The TRK1 Potassium Transporter Is the Critical Effector for Killing of Candida albicans by the Cationic Protein, Histatin 5

• Published in: The Journal of biological chemistry Vol. 279; no. 53; pp. 55060 - 55072
• Main Authors: Baev, Didi, Rivetta, Alberto, Vylkova, Slavena, Sun, Jianing N., Zeng, Ge-Fei, Slayman, Clifford L., Edgerton, Mira
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 31-12-2004; American Society for Biochemistry and Molecular Biology
• Subjects: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid - chemistry; Adenosine Triphosphate - chemistry; Alleles; Anions; Antifungal Agents - pharmacology; Antimicrobial Cationic Peptides - chemistry; Blotting, Western; Candida albicans; Candida albicans - metabolism; Cation Transport Proteins - chemistry; Cation Transport Proteins - physiology; Cations; Cell Membrane - metabolism; Cell Separation; Chloride Channels - chemistry; Chlorides - chemistry; Cytoplasm - metabolism; DNA Primers - chemistry; DNA, Complementary - metabolism; Dose-Response Relationship, Drug; Electrophysiology; Escherichia coli - metabolism; Flow Cytometry; Gene Deletion; Genetic Complementation Test; Histatins; Histidine - chemistry; Models, Chemical; Models, Genetic; Oligonucleotides - chemistry; Open Reading Frames; Patch-Clamp Techniques; Plasmids - metabolism; Potassium - chemistry; Protease Inhibitors - pharmacology; Protein Binding; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; RNA - chemistry; Rubidium - chemistry; Saccharomyces cerevisiae Proteins - chemistry; Salivary Proteins and Peptides - chemistry; Salivary Proteins and Peptides - physiology; Time Factors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M411031200

The principal feature of killing of Candida albicans and other pathogenic fungi by the catonic protein Histatin 5 (Hst 5) is loss of cytoplasmic small molecules and ions, including ATP and K+, which can be blocked by the anion channel inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid. We constructed C. albicans strains expressing one, two, or three copies of the TRK1 gene in order to investigate possible roles of Trk1p (the organism's principal K+ transporter) in the actions of Hst 5. All measured parameters (Hst 5 killing, Hst 5-stimulated ATP efflux, normal Trk1p-mediated K+ (86Rb+) influx, and Trk1p-mediated chloride conductance) were similarly reduced (5–7-fold) by removal of a single copy of the TRK1 gene from this diploid organism and were fully restored by complementation of the missing allele. A TRK1 overexpression strain of C. albicans, constructed by integrating an additional TRK1 gene into wild-type cells, demonstrated cytoplasmic sequestration of Trk1 protein, along with somewhat diminished toxicity of Hst 5. These results could be produced either by depletion of intracellular free Hst 5 due to sequestered binding, or to cooperativity in Hst 5-protein interactions at the plasma membrane. Furthermore, Trk1p-mediated chloride conductance was blocked by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid in all of the tested strains, strongly suggesting that the TRK1 protein provides the essential pathway for ATP loss and is the critical effector for Hst 5 toxicity in C. albicans.