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AbstractTRK 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. 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. 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. , IntroductionWhereas coupled exchange of potassium (K +) for sodium (Na +), mediated by a P-type ATPase in cell plasma membranes, is the principal means for K + accumulation by animal cells, several quite different kinds of transporters impel K + accumulation in plants, fungi, and bacteria. The fact that resting membrane voltages ( V m) in non-animal systems are often very negative to the K + equilibrium voltage ( E K; see ) means that pure channel structures can facilitate net K + uptake and accumulation in many circumstances. ATP-coupled K +-influx pumps also exist, for example the Kdp system in Escherichia coli (; ), but the major devices for K + accumulation are gradient-driven coupled-ion transporters and uniporters.

Best known of these are the so-called TRK and HAK proteins, which – in plants and fungi – are homologues of bacterial Ktr and Kup transporters, respectively.The TRK proteins had been assumed to underlie high-affinity K + accumulation in fungi such as Neurospora (;; but see ). They were also recognized, 10 years ago, as having sequence homology with bona fide K + channels (;; ), and were subsequently demonstrated to fold as internal tetramers, thus forming a channel-like pathway for K + transit (;; ).

The selectivity of this pathway, as explored in both higher plants ( Arabidopsis thaliana:; ) and bacteria ( Vibrio alginolyticus: ) has been shown to depend critically on specific amino-acid residues, whose counterparts in KcsA – the crystallized K + channel from Streptomyces lividans – contribute to actual K +-binding sites.Comparisons among the first few fungal TRK sequences emerging from the genome data revealed an unexpected degree of conservation for residues expected to reside at the surface of the folded structure. This led to suggest oligomerization of folded monomers into tetrads, within cell plasma membranes, resulting in an overall configuration similar to that for aquaporins.Subsequent patch-clamp experiments, on the yeast Saccharomyces cerevisiae and several mutant strains thereof (; ), identified strange ionic currents mediated via the two TRK proteins in that organism (Trk1p and Trk2p; S. Cerevisiae has no homologue of the HAK gene).

These currents are not visible as single-channel events, but do display macroscopic channel-like properties in whole-cell records: they are strongly dependent on extracellular pH (pH o), with a ‘gating’ voltage of −267 mV at pH o=7.5 and −157 mV at pH o=4.5; they are very small for V m's positive to −100 mV, but can be more than 10-fold larger than expected transporter currents at large negative voltages; and they have proven proportional to the intracellular (pipette) chloride (Cl −) concentration at all values of pH o. These currents are evidently carried by Cl − efflux, and detailed kinetic analysis has suggested that they flow through the central ‘pore’ in the assembled tetrads of Trk1p/Trk2p (; ).Because of the direct medical importance of the yeast Candida albicans, particularly for immunocompromised patients, and because of the importance of K + regulation for multiple cellular functions, we undertook to clone the TRK gene(s) in Candida by expression in a double-knockout strain of Saccharomyces, and to characterize the TRK protein(s) in Candida itself. Two related events occurred in the same time-frame: (1) sequencing of the Candida genome, which yielded a defective sequence for the single TRK gene , and (2) discovery that this K + transporter is a critical element in the killing of Candida by the oral antimicrobial peptide, histatin 5. In the present study, we report analysis of the TRK1 gene sequence, comparative analysis of the protein sequence across fungal species, and a partial physiological characterization of the protein CaTrk1p, in C.

Materials and methods Strains and maintenanceHY483, a double-TRK knockout strain of S. Cerevisiae ( MATα leu2-3,112 ura3-1 trp1-1 his3-11,15 ade2-1 can1-100 GAL + SUC2 + trk1Δ∷ HIS3 trk2Δ∷ HIS3; S288C background; ) was used for expression cloning of the Candida TRK1 gene.

The strain was maintained routinely on plates in YPAD +100 mM KCl at 30 °C, and was grown for transformation in liquid YPAD +50 mM KCl (; ). A standard C. Albicans library, prepared from strain American Type Culture Collection (ATCC) 10261 in the centromeric vector YCp50 (; ) was amplified in E.

Coli strain DH5αf1. Transformation of HY483 was carried out with the BIO-101 kit for yeast (MP Biochemicals, Irvine, CA) plus 10 μg of the C. Albicans library DNA. The plasmid DNA from recovered colonies was reisolated using the yeast Teeny-prep protocol. After functional confirmation (growth on low K +), the TRK1 insert was subcloned into YCplac33 , for later use.Other yeast strains, used for functional comparisons, were S. Cerevisiae PLY232 ( MAT a his3-Δ 200 leu2-3,112 trp1-Δ 901 ura3-52 suc2-Δ 9; ), BS202 ( MAT a ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 lys2-Δ NheI; ); and C.

Albicans SC5314 (provided by Dr P.T. Magee, University of Minnesota), CAI4 (Δ ura3∷ imm434/Δ ura3∷ imm434;), SGY243 ( ade2/ ade2Δ ura3∷ ADE2/Δ ura3∷ ADE2; ), CaTK1 (Δ ura3∷ imm434/Δ ura3∷ imm434Δ trk1/TRK1; ), and DBT3 (Δ ura3∷ imm434/Δ ura3∷ imm434 Δ tok1/Δ tok1; ). Ion flux measurementsFunctional characterization of the endogenous C. Albicans TRK protein was carried out in two ways: first, by measurement of chemical fluxes in suspensions of intact C. Albicans yeast cells, using rubidium (especially 86Rb +) as a plausible label for K + influx (;;;; ) and second, by measurement of TRK-dependent ion currents via patch-clamping of yeast-cell spheroplasts.For chemical flux measurements, C.

Albicans (strain CAI4) was grown in shaking cultures at 37 °C to OD 600 nm≈1 ( c. 3 × 10 7 cells mL −1), in commercial YNB medium (QBIOgene, Irvine, CA) plus 200 μM uridine and 140 mM KCl. The resulting log-phase cells were harvested by centrifugation (500 g for 5 min), washed twice with glass-distilled water, and then subjected to a period of general starvation, patterned on that formerly used to ‘stabilize’ S. Cerevisiae for ion-flux experiments (; ).

The washed cells were resuspended (at OD≈1) in 140 mM glucose plus 1 M sorbitol and incubated at room temperature ( c. 23 °C) for 5 h on a rotary shaker (250 r.p.m.).

The resulting starved cells were washed twice, resuspended in transport buffer (50 mM Tris-succinate, pH 5.9, plus 140 mM glucose) at a density of 5 × 0 8 cells mL −1, and equilibrated for 15 min. Rubidium uptake was initiated by injecting 1 mL of this suspension with 25 μL of transport buffer containing 43 mM RbCl (final concentration=1.07 mM) and 0.1 μCi of 86Rb +. Labeled cells were then harvested at intervals, in 200-μL aliquots, by rapid filtration on Durapore membranes (0.45 mm pore diameter; Millipore Corp., Bedford, MA), and rinsed three times with 2 mM MgCl 2 to flush out the extracellular 86Rb +. Pellets and filters were immersed in Ecoscint fluid (National Diagnostics, Atlanta, GA) and counted on a Beckman-Coulter scintillation counter (model LS6500; Fullerton, CA).

Data were collected as counts min −1 per 10 8 cells, and converted to mM (mmol L −1 cell volume) via the measured specific activity plus a standard cell volume of 47 fL per cell.Transport (influx) proved essentially linear for the first c. 5 min of sampling, and sampling was routinely carried out for 3 min. Patch-clamp measurementsThe whole-cell ‘patch’-clamp technique was used, slightly modified from the standard methods for Saccharomyces (; ). Cells were grown in log-phase cultures as described above, but in YPD medium, washed twice, and resuspended (at OD≈1) in 3 mL of 50 mM KH 2PO 4 brought to pH 7.2 with KOH, plus 25 mM β-mercaptoethanol. These suspensions were incubated on a slow orbital shaker (64 r.p.m.), for 30 min at 30 °C, then recentrifuged, and resuspended in 6 mL of the same buffer, plus 3.6 U of zymolyase 20T (ICN Biomedicals Inc., Irvine, CA), and incubated for 45 min at 30 °C. The resulting spheroplasts were spun down (500 g for 5 min), gently resuspended in stabilizing buffer 1 and incubated stationary, at room temperature ( c.

23 °C) until use. A single batch of spheroplasts could be used for patch recording over a 6–8-h period. For actual recording, 1–10 μL of stabilizing suspension was injected into c. 700 μL of sealing buffer 2, gently mixed, and then allowed to settle for 10 min in the recording chamber, so that a small number of spheroplasts adhered lightly to the chamber bottom.Patch pipettes were manufactured as described for Saccharomyces and filled with an artificial intracellular buffer 3. A reference electrode, consisting of a chlorided silver wire (Ag–AgCl) immersed in 1 M KCl, was connected to the efflux-end of the recording chamber via a 1 M KCl–agar bridge.Light suction on a patch pipette, placed near a clean spheroplast on the chamber bottom, would usually draw the cell onto the pipette tip. With very light further suction, a seal of 10–35 GΩ would normally develop within 4–6 min. The whole-cell configuration was obtained by breaking the membrane patch in the pipette tip, via a brief high-voltage pulse ( c.

750 mV for 100 μs). Drop test to demonstrate that TRK1 from Candida albicans complements the K +-transport deficit in Saccharomyces cerevisiae deleted of both TRK1 and TRK2. The central experiment is represented in columns 3, 4, and 5. Strain HY483 is the TRK1,2ΔΔ strain provided.

Untransformed (column 5), or transformed by the empty vector (YCplac33; column 4), HY483 does not grow robustly at K + o. Drop test to demonstrate that TRK1 from Candida albicans complements the K +-transport deficit in Saccharomyces cerevisiae deleted of both TRK1 and TRK2. The central experiment is represented in columns 3, 4, and 5. Strain HY483 is the TRK1,2ΔΔ strain provided. Untransformed (column 5), or transformed by the empty vector (YCplac33; column 4), HY483 does not grow robustly at K + o.

Table 1 Summary of polymorphisms in Candida albicans TRK1A measure of ‘typical’ nucleotide variability, for referencing, was obtained by comparing a region of the genome-database sequences for SC5314 and WO-1, spanning from 6000 bases upstream through 6000 bases downstream of TRK1. This region in chromosome R, described especially for SC5314, includes five more putative ORFs with a total coding sequence of 8087 bases, five noncoding intervals with 3250 bases, and 88% of the centromere (3945 bases). Single-base changes are found at 1% of residues in the noncoding intervals, which is not significantly different from 2% in the TRK1 UTRs, combined for the three strains. (The centromere region is more variable, however, with 2.8% of sites differing between SC5314 and WO-1.)Within the coding sequence itself, SNPs are less frequent, occurring at 26 sites out of 3180 bases, or c.

0.8%, which compares with 0.5% of residues in all six ORFs, between SC5314 and WO-1. Furthermore, the SNP variations within the TRK1 ORF are nonrandomly distributed in at least two respects. First, from strain to strain: four changes from majority in WO-1, four in ATCC 10261, and 18 in SC5314. Second, location within the gene: 25 of the 26 identified SNPs occur in codons for putative cytoplasmic residues (viz., 693 amino acids out of 1059 total), regions that are very poorly conserved across fungal species. The three-amino acid deletion in SC5314 (486-Asp.Asp.Asp.-488) also maps to the major cytoplasmic loop of the protein. Only a single SNP, 2364T in WO-1, maps within the transmembrane or extracellular segments of the protein, which are well conserved across fungal species. Three apparent SNPs in SC5314 have proven to be sequencing errors in the genome database (see boxed residues in; Fig.

Only four SNPs in SC5314 TRK1 and two in WO-1 TRK1 are nonsilent mutations. All these map to unconserved cytoplasmic segments of the protein, where they are predicted to have little or no effect on function. Finally, the silent mutation at base 465 (A/G, ) has been identified as SNP marker 1772/2368 in the Candida SNP map constructed. Amino acid conservation across speciesAs noted in the, TRK proteins in plants, fungi, and bacteria are sequence-similar to the selectivity-filter core of K + channels, and have been postulated to fold in a similar manner. This folding is shown in the bead diagram of, by the clusters just below the membrane–pore–membrane (MPM) numbers (#1, #2, #3, and #4). The index of sequence mutability (μ) across fungal species (calculated by Dr H.R. Guy, National Cancer Institute) is represented in by colors according to the figure key, with red designating best conserved (least mutable), gray designating very poorly conserved, and colorless designating the absence of conservation.

Representation of high sequence conservation within the MPM segments of TRK proteins. Whole-protein alignments and index-of-conservation calculations were carried out by H.R.

Guy according to the procedures described by and, for the first 19 fungal TRK sequences obtained from genomic data: Candida albicans, Aspergillus nidulans (two isoforms), Debaryomyces occidentalis, Ashbya gossypii, Gibberella zeae (three isoforms), Kluyveromyces lactis, Magnaporthe grisea (three isoforms), Neurospora crassa, Podospora anserina, Schizosaccharomyces pombe (two isoforms), Saccharomyces cerevisiae (two isoforms), and Saccharomyces uvarum. The triplet diagonal arrays designate α helices, and the extended doublets designate β strands, predicted by means of the predict protein software, available at. The bead clusters directly below each MPM number (#1, #2, #3, and #4) represent the pore loops, with each α-helical segment on the left and each filter sequence on the right, just above P1, P2, P2, and P4. In the intact, folded protein, the four filter sequences would cluster radially around a pore, thus forming several binding sites for K + ions being transported. Representation of high sequence conservation within the MPM segments of TRK proteins.

Whole-protein alignments and index-of-conservation calculations were carried out by H.R. Guy according to the procedures described by and, for the first 19 fungal TRK sequences obtained from genomic data: Candida albicans, Aspergillus nidulans (two isoforms), Debaryomyces occidentalis, Ashbya gossypii, Gibberella zeae (three isoforms), Kluyveromyces lactis, Magnaporthe grisea (three isoforms), Neurospora crassa, Podospora anserina, Schizosaccharomyces pombe (two isoforms), Saccharomyces cerevisiae (two isoforms), and Saccharomyces uvarum. The triplet diagonal arrays designate α helices, and the extended doublets designate β strands, predicted by means of the predict protein software, available at.

The bead clusters directly below each MPM number (#1, #2, #3, and #4) represent the pore loops, with each α-helical segment on the left and each filter sequence on the right, just above P1, P2, P2, and P4. In the intact, folded protein, the four filter sequences would cluster radially around a pore, thus forming several binding sites for K + ions being transported.The majority of cytoplasmically localized residues, including the N terminus, the C terminus, and the long hydrophilic loop (L23) show little conservation, whereas the transmembrane helices tend to be well conserved, especially the so-called pore loops (P1, P2, P3, and P4). Indeed, the ‘signature’ glycine residues within the putative filter sequences, QA GLN, DL GLT, TV GFS, and TV GMS, appear to be absolutely conserved, not only between species, but also among the separate MPM motifs within each species. More broadly, among the four MPM motifs, the segment TM7 through TM8 is the best conserved.A detailed view of these results, extended to 48 TRK sequences that are now complete in the fungal (ascomycete) genome databases, is provided in the Fig.

This information is analyzed and summarized in, via phylogenetic trees for the four separate MPM motifs. The colors designate seven distinct clades, which are roughly conserved in the four MPM motifs. Albicans (marked by a white dot) relates most closely with the same six TRK proteins (the red block) in all four MPMs, for Ashbya gossypii, Debaryomyces hansenii, Debaryomyces occidentalis, Pichia guilliermondii, Pichia stipitis, and Yarrowia lipolytica, although nearest neighbor arrangements within that group differ considerably among the four MPM motifs. The closest adjacent clade (the blue block), containing S.

Cerevisiae (two isoforms), Saccharomyces uvarum, Candida glabrata, Vandervaltozyma polyspora (two isoforms), and Kluyveromyces lactis, is also consistent in all four MPM motifs. Despite the obvious variance, these distributions of sequence are approximately compatible with the current understanding of phylogenetic relationships among the ascomycete fungi (;; ). They also emphasize that residue dispersion across species has occurred at very different rates in different portions of the TRK molecule; in particular, MPM4 has been much more stable than the other three MPM motifs, requiring c. 50% fewer nucleotide substitutions to source the entire set of 48 fungal sequences.

The possible significance of this finding is treated further in the. Comparison of the separately computed phylogenetic trees of the four MPM motifs in TRK proteins from ascomycete fungi. Sequence data assembled in Fig. S2, aligned via the Clustal V algorithm. Trees constructed via the MegAlign algorithm in the lasergene software (DNASTAR Inc., Madison, WI). Note that distances (hundreds of nucleotide substitutions) are reckoned from the common trunk, rather than from the present, and that the scale of major branches, earlier than 8000 substitutions, is compressed fourfold for MPM1, MPM2, and MPM3. The full list of species names, abbreviated in each panel above, is given in the legend of Fig.

Comparison of the separately computed phylogenetic trees of the four MPM motifs in TRK proteins from ascomycete fungi. Sequence data assembled in Fig.

S2, aligned via the Clustal V algorithm. Trees constructed via the MegAlign algorithm in the lasergene software (DNASTAR Inc., Madison, WI). Note that distances (hundreds of nucleotide substitutions) are reckoned from the common trunk, rather than from the present, and that the scale of major branches, earlier than 8000 substitutions, is compressed fourfold for MPM1, MPM2, and MPM3. The full list of species names, abbreviated in each panel above, is given in the legend of Fig.

The primary function of CaTrk1p: K + uptakeTransport functions at yeast plasma membranes are well demonstrated to be stabilized by preconditioning of the cells under generalized starvation, for example incubation for several hours in distilled water or lightly buffered glucose solution (; ). Influx measurements on such preconditioned cells of Candida were routinely initiated by injecting cell suspensions with 86Rb + in c. 1 mM extracellular chemical Rb + (but nominally zero K +). Averaged results from six experiments are displayed in, showing a bound component of 2.6±1.5 mM (ordinate intercept ±1 SE) and a stable influx (slope) of 6.4±1.2 mM min −1. The dependence of this influx upon the TRK gene/protein was demonstrated previously by means of severe haploid insufficiency: deletion of only one of the two alleles of CaTRK1 reduced Rb + influx by fivefold. Parameters of K + uptake by Trk1p in Candida albicans. (a) Average results for six independent experiments at 1 mM extracellular RbCl.

(b) Separate experiment for kinetic parameters, using three different extracellular concentrations of RbCl. Experimental details are given in.Concentration dependence of the uptake process was assessed from similar measurements made with 10 μM, 100 μM, and 1 mM extracellular rubidium, as shown in, from which the linear slopes describe a simple saturation function having a maximal transport velocity ( V max) of 19.0 mM min −1, and a Michaelis constant ( K 0.5) of 0.64 mM. These results place the normal function of the Trk1 protein in Candida in almost the same physiological range as the combined actions of Trk1p and Trk2p in Saccharomyces, for cells of that species similarly preconditioned.

The two are directly compared in (lower two curves), with kinetic parameters in Saccharomyces of V max=16.2 mM min −1 and K 0.5=0.56 mM. K +-limited growth induces more vigorous TRK-dependent transport than does generalized starvation: comparison of Candida and Saccharomyces. Docx viewer mac os. Data sources: curve 1 ( Candida albicans),; curve 2 ( Saccharomyces cerevisiae), (measured flux of 42K +, not 86Rb +); curve 3,; curve 4,.

Low-K + growth medium contained 10 mM arginine brought to pH 6.5 with phosphoric acid, 2 mM MgSO 4, 0.2 mM CaCl 2, 110 mM glucose, standard vitamins+trace elements, and 20 μM K +. When K + O had fallen to 2 μM, cells were harvested and prepared for the Rb + influx measurements represented in curves 3 and 4. Kinetic parameters, for curves 1–4, respectively: K 0.5=0.64, 0.56, 0.086, and 0.078 mM; V max=19.0, 16.2, 28.4, and 27.5 mM min −1. K +-limited growth induces more vigorous TRK-dependent transport than does generalized starvation: comparison of Candida and Saccharomyces. Data sources: curve 1 ( Candida albicans),; curve 2 ( Saccharomyces cerevisiae), (measured flux of 42K +, not 86Rb +); curve 3,; curve 4,.

Low-K + growth medium contained 10 mM arginine brought to pH 6.5 with phosphoric acid, 2 mM MgSO 4, 0.2 mM CaCl 2, 110 mM glucose, standard vitamins+trace elements, and 20 μM K +. When K + O had fallen to 2 μM, cells were harvested and prepared for the Rb + influx measurements represented in curves 3 and 4.

Kinetic parameters, for curves 1–4, respectively: K 0.5=0.64, 0.56, 0.086, and 0.078 mM; V max=19.0, 16.2, 28.4, and 27.5 mM min −1.A long-recognized additional property of the yeast TRK system(s), however, is that its detailed kinetic behavior depends significantly on the regimen of preconditioning, in a manner which defies simple separation into functionally high-affinity and low-affinity systems (,;;; ). Thus, for Saccharomyces cells grown overnight in medium limited only by low K +, uptake of K + (or Rb +) occurred with roughly 10-fold higher affinity ( K 0.5∼0.08 mM) and twofold higher velocity ( V max=28 mM min −1) than for cells stabilized by preincubation in distilled water. The explicit comparison is made in, between the upper two curves for Saccharomyces, and the bottom curve, all representing data from the established literature.

It is not known with certainty whether the detailed conditions for K + starvation similarly affect the kinetics of transport in Candida, but that would be expected, as a mechanism to optimize resources under conditions of varying nutrient stress. With regard to other members of the C. Albicans clade (red block in ), data on TRK-mediated K + fluxes in K.

Lactis and D. Hansenii qualitatively resemble those for CaTrk1p and ScTRK1,2p, but do not address the quantitative impact of varying methods of starvation. Characteristic secondary function of CaTrk1p: Cl − channelingFor cells the size of C. Albicans, chemical fluxes of K + or Rb + such as reported in and would imply ionic currents in the range of 1–2 pA per cell, only marginally large enough to be measured – as steady currents – by whole-cell patch-clamp techniques. However, early patch-clamp studies of Saccharomyces identified the ScTRK proteins with significantly larger currents, which were peculiarly insensitive to extracellular K + (; ). Those currents were shown to arise from a stable anion permeability in both Trk1p and Trk2p, plus the action of Cl − ions introduced to cytoplasm by the pipette-filling solution (; ). Patch-clamp studies on Candida have now demonstrated a similar Cl − permeability in that organism, dependent upon the CaTRK1 protein.shows a typical set of whole-cell patch-clamp records from a single cell of C.

Albicans, wild-type strain SGY243. Each of the superimposed traces represents current required to clamp the membrane voltage suddenly from the reference value of −40 mV to test values of +100 mV (top trace), +80, +60, −160, −180 mV (bottom trace).

Depicts the actual voltage-clamp pulses (also superimposed), each lasting for 2.5 s, after a 0.5-s ‘hold’ at the reference value. The upward (outward) currents reflect K + efflux through Candida's plasma-membrane K + channel, Tok1p, and – as shown in – those currents disappeared when both alleles of the TOK1 gene were deleted. The currents activated with time constants of c. 120 ms (half times of c. 85 ms read from the left end of each trace) at the onset of each voltage pulse, reflecting molecular conformation changes that are customarily referred to as ‘gating movements’ in bona fide channel proteins. The currents deactivated very much faster when the clamp voltage was returned to its reference value (see right end of each trace).

Voltage pulses trigger outward currents via Tok1p and inward currents via Trk1p in the Candida plasma membrane. Patch-clamp traces from whole-cell records, using 2.5-s voltage pulses from a holding value of −40 mV, as shown superimposed in (b).

(a and e) Wild-type strain SGY243; (c) TOK1-knockout strain DBT3; (d) TRK1-single-allele knockout strain CaTK1. Standard extracellular buffer (sealing buffer, pH=7.5) was used throughout, as described in. Standard intracellular (pipette) buffer, containing 183 mM Cl −, was used in the experiments of (a), (c), and (d); Cl − was replaced by gluconate, for the experiment of (e). Voltage pulses trigger outward currents via Tok1p and inward currents via Trk1p in the Candida plasma membrane. Patch-clamp traces from whole-cell records, using 2.5-s voltage pulses from a holding value of −40 mV, as shown superimposed in (b). (a and e) Wild-type strain SGY243; (c) TOK1-knockout strain DBT3; (d) TRK1-single-allele knockout strain CaTK1.

Standard extracellular buffer (sealing buffer, pH=7.5) was used throughout, as described in. Standard intracellular (pipette) buffer, containing 183 mM Cl −, was used in the experiments of (a), (c), and (d); Cl − was replaced by gluconate, for the experiment of (e).The downward (inward) current traces in reflect ion flow associated with Trk1p, the K + transporter protein, and these were nearly abolished by deletion of a single TRK1 allele, as demonstrated in.

This finding is fully compatible with the severe reduction of cation influx, produced by single-allele deletion ( 86Rb +; ), in this diploid organism. (Ca TRK1 appears to be an essential gene, and C. Albicans does not grow, even on K +-rich medium, when both alleles have been deleted.) As had been found in Saccharomyces, however, these inward currents proved insensitive to extracellular K + (data not shown) and were roughly proportional to intracellular chloride (Cl − in the pipette solutions). Demonstrates the nearly complete disappearance of inward currents when Cl − i was reduced to submillimolar levels.More detailed experiments, however, have revealed several modes in which the Cl − currents, mediated by CaTrk1p, differ very significantly from those mediated by the two TRK proteins in Saccharomyces.

Most conspicuous is a large difference in rates of activation during hyperpolarizing voltage pulses. As shown in, in Saccharomyces the inward currents jumped (downward) essentially as fast as the voltage clamp pulses were imposed.

More specifically, the maximal currents for each pulse were attained within a single sampling interval, 63 μs, for all of the records in., closely resembling the records of, displays much slower activation of the CaTrk1 currents, with time constants of c. The traces in also display much larger amplitude noise at low frequencies than is apparent for Saccharomyces (in ). Taken together, the slow activation and relatively large low-frequency noise suggest that bursts of Cl − ions are admitted through CaTrk1p by typical channel gating movements. For ScTrk1p and ScTrk2p, by contrast, the nearly instantaneous activation and low noise level are more readily compatible with single-ion jumps through the protein, viz., simple Eyring-barrier events.

TRK-dependent Cl − currents are larger, slower, noisier, less pH sensitive, and more sensitive to 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS) in Candida than in Saccharomyces. Procedures as in, except that voltage–pulse durations were only 1.5 s for some experiments with Saccharomyces. Extracellular buffer at pH 5.5 was similar to sealing buffer, except that acidic MES was titrated only as far as pH 5.5, with Tris base. For the experiments of (e) and (f), carried out at pH O=5.5, 0.1 mM DIDS was injected into the pipette solution; similar results were obtained with 1 mM extracellular DIDS. Wild-type strain BS202 of Saccharomyces cerevisiae (a, c, and e), and strain SGY243 of Candida albicans (b, d, and f). TRK-dependent Cl − currents are larger, slower, noisier, less pH sensitive, and more sensitive to 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS) in Candida than in Saccharomyces.

Procedures as in, except that voltage–pulse durations were only 1.5 s for some experiments with Saccharomyces. Extracellular buffer at pH 5.5 was similar to sealing buffer, except that acidic MES was titrated only as far as pH 5.5, with Tris base.

For the experiments of (e) and (f), carried out at pH O=5.5, 0.1 mM DIDS was injected into the pipette solution; similar results were obtained with 1 mM extracellular DIDS. Wild-type strain BS202 of Saccharomyces cerevisiae (a, c, and e), and strain SGY243 of Candida albicans (b, d, and f).Three other properties distinguishing the Cl − currents through CaTrk1p from those through the Saccharomyces proteins are their larger amplitude, their pH insensitivity, and their ready blockade by anion-channel inhibitors. Despite the fact that C.

Albicans cells routinely selected for patch-clamp experiments were significantly smaller than those of S. Cerevisiae (diameters of 5–7 vs.

6–8 μm), the measured TRK-mediated currents were conspicuously larger in Candida, as is readily seen in (cf. The effects of elevating the pH o from 5.5 to 7.5 are also demonstrated in: that is, a fourfold reduction of current amplitude in Saccharomyces but no change of amplitude in Candida (cf. With, and with ). (However, the rate of activation was slowed in Candida by about threefold.) Finally, the classic anion-channel inhibitor 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid had little effect on Cl − currents through ScTrk1p+ScTrk2p (cf. With ), but nearly completely blocked the currents through CaTrk1p (cf.

With; see also ). These results are further evidence that the molecular events determining Cl − permeability of the TRK protein in Candida differ significantly from those in Saccharomyces. Discussion Implications from sequenceComparison of the TRK gene among strains of C. Albicans, as summarized in, shows the strain ATCC 10261 to be more closely related to WO-1 than to SC5314 (which was selected first for Candida genome sequencing), as judged by the frequency of SNP variations in the coding region plus c. 800-base flanks. While the overall incidence of SNPs is consistent with random single events, their actual distribution is clearly nonrandom; as is generally to be expected, SNPs occur in coding regions at only about half of the rate observed in noncoding flanks.

But, among the three strains, DNA sequences that correspond to the ‘channel-forming’ MPM motifs – viz. 35% of the TRK protein – contain only 4% of SNPs (1/26) identified in the whole coding region. On this basis alone, selection has clearly occurred for structural stability in those domains of the protein that are directly involved in K + transport.The same conclusion has emerged more conventionally by comparison of amino-acid sequences among homologues of CaTrk1p, across fungal species (see Fig.

This information reveals MPM4, the most C-ward component of the protein that is folded into the transport structure, to be especially strongly conserved ( and ), accumulating fewer than half the mutations across species as in the other three MPM segments. One possible interpretation of this finding is that MPM1,2,3 have evolved separately from MPM4; but that seems unlikely, because the primary function of TRK proteins in fungi – K + accumulation – is regarded as essential. However, if MPM4, but not the other three MPM motifs, were involved in a separate function (such as Cl − channeling), simultaneous imposition of two selective pressures could retard its evolution. A relevant additional point may be that the selectivity of the actual ionic pathway through TRK proteins, for K + ions relative to Na + or other monovalent cations, is only modest compared with the selectivity of canonical K + channels, for example.A particularly surprising feature of interspecies sequence comparisons for MPM4, as originally noted by, is conservation of residues along TM7 and TM8, which ‘should’ be buried rather nonspecifically in the plasma membrane's phospholipid bilayer. This observation led to a structural picture (see Modeling the unexpected, below) which cogently anticipated the observed secondary function of fungal TRK proteins. Functions of Trk1p in CandidaSerious functional comparisons of Candida Trk1p can be made with proteins from only one other yeast species thus far, S.

As demonstrated in for all four MPM motifs, sequence identity is nearly 60% between CaTrk1p and both Saccharomyces proteins, and similarity is near 75% when conservative substitutions are included. The numbers for MPM4 itself are close to 65% and 85%. While many factors determine the actual functional capability of a protein in situ– including other (binding) proteins and small molecules that may differ from organism to organism – extended identity/similarity between proteins in two separate species is normally expected to reflect a quantitatively similar function. This expectation was certainly satisfied by the data on K + transport per se , when C. Albicans and S.

Cerevisiae were similarly preconditioned by generalized starvation. Table 2 Summary of identities and similarities of primary structure, between CaTrk1p and the two Saccharomyces proteins, ScTrk1p and ScTrk2pThe effect of pure K + starvation (growth in rich medium containing only μM K +) still needs to be explored in Candida, for comparison with data from Saccharomyces (upper two curves of ). Another important property remaining to be explored, in both organisms, is the effect of small changes of sequence on cation selectivity in transport, particularly with respect to the selectivity-filter motifs (QA GLN, DL GLT, TV GFS, TV GMS). Studies on bacterial and plant TRK proteins have shown that cation permeability varies greatly with sequence changes in these motifs, as is to be expected from the large literature on bona fide K +-channel molecules.

In KtrB of V. Alginolyticus, for example, conversion of any of the four ‘signature’ glycine residues to alanine, serine, or aspartate greatly reduced the absolute transport rates of the protein, and conversion specifically to serine resulted in preferential transport of Na + rather than K +.The secondary function of TRK proteins, outward conduction of Cl − ions , also confirms general expectation based on similarity of sequence. However, the observed quantitative differences from this function in Saccharomyces are particularly interesting. As shown in, the Cl − currents associated with CaTrk1p are slowly activating (in response to voltage shifts), large, noisy, insensitive to changes of extracellular pH, and very sensitive to anion channel blockers. Such differences might arise from any of several general causes: detailed sequence differences between the Saccharomyces and Candida proteins, differences of the membrane environment in the two species, or different cytoplasmic binding proteins and regulatory pathways.Although this secondary function of fungal TRK proteins may have been an important factor in the strong interspecies sequence conservation of the MPM4 segment, the essential physiological role of such Cl − channeling is still speculative. Glycophilic fungi seem to need only trace amounts of Cl −, and intracellular concentrations should be kept low – compared with the extracellular solutions – by large steady-state membrane voltages (viz., in the range of −200 mV). In this context, a TRK-mediated Cl − pathway should serve as a Cl − escape route, perhaps even too efficiently, because Saccharomyces, at least, appears to concentrate Cl − (weakly) by means of a formate transporter.

But in yeasts that can adapt to very salty environments, this pathway could become essential to Cl − detoxification. The clade containing C. Albicans (red block, ) is rich in halophilic species, including D. Occidentalis, P.

Guilliermondii, P. Stipidis, and Y. Whether the pathway plays that same role in sustaining C. Albicans on mammalian epithelial surfaces, or in physiological saline solutions such as saliva (with.

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The complexity of computer graphics illumination models and the associated need to find ways of reducing evaluation time has led to the use of two methods for simplifying the spectral data needed for an exact solution. The first method, where spectral data is sampled at a number of discrete points, has been extensively investigated and bounds for the error are known. Unfortunately, the second method, where spectral data is replaced with tristimulus values (such as RGB values), is very little understood even though it is widely used. In this paper we examine the error incurred by the use of this method by investigating the problem of approximating the tristimulus coordinates of light reflected from a surface from those of the source and the surface. A variation on a well known and widely used approximation is presented. This variation used the XYZ primaries which have unique properties that yield straightforward analytic bounds for the approximation error. This analysis is important because it gives a sound mathematical footing to the widely used method of trichromatic approximation. The error bounds will give some insights into the factors that affect accuracy and will indicate why this method often works quite well in practice.

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3. 3.HALL, R., AND GREENBErtG, D. A testbed for realistic image synthesis. IEEE CGg.4A (November 1983), 10-20.Google Scholar
4. 4.KREYSZIO, E. Introductory Functional Analysis with Applications. John Wiley & Sons, 1978.Google Scholar
5. 5.MEYER, G. Wavelength selection for synthetic image generation. Compuier Vision, Graphics, and Image Processing (January 1988), 57-79. Google ScholarDigital Library
6. 6.MEYER, G., AND GrtEENB~.rtO, D. Colorimetry and computer graphics. In SIGGRAPH- State of the Art Tutorial on Color Spaces (1984).Google Scholar
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1. Trichromatic approximation for computer graphics illumination models

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  DOI:10.1145/122718

  • DOI:10.1145/127719

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