Staphylococcal Pore-forming Leukotoxins: Opening of Ca<sup>2+</sup>-activated K<sup>+</sup> Channels and Specificity of Membrane Pores in Human Neutrophils

Abstract

Pore-forming toxins are key virulence determinants produced by human bacterial pathogens Staphylococcus aureus, inducing two independent cellular events in neutrophils. Upon a specific binding to membrane receptors, both Panton and Valentin Leukocidin and γ-hemolysin induced an increase of Na⁺ and K⁺ fluxes, likely associated to the activation of preexisting ionic channels or to the membrane pores formation. This was investigated by using, spectrofluorometry techniques and, specific molecular probes in human neutrophils. Interestingly, we found that, in the absence of extracellular Ca²⁺, leukotoxins did form membrane pores, which were large enough to allow a massive entry of ethidium into neutrophils. Simultaneously, sustained Na⁺ influx and K⁺ efflux were observed. Another set of experiments carried out in the presence of extracellular Ca²⁺ did show that, the percentage of pores formed by leukotoxins was significantly, reduced due to the Ca²⁺ effect to eventually protect cells from lysis. The simultaneous recording of Na⁺ and K⁺ movements showed a significant increase of the K⁺ efflux although, the Na⁺ influx was reduced. By using potassium channels blockers, we found that, the potassium efflux enhanced by the presence of extracellular Ca²⁺, was markedly, inhibited in apamin-, charybdotoxin-, tetrodotoxin-, and quinine-pretreatment neutrophils. We also found that, the increase of the K⁺ efflux was reduced by either, thapsigargin or TMB8, potent blockers of the internal Ca²⁺ stores depletion. Consequently, we proposed that, the activation of another potassium pathway by leukotoxins, known as Ca²⁺-activated K⁺ channels following the Ca²⁺ stores depletion. Furthermore, potassium channels blockers did not affect ethidium, Na⁺ and K⁺ movements, in the absence of extracellular Ca²⁺. Moreover, in this condition, no monovalent ions movement was recorded, when the pores formation was altered by tetra-ethyl-ammonium. In the present study, we further highlighted the specificity of membrane pores to Na⁺ and K⁺ ions when, the pores formation was completely blocked by divalent ions blockers (Ca²⁺ and Zn²⁺). Under these conditions, no monovalent ions movement, was recorded although, a significant influx of Ca²⁺ and Zn²⁺ was observed after the leukotoxins application. In conclusion, our data provided an evidence that, staphylococcal leukotoxins induced in human neutrophils: 1) the opening of Ca²⁺-activated K⁺ channels, only in the presence of 1 mM extracellular Ca²⁺; 2) the formation of membrane pores, which exhibited a high specificity to monovalent cations and, 3) an influx of sodium, through a tetrodotoxin not-sensitive pathway ruling out the hypothesis that, Na⁺ channels could be activated by leukotoxins.