The large-conductance Ca2+-activated K+ channel is essential for innate immunity

Abstract

Neutrophil leukocytes have a pivotal function in innate immunity. Dogma dictates that the lethal blow is delivered to microbes by reactive oxygen species (ROS) and halogens, products of the NADPH oxidase, whose impairment causes immunodeficiency. However, recent evidence indicates that the microbes might be killed by proteases, activated by the oxidase through the generation of a hypertonic, K+-rich and alkaline environment in the phagocytic vacuole. Here we show that K+ crosses the membrane through large-conductance Ca2+-activated K+ (BK(Ca)) channels. Specific inhibitors of these channels, iberiotoxin and paxilline, blocked oxidase-induced 86Rb+ fluxes and alkalinization of the phagocytic vacuole, whereas NS1619, a BK(Ca) channel opener, enhanced both. Characteristic outwardly rectifying K+ currents, reversibly inhibited by iberiotoxin, were demonstrated in neutrophils and eosinophils and the expression of the alpha-subunit of the BK channel was confirmed by western blotting. The channels were opened by the combination of membrane depolarization and elevated Ca2+ concentration, both consequences of oxidase activity. Remarkably, microbial killing and digestion were abolished when the BK(Ca) channel was blocked, revealing an essential and unexpected function for this K+ channel in the microbicidal process.

Figure 1

BK_Ca channels influence the pH within the phagocytic vacuole and ⁸⁶Rb⁺ efflux from neutrophils and eosinophils. a, Vacuolar pH at 150 s (means ± s.e.m.; three asterisks, P < 0.001 compared with control). b, Time course of pH changes. Inset, dose response of depression of pH by iberiotoxin and paxilline. c, d, ⁸⁶Rb⁺ release from neutrophils (c) and from eosinophils (d). For control cells the release was 14 ± 3% and was normalized to 1. e, Western blots for α-subunit of BK_Ca channel. Lane 1, eosinophil homogenate; lane 2, neutrophil homogenate; lane 3, cytosol; lane 4, granule membranes; lane 5, plasma membrane; lane 6, plasma membrane plus immunizing peptide. PCR fragment (arrow) for hSlo product from differentiated (lane 9) and undifferentiated (lane 8) HL-60 cells. Lane 7, markers.

Figure 2

BK_Ca currents in granulocytes. a, Representative recordings from two neutrophils showing activation of outward currents by TPA and subsequent reversible inhibition by iberiotoxin (IbTx; upper panels) but not by Zn²⁺ (lower panels) in the presence of TPA. b, Bar graph of pooled data (lane 1, control; lane 2, TPA; lane 3, TPA + iberiotoxin; lane 4, iberiotoxin washout + TPA). Asterisk, P < 0.01; hash sign, P ≤ 0.05 compared with control. Numbers of samples are shown above the columns. c, Representative recordings from three different eosinophils showing activation of outward currents by TPA that were reversibly inhibited by iberiotoxin (top panels) and insensitive to block by Zn³⁺ (middle panels) but were inhibited by DPI (bottom panels). d, Pooled data. Lane 1, control; lane 2, TPA; lane 3, iberiotoxin; lane 4, DPI; lane 5, Zn. Numbers of samples are shown above the columns. e, Characteristic cell-attached single-channel recordings from a TPA-stimulated neutrophil. A single-channel current–voltage relationship (n = 3) is shown below. f, Neither 3 mM Zn²⁺ nor Cd²⁺ inhibited the TPA-stimulated (hatched columns) release of ⁸⁶Rb⁺. g, Zn²⁺ (squares) and Cd²⁺ (triangles) inhibit cytochrome c reduction (open symbols) but not oxygen consumption (filled symbols) by TPA-stimulated (circles) neutrophils and eosinophils. h, Zn²⁺ and Cd²⁺ dismutate ${\mathrm{O}}_2^{-}$ at the same rate as superoxide dismutase (SOD; 1 μg ml⁻¹.

Figure 3

Ca²⁺ and membrane depolarization open BK_Ca channels. a, b, Effects of TPA (hatched) in the presence or absence of other agents on ⁸⁶Rb⁺ release from neutrophils (a) or eosinophils (b). IbTx, iberiotoxin. c, d, Traces of cells loaded with fura-2 and hydroethidine (HEt). TPA increased [Ca²⁺]_c (fura-2) and increased ROS generation (HEt) (means ± s.d. from 60 cells) (c), both of which were suppressed by DPI (d). e, f, Indo-1 as the [Ca²⁺] indicator (upper panels in e) showed a significant increase in [Ca²⁺]_c, which appeared more pronounced under the plasmalemma after TPA (means ± s.e.m.). TPA decreased pH_i (imaged with carboxy-SNARF-1; lower panels in e), particularly under the plasmalemma, as shown with images of a single neutrophil (f). g, FLIPR measurements of [Ca²⁺]_c with Fluo-4 AM. TPA induced a rise in [Ca²⁺]_c (curve 5) that was blocked by DPI (curve 2), CCCP (curve 3) and thapsigargin and EGTA (curve 1) but not by EGTA alone (curve 4). Curve 6 shows the effect of TPA, DPI and A23187; curve 7 shows the effect of acidification of cytosol with NH₄Cl washout (blue arrow). (Agents were applied at the black arrow.)

Figure 4

Inhibitors of BK_Ca channels abolish microbial killing and digestion. a–c, Killing of S. aureus (a; n = 5), S. marcescens (b; n = 2) and C. albicans (c; n = 2) was inhibited by iberiotoxin and paxilline, whereas survival in the presence of 4-AP or NS1619 was no different from that of control neutrophils (circles). d, Digestion of ³⁵S-methionine-labelled S. aureus was prevented by both iberiotoxin (n = 3) and paxilline (n = 3) but was similar to that of the control (n = 3) in the presence of NS1619 (n = 3). e–g, None of these modulators of the BK_Ca channel altered oxygen consumption (e), phagocytosis (f; as fluorescent intensity units (FIU), n = 3, impaired by cytocalasin B (cyto B)) or iodination (g; inhibited by DPI).