The TRK proteins-Trk1p and Trk2p- are the main agents responsible for "active" accumulation of potassium by the yeast Saccharomyces cerevisiae. In previous studies, inward currents measured through those proteins by whole-cell patch-clamping proved very unresponsive to changes of extracellular potassium concentration, although they did increase with extracellular proton concentration-qualitatively as expected for H(+) coupling to K(+) uptake. These puzzling observations have now been explored in greater detail, with the following major findings: a) the large inward TRK currents are not carried by influx of either K(+) or H(+), but rather by an efflux of chloride ions; b) with normal expression levels for Trk1p and Trk2p in potassium-replete cells, the inward TRK currents are contributed approximately half by Trk1p and half by Trk2p; but c) strain background strongly influences the absolute magnitude of these currents, which are nearly twice as large in W303-derived spheroplasts as in S288c-derived cells (same cell-size and identical recording conditions); d) incorporation of mutations that increase cell size (deletion of the Golgi calcium pump, Pmr1p) or that upregulate the TRK2 promoter, can further substantially increase the TRK currents; e) removal of intracellular chloride (e.g., replacement by sulfate or gluconate) reveals small inward currents that are K(+)-dependent and can be enhanced by K(+) starvation; and f) finally, the latter currents display two saturating kinetic components, with preliminary estimates of K(0.5) at 46 micro M [K(+)](out) and 6.8 m M [K(+)](out), and saturating fluxes of approximately 5 m M/min and approximately 10 m M/min (referred to intracellular water). These numbers are compatible with the normal K(+)-transport properties of Trk1p and Trk2p, respectively.

Nigericin is an ionophore commonly used at the end of experiments to calibrate intracellularly trapped pH-sensitive dyes. In the present study, we explore the possibility that residual nigericin from dye calibration in one experiment might interfere with intracellular pH (pHi) changes in the next. Using the pH-sensitive fluorescent dye 2', 7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF), we measured pHi in cultured rat renal mesangial cells. Nigericin contamination caused: (i) an increase in acid loading during the pHi decrease elicited by removing extracellular Na+, (ii) an increase in acid extrusion during the pHi increase caused by elevating extracellular [K+], and (iii) an acid shift in the pHi dependence of the background intracellular acid loading unmasked by inhibiting Na-H exchange with ethylisopropylamiloride (EIPA). However, contamination had no effect on the pHi dependence of Na-H exchange, computed by adding the pHi dependencies of total acid extrusion and background acid loading. Nigericin contamination can be conveniently minimized by using a separate line to deliver nigericin to the cells, and by briefly washing the tubing with ethanol and water after each experiment.

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 toxin's 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.Antimicrobial defense mechanisms in the oral cavity are provided by innate nonimmune proteins and peptides of salivary gland origin, which include lactoperoxidase, lysozyme, lactoferrin, cathelicidins, and histatins (Hst's). These agents have overlapping antimicrobial activities and are key initial host defense molecules in limiting oral infections (15,26). The Hst's form a family of small, histidine-rich, cationic peptides secreted by human parotid and submandibular glands (27). Of this family, Hst 5 has the highest antifungal activity for Candida albicans and related yeasts (38). Oral candidiasis (thrush) is a common mucosal infection caused by C. albicans in patients undergoing chemotherapy or organ transplantation and in persons with immunodeficiency virus infections, underlying systemic disease states, or medications which reduce salivary flow.The spectrum of innate eukaryotic antifungal molecules includes a large array of peptides and proteins, varying widely in both structure and molecular mechanism of action. Many of these agents, but not all, directly target the plasma membrane and/or organellar membranes for disruption; others kill fungi via multitarget, multistep pathways. Chromofungin is an antifungal peptide derived ...

TRK proteins - essential potassium (K(+)) transporters in fungi and bacteria, as well as in plants - are generally absent from animal cells, which makes them potential targets for selective drug action. Indeed, in the human pathogen Candida albicans, the single TRK isoform (CaTrk1p) has recently been demonstrated to be required for activity of histidine-rich salivary antimicrobial peptides (histatins). Background for a detailed molecular investigation of TRK-protein design and function is provided here in sequence analysis and quantitative functional comparison of CaTrk1p with its better-known homologues from Saccharomyces cerevisiae. Among C. albicans strains (ATCC 10261, SC5314, WO-1), the DNA sequence is essentially devoid of single nucleotide polymorphisms in regions coding for evolutionarily conserved segments of the protein, meaning the four intramembranal [membrane-pore-membrane (MPM)] segments thought to be involved directly with the conduction of K(+) ions. Among 48 fungal (ascomycete) TRK homologues now described by complete sequences, clades (but not the detailed order within clades) appear conserved for all four MPM segments, independently assessed. The primary function of TRK proteins, 'active' transport of K(+) ions, is quantitatively conserved between C. albicans and S. cerevisiae. However, the secondary function, chloride efflux channeling, is present but poorly conserved between the two species, being highly variant with respect to activation velocity, amplitude, flickering (channel-like) behavior, pH dependence, and inhibitor sensitivity.

Using the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), we examined the effect of hyperosmolar solutions, which presumably caused cell shrinkage, on intracellular pH (pHi) regulation in mesangial cells (single cells or populations) cultured from the rat kidney. The calibration of BCECF is identical in shrunken and unshrunken mesangial cells if the extracellular K+concentration ([K+]) is adjusted to match the predicted intracellular [K+]. For pHi values between ∼6.7 and ∼7.4, the intrinsic buffering power in shrunken cells (600 mosmol/kgH2O) is threefold larger than in unshrunken cells (∼300 mosmol/kgH2O). In the nominal absence of CO2/[Formula: see text], exposing cell populations to a HEPES-buffered solution supplemented with ∼300 mM mannitol (600 mosmol/kgH2O) causes steady-state pHi to increase by ∼0.4. The pHi increase is due to activation of Na+/H+exchange because, in single cells, it is blocked in the absence of external Na+ or in the presence of 50 μM ethylisopropylamiloride (EIPA). Preincubating cells in a Cl−-free solution for at least 14 min inhibits the shrinkage-induced pHi increase by 80%. We calculated the pHi dependence of the Na+/H+exchange rate in cell populations under normosmolar and hyperosmolar conditions by summing 1) the pHi dependence of the total acid-extrusion rate and 2) the pHi dependence of the EIPA-insensitive acid-loading rate. Shrinkage alkali shifts the pHi dependence of Na+/H+exchange by ∼0.7 pH units.