Alberto Rivetta

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

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 organisms 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.

Activation of an essential calcium signaling pathway in Saccharomyces cerevisiae by Kch1 and Kch2, putative low-affinity potassium transporters.

ABSTRACT In the budding yeast Saccharomyces cerevisiae, mating pheromones activate a high-affinity Ca2+ influx system (HACS) that activates calcineurin and is essential for cell survival. Here we identify extracellular K+ and a homologous pair of transmembrane proteins, Kch1 and Kch2 (Prm6), as necessary components of the HACS activation mechanism. Expression of Kch1 and especially Kch2 was strongly induced during the response to mating pheromones. When forcibly overexpressed, Kch1 and Kch2 localized to the plasma membrane and activated HACS in a fashion that depended on extracellular K+ but not pheromones. They also promoted growth of trk1 trk2 mutant cells in low K+ environments, suggesting they promote K+ uptake. Voltage-clamp recordings of protoplasts revealed diminished inward K+ currents in kch1 kch2 double-mutant cells relative to the wild type. Conversely, heterologous expression of Kch1 in HEK293T cells caused the appearance of inwardly rectifying K+ currents. Collectively, these findings suggest that Kch1 and Kch2 directly promote K+ influx and that HACS may electrochemically respond to K+ influx in much the same way as the homologous voltage-gated Ca2+ channels in most animal cell types.

Killing of Candida albicans by Human Salivary Histatin 5 Is Modulated, but Not Determined, by the Potassium Channel TOK1

ABSTRACT Salivary histatin 5 (Hst 5), a potent toxin for the human fungal pathogen Candida albicans, induces noncytolytic efflux of cellular ATP, potassium, and magnesium in the absence of cytolysis, implicating these ion movements in the toxins fungicidal activity. Hst 5 action on Candida resembles, in many respects, the action of the K1 killer toxin on Saccharomyces cerevisiae, and in that system the yeast plasma membrane potassium channel, Tok1p, has recently been reported to be a primary target of toxin action. The question of whether the Candida homologue of Saccharomyces Tok1p might be a primary target of Hst 5 action has now been investigated by disruption of the C. albicans TOK1 gene. The resultant strains (TOK1/tok1) and (tok1/tok1) were compared with wild-type Candida (TOK1/TOK1) for relative ATP leakage and killing in response to Hst 5. Patch-clamp measurements on Candida protoplasts were used to verify the functional deletion of Tok1p and to provide its first description in Candida. Tok1p is an outwardly rectifying, noisily gated, 40-pS channel, very similar to that described in Saccharomyces. Knockout of CaTOK1 (tok1/tok1) completely abolishes the currents and gating events characteristic of Tok1p. Also, knockout (tok1/tok1) increases residual viability of Candida after Hst 5 treatment to 27%, from 7% in the wild type, while the single allele deletion (TOK1/tok1) increases viability to 18%. Comparable results were obtained for Hst-induced ATP efflux, but quantitative features of ATP loss suggest that wild-type TOK1 genes function cooperatively. Overall, very substantial killing and ATP efflux are produced by Hst 5 treatment after complete knockout of wild-type TOK1, making clear that Tok1p channels are not the primary site of Hst 5 action, even though they do play a modulating role.

Yeast Fex1p Is a Constitutively Expressed Fluoride Channel with Functional Asymmetry of Its Two Homologous Domains

Background: Fluoride is broadly toxic, and organisms use fluoride export (FEX) proteins to expel it. Results: FEX is a constitutively expressed fluoride channel, and mutations to the C- and N-terminal domains have asymmetric effects. Conclusion: Protection from fluoride is constantly needed, and a positive residue in the membrane is required. Significance: Understanding FEX furthers our knowledge of fluoride resistance mechanisms. Fluoride is a ubiquitous environmental toxin with which all biological species must cope. A recently discovered family of fluoride export (FEX) proteins protects organisms from fluoride toxicity by removing it from the cell. We show here that FEX proteins in Saccharomyces cerevisiae function as ion channels that are selective for fluoride over chloride and that these proteins are constitutively expressed at the yeast plasma membrane. Continuous expression is in contrast to many other toxin exporters in yeast, and this, along with the fact that two nearly duplicate proteins are encoded in the yeast genome, suggests that the threat posed by fluoride ions is frequent and detrimental. Structurally, eukaryotic FEX proteins consist of two homologous four-transmembrane helix domains folded into an antiparallel dimer, where the orientation of the two domains is fixed by a single transmembrane linker helix. Using phylogenetic sequence conservation as a guide, we have identified several functionally important residues. There is substantial functional asymmetry in the effect of mutation at corresponding sites in the two domains. Specifically, mutations to residues in the C-terminal domain proved significantly more detrimental to function than did similar mutations in the N-terminal domain. Our data suggest particular residues that may be important to anion specificity, most notably the necessity of a positive charge near the end of TMH1 in the C-terminal domain. It is possible that a cationic charge at this location may create an electrostatic well for fluoride ions entering the channel from the cytoplasm.

Coordination of K+ Transporters in Neurospora: TRK1 Is Scarce and Constitutive, while HAK1 Is Abundant and Highly Regulated

ABSTRACT Fungi, plants, and bacteria accumulate potassium via two distinct molecular machines not directly coupled to ATP hydrolysis. The first, designated TRK, HKT, or KTR, has eight transmembrane helices and is folded like known potassium channels, while the second, designated HAK, KT, or KUP, has 12 transmembrane helices and resembles MFS class proteins. One of each type functions in the model organism Neurospora crassa, where both are readily accessible for biochemical, genetic, and electrophysiological characterization. We have now determined the operating balance between Trk1p and Hak1p under several important conditions, including potassium limitation and carbon starvation. Growth measurements, epitope tagging, and quantitative Western blotting have shown the gene HAK1 to be much more highly regulated than is TRK1. This conclusion follows from three experimental results: (i) Trk1p is expressed constitutively but at low levels, and it is barely sensitive to extracellular [K+] and/or the coexpression of HAK1; (ii) Hak1p is abundant but is markedly depressed by elevated extracellular concentrations of K+ and by coexpression of TRK1; and (iii) Carbon starvation slowly enhances Hak1p expression and depresses Trk1p expression, yielding steady-state Hak1p:Trk1p ratios of ∼500:1, viz., 10- to 50-fold larger than that in K+- and carbon-replete cells. Additionally, it appears that both potassium transporters can adjust kinetically to sustained low-K+ stress by means of progressively increasing transporter affinity for extracellular K+. The underlying observations are (iv) that K+ influx via Trk1p remains nearly constant at ∼9 mM/h when extracellular K+ is progressively depleted below 0.05 mM and (v) that K+ influx via Hak1p remains at ∼3 mM/h when extracellular K+ is depleted below 0.1 mM.