflux mediated by 1-EBIO, we found that this SK opener increased apoptosis of PLB-985 cells. Together, these findings suggest a previously uncharacterized mechanism for the regulation of neutrophil ROS production and programmed cell death.N eutrophils, as key players in the innate immune response, must be able to identify, phagocytose, and neutralize a broad array of pathogenic microbes (1). Killing usually involves isolation of bacteria or fungi within the phagosome, although neutrophils can release toxic contents into their surroundings to kill extracellular microbes (1). These toxic contents include reactive oxygen species (ROS), such as hydrogen peroxide and hypochlorous acid, as well as proteases and antimicrobial peptides. Because these toxins also can damage host cells, limiting their release and clearing apoptotic neutrophils ultimately are necessary for resolving an infection (2). Understanding how neutrophils regulate the production of oxidants is, therefore, important for both resisting pathogens and controlling inflammation.Although ion channels are best known for their roles in neuronal and cardiac action potentials, their importance in some immune cells, particularly neutrophils, has begun to emerge over the past few years (1, 3-5). The NADPH oxidase of neutrophils produces large amounts of superoxide at the plasma and phagosomal membranes, resulting in an electrogenic efflux of electrons from the cytoplasm (6). An extensive literature indicates that a proton channel, whose molecular identity has recently been identified (7,8), carries a compensating outward current that prevents depolarization of the cell membrane to potentials that inhibit the NADPH oxidase...

Probing the equatorial groove of the hookworm protein and vaccine candidate antigen, Na-ASP-2

“…Intriguingly, Na-ASP-2 is able to induce neutrophil recruitment into areas of acute inflammation, and it has been speculated that this may act as a promoting factor of inflammation during tissue migration to increase tissue permeability (Bower et al, 2008). SK channels mediate the production of reactive oxygen species that may be used for oxidative destruction of pathogens (Fay et al, 2006). Thus, it is tempting to speculate that Na-ASP-2 excreted/secreted by the migrating larvae could interfere with this process by modulating the SK3 ion channel.…”

“…Thus, it is tempting to speculate that Na-ASP-2 excreted/secreted by the migrating larvae could interfere with this process by modulating the SK3 ion channel. Although SK channels have been well characterised in neurons (Blatz & Magleby, 1986;Bond et al, 2004;Köhler et al, 1996) and endocrine cells (Nagayama et al, 2000;Tamarina et al, 2003;Uceda et al, 1992), they have been investigated only recently in non-excitable cells such as immune cells (Fay et al, 2006;Jäger et al, 2000;Quesada et al, 2001). Biologically, SK channels can be blocked by the cyclic peptide apamin found in bee venom toxin (Habermann, 1984).…”

“…Ion channels are of interest as novel targets of a diverse range of therapeutics because they modulate calcium signaling cascades and membrane potential in both excitable and non-excitable cells (Dilly et al, 2011;Wulff et al, 2007).While ion channels are best known for their roles in neuronal and cardiac action potentials, their importance in immune cells is less well characterised, but a role in mediating reactive oxygen species (ROS) production and apoptosis in granulocytes has been described (Fay et al, 2006). An activator of SK channels (1-EBIO) causes production of ROS by neutrophils and granulocyte-differentiated PLB-985 neutrophil-like cells, and this response can be blocked by the bee venom SK3-binding protein, apamin (Fay et al, 2006).…”

“…An activator of SK channels (1-EBIO) causes production of ROS by neutrophils and granulocyte-differentiated PLB-985 neutrophil-like cells, and this response can be blocked by the bee venom SK3-binding protein, apamin (Fay et al, 2006). It is tempting to suggest that Na-ASP-2 has a similar function and blocks the production of reactive oxygen species that are toxic to invading hookworm larvae as they migrate through host tissues on their sohourn towards the gastrointestinal tract.…”

The Relevance of Structural Biology in Studying Molecules Involved in Parasite–Host Interactions: Potential for Designing New Interventions

“…Recent reports indicate that SK channels play a role in mediating reactive oxygen species (ROS) production. [66] Putatively, ASPs may act similarly to the bee venom apamin and block the production of ROS [66] that are toxic to invading hookworm larvae as they migrate through host tissues to the gastrointestinal tract. …”