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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 <i>TOK1</i>-disrupted strains. K⁺-depletion was necessary but not sufficient to induce RCD as inferred from the effects of valinomycin, NH₄Cl 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.