Conservation and dispersion of sequence and function in fungal TRK potassium transporters: focus on Candida albicans

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.     

TRK1 and TRK2 encode structurally related K+ transporters in Saccharomyces cerevisiae.

We describe the cloning and molecular analysis of TRK2, the gene likely to encode the low-affinity K+ transporter in Saccharomyces cerevisiae. TRK2 encodes a protein of 889 amino acids containing 12 putative membrane-spanning domains (M1 through M12), with a large hydrophilic region between M3 and M4. These structural features closely resemble those contained in TRK1, the high-affinity K+ transporter. TRK2 shares 55% amino acid sequence identity with TRK1. The putative membrane-spanning domains of TRK1 and TRK2 share the highest sequence conservation, while the large hydrophilic regions between M3 and M4 exhibit the greatest divergence. The different affinities of TRK1 trk2 delta cells and trk1 delta TRK2 cells for K+ underscore the functional independence of the high- and low-affinity transporters. TRK2 is nonessential in TRK1 or trk1 delta haploid cells. The viability of cells containing null mutations in both TRK1 and TRK2 reveals the existence of an additional, functionally independent potassium transporter(s). Cells deleted for both TRK1 and TRK2 are hypersensitive to low pH; they are severely limited in their ability to take up K+, particularly when faced with a large inward-facing H+ gradient, indicating that the K+ transporter(s) that remains in trk1 delta trk2 delta cells functions differently than those of the TRK class.     

Small conductance potassium channels drive ATP-activated chloride secretion in the oviduct

The molecular mechanisms controlling fluid secretion within the oviduct have yet to be determined. As in other epithelia, both secretory and absorptive pathways are likely to work in tandem to drive appropriate ionic movement to support fluid movement across the oviduct epithelium. This study explored the role of potassium channels in basolateral extracellular ATP (ATP(e))-stimulated ion transport in bovine oviduct epithelium using the Ussing chamber short-circuit current (I(SC)) technique. Basal I(SC) in bovine oviduct epithelium comprises both chloride secretion and sodium absorption and was inhibited by treatment with basolateral K(+) channel inhibitors tetrapentlyammonium chloride (TPeA) or BaCl(2). Similarly, ATP-stimulated chloride secretion was significantly attenuated by pretreatment with BaCl(2,) tetraethylammonium (TEA), tolbutamide, and TPeA. Basolateral K(+) current, isolated using nystatin-perforation technique, was rapidly activated by ATP(e), and pretreatment of monolayers with thapsigargin or TPeA abolished this ATP-stimulated K(+) current. To further investigate the type of K(+) channel involved in the ATP response in the bovine oviduct, a number of specific Ca(2+)-activated K(+) channel inhibitors were tested on the ATP-induced ΔI(SC) in intact monolayers. Charbydotoxin, (high conductance and intermediate conductance inhibitor), or paxilline, (high conductance inhibitor) did not significantly alter the ATP(e) response. However, pretreatment with the small conductance inhibitor apamin resulted in a 60% reduction in the response to ATP(e). The presence of small conductance family member KCNN3 was confirmed by RT-PCR and immunohistochemistry. Measurements of intracellular calcium using Fura-2 spectrofluorescence imaging revealed the ability of ATP(e) to increase intracellular calcium in a phospholipase C-inositol 1,4,5-trisphosphate pathway-sensitive manner. In conclusion, these results provide strong evidence that purinergic activation of a calcium-dependent, apamin-sensitive potassium conductance is essential to promote chloride secretion and thus fluid formation in the oviduct.     

TRK2 is not a low-affinity potassium transporter in Saccharomyces cerevisiae.

TRK1 and TRK2 encode proteins involved in K+ uptake in Saccharomyces cerevisiae. A kinetic study of Rb+ influx in trk1 TRK2, trk1 TRK2D, and trk1 trk2 mutants reveals that TRK2 shows moderate affinity for Rb+. K(+)-starved trk1 delta TRK2 cells show a low-affinity component accounting for almost the total Vmax of the influx and a moderate-affinity component exhibiting a very low Vmax. Overexpression of TRK2 in trk1 delta TRK2D cells increases the Vmax of the moderate-affinity component, and this component disappears in trk1 delta trk2 delta cells. In contrast, the low-affinity component of Rb+ influx in trk1 delta TRK2 cells is not affected by mutations in TRK2. Consistent with the different levels of activity of the moderate-affinity Rb+ influx, trk1 delta TRK2 cells grow slowly in micromolar K+, trk1 delta TRK2D cells grow rapidly, and trk1 delta trk2 delta cells fail to grow. The existence of a unique K+ uptake system composed of several proteins is also discussed.     

Causes of conductance change in yeast cultures

The conductance change due to growth of Saccharomyces cerevisiae Y112, Zygosaccharomyces bailii M and Rhodotorula rabra NCYC 63 in culture media containing glucose, tartrate pH buffer and ammonium ions as sole nitrogen source was compared with that in a medium containing L-asparagine as sole nitrogen source. Decreases in conductance were observed in glucose-ammonium cultures of all three yeasts while little change occurred in cultures with L-asparagine as sole nitrogen source. This supports the hypothesis that the metabolic activity primarily responsible for conductance change in yeast cultures is the uptake of charged ammonium ions as nitrogen source and the reaction of protons with pH buffer compounds.
Rhodotorula rubra cultures with L-asparagine as sole carbon source caused large increases in conductance with growth. Chemical analyses of culture filtrates showed that this increase in conductance was due to use of L-asparagine as carbon source and the excretion of nitrogen surplus to biosynthetic needs as ammonium. In addition, the production of aspartate, acetate and bicarbonate contributed to the increase in conductance.     

Calcium-activated potassium conductance noise in snail neurons

Current fluctuations were measured in small, 3-6 micrometers-diameter patches of soma membrane in bursting neurons of the snail, Helix pomatia. The fluctuations dramatically increased in magnitude with depolarization of the membrane potential under voltage clamp conditions. Two components of conductance noise were identified in the power spectra calculated from the membrane currents. One component had a corner frequency which increased with depolarization. This component was blocked by intracellular injection of TEA and was relatively insensitive to extracellular calcium levels (as long as the total number of effective divalent cations remained constant). It was identified as fluctuations of the voltage-dependent component of delayed outward current. The second component of conductance noise had a corner frequency which decreased with depolarization. It was relatively unaffected by TEA injection and was reversibly blocked by substitution of extracellular calcium with magnesium, cobalt, or nickel. This second component of noise was identified as fluctuations of the calcium-dependent potassium current. The results suggest that the two components of delayed outward current are conducted through physically distinct channels.     

Electrostatic tuning of ion conductance in potassium channels

Members of the K(+) channel family display remarkable conservation of sequence and structure of the ion selectivity filter, whereas the rates of K(+) turnover vary widely within the family. Here we show that channel conductance is strongly influenced by charge at the channel's intracellular mouth. Introduction of a ring of negative charges at this position in KcsA, a bacterial K(+) channel, augments the conductance in a pH-dependent manner. These results are explained by a simple electrostatic effect based on known channel structures, where the negative charges serve to alter the electrical potential at the inner mouth and, thus, to increase the local K(+) concentration. In addition, removal of the conserved negative charges at equivalent positions in a high-conductance eukaryotic K(+) channel leads to a decrease in conductance.     

Analysis of potassium conductance in squid giant axons

Assuming a model of facilitated ionic transport across axonal membranes proposed by McIlroy (1975) and extended by McIlroy and Hahn (1978), it is shown that if the selectivity coefficient, π_K, of the potassium conducting system ?59 the permeabilityP _Ks, of the periaxonal barrier of the squid giant axon for K⁺ ions?(1.2±0.44)×10^?4 cm sec^?1 and the thickness of the periaxonal space ?477±168 Å. Using a value (10^?4 cm sec^?1) ofP _Ks in the foregoing range the experimental curves for the steady state membrane ionic conductance versus measured membrane potential difference (p.d.), ?, of Gilbert and Ehrenstein (1969) are corrected for the effect of accumulation of K⁺ in the periaxonal space. This correction is most marked for the axon immersed in a natural ionic environment, whose conductance curve is shifted ?70mV along the voltage axis in the hyperpolarization direction. By assuming that the physico-chemical connection between a depolarization of the axonal membrane and the consequent membrane conductance changes is a Wien dissociative effect of the membrane's electric field on a weak electrolyte situated in the axolemma, the position of the peaks of the corrected conductance versus ? curves can be identified with zero membrane electric field and hence with zero p.d.across the axolemma. A set of values for the double-layer p.d.s at the axonal membrane interfaces with the external electrolytes in the vicinity of the K⁺ conducting pores can therefore be deduced for the various external electrolytes employed by Gilbert and Ehrenstein. A model of these double-layer p.d.s in which the membrane interfaces are assumed to possess fixed monovalent negatively charged sites, at least in the neighbourhood of the K⁺ conducting pores, is constructed. It is shown that, using the previously deduced values for the doublelayer p.d.s, such a model has a consistent, physically realistic solution for the distance between the fixed charged sites and for the dissociation constants of these sites in their interaction with the ions of the extramembrane electrolytes.     

Causes of conductance change in yeast cultures.

The conductance change due to growth of Saccharomyces cerevisiae Y112, Zygosaccharomyces bailii M and Rhodotorula rubra NCYC 63 in culture media containing glucose, tartrate pH buffer and ammonium ions as sole nitrogen source was compared with that in a medium containing L-asparagine as sole nitrogen source. Decreases in conductance were observed in glucose-ammonium cultures of all three yeasts while little change occurred in cultures with L-asparagine as sole nitrogen source. This supports the hypothesis that the metabolic activity primarily responsible for conductance change in yeast cultures is the uptake of charged ammonium ions as nitrogen source and the reaction of protons with pH buffer compounds. Rhodotorula rubra cultures with L-asparagine as sole carbon source caused large increases in conductance with growth. Chemical analyses of culture filtrates showed that this increase in conductance was due to use of L-asparagine as carbon source and the excretion of nitrogen surplus to biosynthetic needs as ammonium. In addition, the production of aspartate, acetate and bicarbonate contributed to the increase in conductance.     

A potassium conductance activated by hyperpolarization in paramecium

Summary Voltage clamp studies show that the wild-type membrane ofParamecium tetraurelia contains a conductance component which is sensitive to hyperpolarization. This component manifests itself as anomalous, or inward going, rectification of membrane voltage in response to applied constant current pulses and as a hyperpolarizing spike when no K is added to the external solution (Y. Satow, C. Kung, 1977.J. Comp. Physiol. 11999). Like the conductances which underlie anomalous rectification in other cells, the hyperpolarization-sensitive conductance inParamecium is specific for K, and the magnitude of the voltage-dependent conductance change depends not only on voltage but also on external potassium concentration. The internal potassium ion concentration ofParamecium is calculated to be between 17 and 18mm.     

Aldosterone and chloride conductance of amphibian skin

Chloride influx (JCl) across the skin of toads maintained in dilute MgCl2 or Na2SO4 was determined after overnight incubation with(out) aldosterone, and related to mitochondria-rich cell (MRC) density of the preparations. Adaptation to MgCl2 vs. Na2SO4 was reflected by higher plasma aldosterone in the former group (17 vs. 3 nmol/l, respectively) while JCl was lower, even after overnight incubation (172 vs. 318 pmol cm-2 s-1). Incubation with aldosterone induced a more pronounced increase in JCl in the case of Na2SO4- vs. MgCl2-adapted toads (delta JCl: 242 vs. 25 pmol cm-2 s-1, respectively), which could be related to difference in MRC density between these two groups (1078 vs. 615 cells/mm2, respectively). On the other hand, the in vitro effect of aldosterone on Na+ transport (assessed by Isc) was equally pronounced in both groups, and thus independent of MRC density. These data suggest that aldosterone, rather than being involved in MRC proliferation, stimulates Cl- conductance by influencing the functional state of MRC.     

Genotoxic effects of potassium dichromate, sodium arsenite, cobalt chloride and lead nitrate in diploid yeast

4 metal salts, potassium dichromate, sodium arsenite, cobalt chloride and lead nitrate were tested for their genotoxic effects in Saccharomyces cerevisiae. Potassium dichromate was the most potent agent for induction of gene conversion and reverse mutation. Sodium arsenite was virtually ineffective as a convertogen but gave a positive result for reversion. Cobalt chloride was the least toxic, exhibited a convertogenic activity but was only marginally active for reverse mutation. Lead nitrate was the most toxic salt but was genetically inactive.     

A voltage-gated chloride conductance in rat cultured astrocytes

Large voltage-dependent outward currents are recorded with the whole-cell patch-clamp technique from rat cultured astrocytes under conditions where an outward movement of potassium ions is excluded (either by blockage of the potassium channels pharmacologically or by replacement of the internal potassium by the impermeant large organic cation N-methyl-(+)-glucamine). The current, which is activated at potentials more positive than -40 to -50 mV, is normally carried by an inward movement of chloride ions. Its reversal potential is the same as the chloride equilibrium potential. With depolarization to +60 mV (for 225 ms) little or no inactivation of the current occurs: with depolarizations to +90 to +110 mV a time-dependent decay is seen. The current, which is often not marked immediately after formation of the whole-cell clamp, generally increases over a period of a few minutes to a maximum (after which it usually declines), as if some as yet unknown intracellular factor keeping the channels closed were being washed away from the membrane. The time course of this phenomenon is not affected by changing of the internal free calcium concentration (from 10(-8)M to 10(-6)M) or by an intracellular mixture of cyclic AMP (1 mM), ATP (4 mM) and Mg+ (2 mM). The conductance is slightly increased when the chloride of the bathing medium is replaced by bromide; is much reduced on replacement by methylsulphate, sulphate, isethionate, or acetate; and is virtually abolished on replacement by the large anion gluconate. The outward current is inhibited by the disulphonate stilbenes DIDS and SITS; this blocking action was initially partly reversible, although never completely so. It is suggested that the chloride conductance plays a role in the spatial buffering of potassium by astrocytes.     

Diurnal modulation of photoreceptor potassium conductance in the locust

Single electrode clamp techniques demonstrated diurnal changes in photoreceptor membrane conductance, recorded intracellularly in the intact, dark-adapted retina of the locust Schistocerca gregaria. In the day, locust photoreceptors exhibited the membrane properties of fast cells, as previously defined in rapidly moving diurnal Diptera. Depolarization activated a powerful potassium conductance with two kinetic components, one rapidly activating close to resting potential and the other activating more slowly when further depolarized, giving a pronounced delayed rectification. There was little inactivation. At night, locust photoreceptors resembled slow cells, as defined in weakly flying crepuscular and nocturnal Diptera. Depolarization rapidly activated an outward current which then inactivated over 100 ms to reduce rectification. The change from day to night state was mimicked by applying 10 mM serotonin extracellularly to the retina. We conclude that the potassium conductances of locust photoreceptor membranes are modulated according to a diurnal rhythm, possibly by serotonin. This neuromodulation is used to match photoreceptor membrane properties to photic habitat. Our findings suggest a definite and potentially widespread function for serotonin as a mediator of diurnal changes in the insect visual system.     

TRK1 encodes a plasma membrane protein required for high-affinity potassium transport in Saccharomyces cerevisiae.

We identified a 180-kilodalton plasma membrane protein in Saccharomyces cerevisiae required for high-affinity transport (uptake) of potassium. The gene that encodes this putative potassium transporter (TRK1) was cloned by its ability to relieve the potassium transport defect in trk1 cells. TRK1 encodes a protein 1,235 amino acids long that contains 12 potential membrane-spanning domains. Our results demonstrate the physical and functional independence of the yeast potassium and proton transport systems. TRK1 is nonessential in S. cerevisiae and maps to a locus unlinked to PMA1, the gene that encodes the plasma membrane ATPase. Haploid cells that contain a null allele of TRK1 (trk1 delta) rely on a low-affinity transporter for potassium uptake and, under certain conditions, exhibit energy-dependent loss of potassium, directly exposing the activity of a transporter responsible for the efflux of this ion.     

A synthetic chloride channel restores chloride conductance in human cystic fibrosis epithelial cells

Mutations in the gene-encoding cystic fibrosis transmembrane conductance regulator (CFTR) cause defective transepithelial transport of chloride (Cl(-)) ions and fluid, thereby becoming responsible for the onset of cystic fibrosis (CF). One strategy to reduce the pathophysiology associated with CF is to increase Cl(-) transport through alternative pathways. In this paper, we demonstrate that a small synthetic molecule which forms Cl(-) channels to mediate Cl(-) transport across lipid bilayer membranes is capable of restoring Cl(-) permeability in human CF epithelial cells; as a result, it has the potential to become a lead compound for the treatment of human diseases associated with Cl(-) channel dysfunction.     

Oxidative regulation of large conductance calcium-activated potassium channels

Reactive oxygen/nitrogen species are readily generated in vivo, playing roles in many physiological and pathological conditions, such as Alzheimer's disease and Parkinson's disease, by oxidatively modifying various proteins. Previous studies indicate that large conductance Ca(2+)-activated K(+) channels (BK(Ca) or Slo) are subject to redox regulation. However, conflicting results exist whether oxidation increases or decreases the channel activity. We used chloramine-T, which preferentially oxidizes methionine, to examine the functional consequences of methionine oxidation in the cloned human Slo (hSlo) channel expressed in mammalian cells. In the virtual absence of Ca(2+), the oxidant shifted the steady-state macroscopic conductance to a more negative direction and slowed deactivation. The results obtained suggest that oxidation enhances specific voltage-dependent opening transitions and slows the rate-limiting closing transition. Enhancement of the hSlo activity was partially reversed by the enzyme peptide methionine sulfoxide reductase, suggesting that the upregulation is mediated by methionine oxidation. In contrast, hydrogen peroxide and cysteine-specific reagents, DTNB, MTSEA, and PCMB, decreased the channel activity. Chloramine-T was much less effective when concurrently applied with the K(+) channel blocker TEA, which is consistent with the possibility that the target methionine lies within the channel pore. Regulation of the Slo channel by methionine oxidation may represent an important link between cellular electrical excitability and metabolism.     

A conductance maximum observed in an inward-rectifier potassium channel

One prediction of a multi-ion pore is that its conductance should reach a maximum and then begin to decrease as the concentration of permeant ion is raised equally on both sides of the membrane. A conductance maximum has been observed at the single-channel level in gramicidin and in a Ca(2+)-activated K+ channel at extremely high ion concentration (> 1,000 mM) (Hladky, S. B., and D. A. Haydon. 1972. Biochimica et Biophysica Acta. 274:294-312; Eisenmam, G., J. Sandblom, and E. Neher. 1977. In Metal Ligand Interaction in Organic Chemistry and Biochemistry. 1-36; Finkelstein, P., and O. S. Andersen. 1981. Journal of Membrane Biology. 59:155-171; Villarroel, A., O. Alvarez, and G. Eisenman. 1988. Biophysical Journal. 53:259a. [Abstr.]). In the present study we examine the conductance-concentration relationship in an inward-rectifier K+ channel, ROMK1. Single channels, expressed in Xenopus oocytes, were studied using inside-out patch recording in the absence of internal Mg2+ to eliminate blockade of outward current. Potassium, at equal concentrations on both sides of the membrane, was varied from 10 to 1,000 mM. As K+ was raised from 10 mM, the conductance increased steeply and reached a maximum value (39 pS) at 300 mM. The single-channel conductance then became progressively smaller as K+ was raised beyond 300 mM. At 1000 mM K+, the conductance was reduced to approximately 75% of its maximum value. The shape of the conductance-concentration curve observed in the ROMK1 channel implies that it has multiple K(+)-occupied binding sites in its conduction pathway.     

G-protein activators induce a potassium conductance in murine macrophages

The whole-cell patch clamp technique was used to test whether intracellular application of G-protein activators affect ionic currents in murine macrophages. Both the J774.1 macrophage-like cell line and primary bone marrow derived macrophages were used. Cells were bathed in Na Hanks' solution and intracellularly dialyzed (via the patch pipette) with K Hanks (145 mM KCl, < 100 nM Ca) plus or minus the G-protein activators GTP gamma S (10 microM), GppNHp (10 microM), or AIF4- (200 microM AlCl3 + 5 mM KF). In the absence of G-protein activators, only two K currents, an inwardly rectifying K current (Kir) and an outward, inactivating K current (Ko) were observed. In the presence of protein activators, two effects were observed: (i) the Kir conductance, which is stable for up to 30 min under control conditions, decayed twice as fast and (ii) an outwardly rectifying, noninactivating current appeared. The induced outward current appeared < 2 min after attaining the whole-cell patch clamp configuration. The current could be distinguished from the Kir and Ko currents on the basis of its direction of rectification (outward), barium sensitivity (> 1 mM), and kinetics (no time-dependent inactivation). Intracellular application of GTP (500 microM), GDP (500 microM), cAMP (100 microM + 0.5 mM ATP), or IP3 (20 microM) did not induce the current; 100 microM ATP gamma S activated a half-maximal amount of current. Induction of outward current by 10 microM GTP gamma S could be prevented by pre-exposing cells to pertussis toxin but not cholera toxin. This current is K selective since (i) its induction was accompanied by hyperpolarization of the cell toward EK, even after Kir had "washed out", (ii) it was present after > 90% of both intracellular and extracellular Cl were replaced by isethionate, and (iii) the induced outward conductance was absent when Ki was completely replaced by Cs, and was reduced by approximately 1/3 when [K]i was reduced by 1/3. Quinidine (1 mM) and 4-aminopyridine (10 mM) inhibited the current, but apamin (1 microM) and charybdotoxin (1 microM) did not.