Cytosolic Acidification Is the First Transduction Signal of Lactoferrin-Induced Regulated Cell Death Pathway

In yeast, we reported the critical role of K(+)-efflux for the progress of the regulated cell death (RCD) induced by human lactoferrin (hLf), an antimicrobial protein of the innate immune system that blocks Pma1p H(+)-ATPase. In the present study, the K(+) channel Tok1p was identified as the K(+) channel-mediating K(+)-efflux, as indicated by the protective effect of extracellular K(+) (≥30 mM), K(+)-channel blockers, and the greater hLf-resistance of TOK1-disrupted strains. K(+)-depletion was necessary but not sufficient to induce RCD as inferred from the effects of valinomycin, NH(4)Cl or nigericin which released a percentage of K(+) similar to that released by lactoferrin without affecting cell viability. Cytosolic pH of hLf-treated cells decreased transiently (~0.3 pH units) and its inhibition prevented the RCD process, indicating that cytosolic acidification was a necessary and sufficient triggering signal. The blocking effect of lactoferrin on Pma1p H(+)-ATPase caused a transitory decrease of cytosolic pH, and the subsequent membrane depolarization activated the voltage-gated K(+) channel, Tok1p, allowing an electrogenic K(+)-efflux. These ionic events, cytosolic accumulation of H(+) followed by K(+)-efflux, constituted the initiating signals of this mitochondria-mediated cell death. These findings suggest, for the first time, the existence of an ionic signaling pathway in RCD.