In the yeast potassium channel, Tok1p, the external ring of aspartate residues modulates both gating and conductance.

The yeast plasma-membrane potassium channel, Tok1p, is a voltage-dependent outward rectifier, the gating and steady-state conductance of which are conspicuously modulated by extracellular [K(+)] ([K(+)](o)). Activation is slow at high [K(+)](o), showing time constants (tau(a)) of approximately 90 ms when [K(+)](o) is 150 mM (depolarizing step to +100 mV), and inactivation is weak (<30%) during sustained depolarization. Lowering [K(+)](o) accelerates activation, increases peak current, and enhances inactivation, so that at 15 mM [K(+)](o) tau(a) is less than 50 ms and inactivation suppresses approximately 60% of peak current. Two negative residues, Asp292 and Asp426, near the mouth of the assembled channel, modulate both kinetics and conductance of the channel. Charge neutralization in the mutant Asp292Asn allows fast activation (tau(a) approximately 20 ms) at high [K(+)](o), peak currents diminishing with decreasing [K(+)](o), and fast, nearly complete, inactivation. The voltage dependence of tau(a) persists in the mutant, but the [K(+)](o) dependence almost disappears. Similar but smaller changes are seen in the Asp426Asn mutant, implying that pore geometry in the functional channel has twofold, not fourfold, symmetry.

Chloride channel function in the yeast TRK-potassium transporters.

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.

The presumed potassium carrier Trk2p in Saccharomyces cerevisiae determines an H+-dependent, K+-independent current.

Ionic currents related to the major potassium uptake systems in Saccharomyces cerevisiae were examined by whole cell patch-clamping, under K+ replete conditions. Those currents have the following properties. They (1) are inward under all conditions investigated, (2) arise instantaneously with appropriate voltage steps, (3) depend solely upon the moderate affinity transporter Trk2p, not upon the high affinity transporter Trk1p. They (4) appear to be independent of the extracellular K+ concentration, (5) are also independent of extracellular Ca2+, Mg2+ and Cl- but (6) are strongly dependent on extracellular pH, being large at low pH (up to several hundred pA at -200 mV and pH 4) and near zero at high pH (above 7.5). They (7) increase in proportion to log[H+]o, rather than directly in proportion to the proton concentration and (8) behave kinetically as if each transporter cycle moved one proton plus one (high pH) or two (low pH) other ions, as yet unidentified. In view of background knowledge on K+ transport related to Trk2p, the new results suggest that the K+ status of yeast cells modulates both the kinetics of Trk2p-mediated transport and the identity of ions involved. That modulation could act either on the Trk2 protein itself or on interactions of Trk2 with other proteins in a hypothetical transporter complex. Structural considerations suggest a strong analogy to the KtrAB system in Vibrio alginolyticus and/or the TrkH system in Escherichia coli.

Electrophysiology in the eukaryotic model cell Saccharomyces cerevisiae.

Since the mid-1980s, use of the budding yeast, Saccharomyces cerevisiae, for expression of heterologous (foreign) genes and proteins has burgeoned for several major purposes, including facile genetic manipulation, large-scale production of specific proteins, and preliminary functional analysis. Expression of heterologous membrane proteins in yeast has not kept pace with expression of cytoplasmic proteins for two principal reasons: (1) although plant and fungal proteins express and function easily in yeast membranes, animal proteins do not, at least yet; and (2) the yeast plasma membrane is generally regarded as a difficult system to which to apply the standard electrophysiological techniques for detailed functional analysis of membrane proteins. Especially now, since completion of the genome-sequencing project for Saccharomyces, yeast membranes themselves can be seen as an ample source of diverse membrane proteins - including ion channels, pumps, and cotransporters - which lend themselves to electrophysiological analysis, and specifically to patch-clamping. Using some of these native proteins for assay, we report systematic methods to prepare both the yeast plasma membrane and the yeast vacuolar membrane (tonoplast) for patch-clamp experiments. We also describe optimized ambient conditions - such as electrode preparation, buffer solutions, and time regimens - which facilitate efficient patch recording from Saccharomyces membranes. There are two main keys to successful patch-clamping with Saccharomyces. The first is patience; the second is scrupulous cleanliness. Large cells, such as provided by polyploid strains, are also useful in yeast patch recording, especially while the skill required for gigaseal formation is being learned. Cleanliness is aided by (1) osmotic extrusion of protoplasts, after minimal digestion of yeast walls; (2) use of a rather spare suspension of protoplasts in the recording chamber; (3) maintenance of continuous chamber perfusion prior to formation of gigaseals; (4) preparation (pulling and filling) of patch pipettes immediately before use; (5) application of a modest pressure head to the pipette-filling solution before the tip enters the recording bath; (6) optical control for debris at the pipette tip; and (7) discarding of any pipette that does not "work" on the first try at gigaseal formation. Other useful tricks toward gigaseal formation include the making of protoplasts from cells grown aerobically, rather than anaerobically; use of sustained but gentle suction, rather than hard suction; and manipulation of bath temperature and/or osmotic strength. Yeast plasma membranes form gigaseals with difficulty, but these tend to be very stable and allow for long-term cell-attached or whole-cell recording. Yeast tonoplasts form gigaseals with ease, but these tend to be unstable and rarely allow recording for more than 15 min. The difference of stability accrues mainly because of the fact that yeast protoplasts adhere only lightly to the recording chamber and can therefore be lifted away on the patch pipette, whereas yeast vacuoles adhere firmly to the chamber bottom and are subsequently stressed by very slight relative movements of the pipette. With plasma membranes, conversion from cell-attached recording geometry to isolated ISO patch (inside-out) geometry is accomplished by blowing a fine stream of air bubbles across the pipette tip; to whole-cell recording geometry, by combining suction and one high-voltage pulse; and from whole-cell to OSO patch (outside-out) geometry, by sudden acceleration of the bath perfusion stream. With tonoplasts, conversion from the vacuole-attached recording geometry to whole-vacuole geometry is accomplished by application of a large brief voltage pulse; and further conversion to the OSO patch geometry is carried out conventionally, by slow withdrawal of the patch pipette from the vacuole, which usually remains attached to the chamber bottom.

NSC1: a novel high-current inward rectifier for cations in the plasma membrane of Saccharomyces cerevisiae.

The plasma membrane of the yeast Saccharomyces cerevisiae possesses a non-specific cation 'channel', tentatively dubbed NSC1, which is blocked by normal (mM) calcium and other divalent metal ions, is unblocked by reduction of extracellular free divalents below approximately 10 microM, and is independent of the identified potassium channel and porters in yeast, Duk1p, Trk1p, and Trk2p. Ion currents through NSC1, observed by means of whole-cell patch recording, have the following characteristics: Large amplitude, often exceeding 1 nA of K+/ cell at -200 mV, in tetraploid yeast, sufficient to double the normal intracellular K+ concentration within 10 s; non-saturation at large negative voltages; complicated activation kinetics, in which approximately 50% of the total current arises nearly instantaneously with a voltage-clamp step, while the remainder develops as two components, with time constants of approximately 100 ms and approximately 1.3 s; and voltage independence of both the activation time constants and the associated fractional current amplitudes.

Physiological characterization of the yeast plasma membrane outward rectifying K+ channel, DUK1 (TOK1), in situ.

The major voltage-dependent ion channel in the plasma membrane of Saccharomyces cerevisiae, a conspicuous outwardly rectifying K+ channel, was first dubbed YPK1 and later renamed according to its registered gene names (DUK1, TOK1). It has proven novel in both structure and function. Whole-cell patch-clamp studies of the channel directly on yeast protoplasts now extend our earlier description obtained from isolated patches of yeast membrane (Bertl & Slayman, 1992; Bertl et al., 1993), and provide new data both on the contributions of channel properties to yeast physiology and on possible contributions of molecular structure of channel properties. Three recording tactics produce completely equivalent results and thereby allow great flexibility in the design of experiments: whole-cell voltage clamp with sustained voltage steps (approximately 2.5 sec), whole-cell voltage clamp with slow voltage ramps (5 sec, -40 to +100 mV), and time-averaging of single-channel currents. Activation of Duk1 channels under steady-state conditions is dependent upon ATP in the cytoplasmic solution, and the absence of ATP results in channel "rundown"--decreasing numbers of activable channels--over periods of 10 min to 1 hr from the start of patch recording. Several putative serine- and threonine-phosphorylation sites, as well as a variant ATP-binding fold, exist in the molecule as potential mediators of the ATP effects. The channel runs down similarly following cytoplasmic acidification, but is almost completely insensitive to extracellular pH changes (8.0 to 5.5 tested). This remarkable asymmetry may depend on the protein's strongly asymmetric distribution of histidine residues, with 10 out of 12 predicted to lie close to the membrane-cytoplasm interface. Further data confirm the well-recognized observation that changes of K+ concentration, intracellular or extracellular, can shift the gating voltage of Duk1p in the direction of EK. Among the other alkali-metal cations tested, extracellular Rb+ and Cs(+)--but not Na(+)--substitute almost completely for K+. Extracellular TEA+ inhibits whole-cell K+ currents through Duk1p with a KI of 2.8 mM, and does so probably by reducing the single-channel current.

Functional comparison of plant inward-rectifier channels expressed in yeast.

Functional expression of plant ion channels in the yeast Saccharomyces cerevisiae is readily demonstrated by the successful screening of plant cDNA libraries for complementation of transport defects in especially constructed strains of yeast. The first experiments of this sort identified two potassium-channel genes from Arabidopsis thaliana, designated KAT1 and AKT1 (Anderson et al., 1992; Sentenac et al., 1992), both of which code for proteins resembling the Shaker superfamily of K(+) channels in animal cells. Patch-clamp analysis, directly in yeast, of the two channel proteins (Kat1 and Akt1) reveals both functional similarities and functional differences: similarities in selectivity and in normal gating kinetics; and differences in time-dependent effects of ion replacement, in the affinities of blocking ions, and in dependence of gating kinetics on extracellular K(+). Kat1, previously described in yeast (Bertl et al., 1995), is about 20-fold more permeable to K(+) than to Na(+) or NH(+)(4), shows K(+)-independent gating kinetics, and is blocked with moderate effectiveness (30-50% at 10 mM) by barium and tetraethylammonium (TEA(+)) ions. Akt1, by contrast, is weakly inhibited by TEA(+), more strongly inhibited by Ba(2+), and very strongly inhibited by Cs(+). Furthermore Na(+) and NH(+)(4), while having about the same permeance to Akt1 as to Kat1, have delayed effects on Akt1: brief replacement of extracellular K(+) by Na(+) enhances by nearly 100% the subsequent K(+) currents after sodium removal; and brief replacement of K(+) by NH(+)(4) reduces subsequent K(+) currents by nearly 75%. Furthermore, lowering of extracellular K(+) concentration, by replacement with osmotically equivalent sorbitol, significantly retards the opening of Akt1 channels; that is, the gating kinetics for Akt1 are clearly influenced by the concentration of permeant ions. In this respect, Akt1 resembles the native yeast outward rectifier, Ypk1 (Duk1; Reid et al., 1996). The data suggest that all of the ions tested bind within the open channels, such that the weakly permeant species (Na(+), NH(+)(4)) are easily displaced by K(+), but the blocking species (Cs(+), Ba(2+), TEA(+)) are not easily displaced. With Akt1, furthermore, the permeant ions bind to a modulator site where they persist after removal from the medium, and through which they can alter the channel conductance. Extracellular K(+) itself also binds to a modulator site, thereby enhancing the rate of opening of Akt1.

Use of Saccharomyces cerevisiae for patch-clamp analysis of heterologous membrane proteins: characterization of Kat1, an inward-rectifying K+ channel from Arabidopsis thaliana, and comparison with endogeneous yeast channels and carriers.

Transport-deficient strains of the yeast Saccharomyces cerevisiae have recently proven useful for cloning, by functional complementation, of cDNAs encoding heterologous membrane transporters: specifically, H(+)-amino acid symporters and K+ channels from the higher plant Arabidopsis thaliana. The present study uses whole-cell patch-clamp experiments to show that yeast strains which grow poorly on submillimolar K+ due to the deletion of two K(+)-transporter genes (TRK1 and TRK2) are in fact missing a prominent K+ inward current present in wild-type cells. Rescue of such strains for growth on low K+ by transformation with a gene (KAT1) encoding an inward-rectifying K+ channel from Arabidopsis is accompanied by the appearance of an inward current whose characteristics are in qualitative agreement with previous studies in the Xenopus oocyte system, but differ in quantitative details. The ability to make such measurements directly on Saccharomyces should facilitate structure-function studies of any electrogenic or electrophoretic ion transporters which can be expressed in the plasma membrane (or tonoplast) of that organism.

The fungal H(+)-ATPase from Neurospora crassa reconstituted with fusicoccin receptors senses fusicoccin signal.

Fusicoccin affects several physiological processes regulated by the plasma membrane H(+)-ATPase in higher plants while other organisms having P-type H(+)-ATPases (e.g., fungi) are fusicoccin-insensitive. We have previously shown that fusicoccin binding to its receptor is necessary for H(+)-ATPase stimulation and have achieved the functional reconstitution into liposomes of fusicoccin receptors and the H(+)-ATPase from maize. In this paper we show that fusicoccin sensitivity can be conferred on the H(+)-ATPase from Neurospora crassa, a fungus insensitive to fusicoccin. In fact, H+ pumping by purified H(+)-ATPase from Neurospora crassa reconstituted into liposomes containing crude or partially purified fusicoccin receptors from maize was markedly enhanced by fusicoccin. The stimulation of H+ pumping by fusicoccin is dependent upon pH, fusicoccin, and protein concentration, as was reported for the system reconstituted with both proteins from maize.

Endosomal accumulation of pH indicator dyes delivered as acetoxymethyl esters.

Intracellular distributions of the putative cytosolic pH indicator dyes BCECF [2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein], C.SNARF [5(and 6)-carboxy-seminaphthorhodafluor-1], and C.SNARF-calcein have been examined in Neurospora crassa and in murine fibroblasts (NIH-3T3 cells) under conditions in which both kinds of cells produce visible microscopic vacuoles. All three dyes were administered in electroneutral forms, with the hydroxyl and carboxyl groups esterified (designated as -AM esters). As judged qualitatively from fluorescence levels, hydrolytic derivatives of the two heavily esterified dyes (BCECF-AM and C.SNARF-calcein-AM) accumulated in the vacuoles after exposures of approximately 15 min or more, while the simpler dye (C.SNARF-AM) and its derivatives were almost excluded from visible vacuoles. Fluorescence from this dye, alone among the three, also washed out of Neurospora rapidly upon removal of extracellular dye. There was no evidence for stable accumulation of any of the dyes in cytosol per se. For BCECF(-AM), comparison of the distribution of fluorescence with the size distribution of vacuoles in Neurospora strongly suggests that the dyes are also accumulated by endomembranal vesicles (EMVs) which lie below the limit of resolution in the light microscope, and the same inference can be drawn for the fibroblasts. Uptake of -AM dyes by EMVs, including frank vacuoles, probably results from the action of intravesicular esterases, following diffusional entry of lipophilic neutral molecules or partially de-esterified anions. Calculations of actual cytosolic pH values, or even changes of pH, based on intracellular fluorescence of these dyes, clearly depend upon quantitative knowledge of the subcellular dye distribution. Therefore, until the problem is reliably solved of how to visualize submicroscopic vesicles in living cells, the safest approach to the use of BCECF, C-SNARF and their congeners for cytosolic pH measurement would be to devise methods for coaxing uptake of the ionic forms of these dyes and to abandon use of the esterified forms.

Small lipid-soluble cations are not membrane voltage probes for Neurospora or Saccharomyces.

Small lipid-soluble cations, such as tetraphenylphosphonium (TPP+) and tetraphenylarsonium (TPA+) are frequently used as probes of membrane voltage (delta psi, or Vm) for small animal cells, organelles, and vesicles. Because much controversy has accompanied corresponding measurements on 'walled' eukaryotic cells (plants, fungi), we studied their transport and relation to Vm in the large-celled fungus Neurospora crassa-where Vm can readily be determined with microelectrodes-as well as in the most commonly used model eukaryotic cell, the yeast Saccharomyces cerevisiae. We found no reasonable conditions under which the distribution of TPP+ or TPA+, between the cytoplasm (i) and extracellular solution (o), can serve to estimate Vm, even roughly, in either of these organisms. When applied at probe concentrations (i.e., < or = 100 microM, which did not depolarize the cells nor deplete ATP), TPP+ stabilized at ratios (i/o) below 30 in both organisms. That would imply apparent Vm values positive to -90 mV, in the face of directly measured Vm values (in Neurospora) negative to -180 mV. When applied at moderate or high concentrations (1-30 mM), TPP+ and TPA+ induced several phases of depolarization and changes of membrane resistance (Rm), as well as depletion of cytoplasmic energy stores. Only the first phase depolarization, occurring within the perfusion-turnover time and accompanied by a nearly proportionate decline of Rm, could have resulted from TPP+ or TPA+ currents per se. And the implied currents were small. Repeated testing, furthermore, greatly reduced the depolarizing effects of these lipid-soluble ions, implicating an active cellular response to decrease membrane permeability.

Cation effluxes associated with the uptake of TPP+, TPA+, and TPMP+ by Neurospora: evidence for a predominantly electroneutral influx process.

Previously observed anomalies in the transport of lipid-soluble cations (LSI's) - presumed voltage-probe ions-by intact fungal cells [1] prompted a systematic investigation of ion exchanges induced by high (millimolar) concentrations of the particular species tetraphenylphosphonium ion (TPP+), tetraphenylarsonium ion (TPA+), and triphenylmethylphosphonium ion (TPMP+). With low extracellular free Ca2+ (no calcium added to the medium), influx of the LSI's was biphasic, indicating rapid entry into the cytoplasm followed by sequestration into a subcompartment. The latter process, especially, was strongly inhibited by extracellular Ca2+ (1 mM). Contrary to the expectation for electrophoretically driven entry of LSI's into fungal cells, no major efflux of protons (acidification of the medium) could be measured; in fact, significant alkalinization of the medium was observed. The major cellular inorganic cations, K+ or Na+ (under different conditions), were released during LSI uptake, but with kinetic behavior which clearly ruled out direct coupling to the uptake of TPP+, TPA+, or TPMP+. The major mechanism for entry of these lipid-soluble cations into Neurospora appears to be electroneutral diffusion in combination with one or more hydrophilic anions. Subsequent penetration of the fungal vacuoles would result in binding of LSI's to storage polyanions (viz., polyphosphate) and concomitant displacement of the normal vacuolar cations, such as basic amino acids and polyamines, thus leading to alkalinization of the extracellular medium. The observed effluxes of cytoplasmic K+ and Na+ should result independently from energetic changes (i.e., uncoupling of the mitochondrial) and are most easily described by simple, but asynchronous, changes in the average rate constants for entry and exit of the alkali-metal cations.

Modes and means of channel behavior: an introduction.

A flush of technical developments and the resultant burgeoning research on channel molecules in biological membranes has, over the past two decades, led to clear operational distinctions between channel behavior and carrier behavior. These have evinced the tacit assumption that the two types of behavior are associated with distinctly different structural motifs (though certainly having some elements in common). Although a full survey of the physiological situation is hampered by low activation probabilities for most channels and by the small currents for most carrier systems (necessitating study of large ensembles of molecules), it is nevertheless clear that many intermediate types of behavior do exist, either in comparison of different channel and carrier molecules or in the responses of a given (class of) molecule at different times and in different membranes. Then in order really to understand the molecular mechanisms of transport, we must explicitly design experiments to isolate and quantify these unconventional modes of behavior.

Inward and outward rectifying potassium currents in Saccharomyces cerevisiae mediated by endogenous and heterelogously expressed ion channels.

Disruption of genes encoding endogenous transport proteins in Saccharomyces cerevisiae has facilitated the recent cloning, by functional expression, of cDNAs encoding K+ channels and amino acid transporters from the plant Arabidopsis thaliana [1-4]. In the present study, we demonstrate in whole-cell patch clamp experiments that the inability of trk1deltatrk2delta mutants of S. cerevisiae to grow on submillimolar K+ correlates with the lack of K+ inward currents, which are present in wild-type cells, and that transformation of the trk1deltatrk2delta double-deletion mutant with KAT1 from Arabidopsis thaliana restores this phenotype by encoding a plasma membrane protein that allows large K+ inward currents. Similar K+ inward currents are induced by transformation of a trk1 mutant with AKT1 from A. thaliana.

Reconstitution of a plasma-membrane H(+)-ATPase into bilayer lipid membrane.

The plasma membrane H(+)-ATPase of Neurospora has been reconstituted into planar lipid bilayer membranes by means of the vesicle-fusion technique described by Finkelstein and his collaborators (Zimmerberg et al., 1980; Cohen et al., 1980, 1984; Akabas et al., 1984). Enzyme was first transferred from isolated plasma membrane fragments into asolectin vesicles by a detergent-dialysis procedure (Perlin et al., 1984). After H(+)-pumping activity had been checked by quenching of acridine orange fluorescence, the vesicles were fused into performed bilayers. Critical features of the fusion process include (i) attachment of the vesicles to the bilayer in the presence of divalent cations (Mg++), and (ii) rapid osmotic swelling, which was enhanced by prior sonication or freeze-thawing of the vesicles, and/or by inclusions of physiologic channels. Enough proton pumps could be thus incorporated into bilayers to achieve ATP-driven, vanadate-sensitive currents of 0.04-0.4 pA. Aqueous solutions of low ionic strength were used to suppress conductance fluctuations due to the channels, and when that precaution was taken, we could demonstrate the proton pump the work against membrane potentials of at least 50 mV.

Gating and conductance in an outward-rectifying K+ channel from the plasma membrane of Saccharomyces cerevisiae.

The plasma membrane of the yeast Saccharomyces cerevisiae has been investigated by patch-clamp techniques, focusing upon the most conspicuous ion channel in that membrane, a K(+)-selective channel. In simple observations on inside-out patches, the channel is predominantly closed at negative membrane voltages, but opens upon polarization towards positive voltages, typically displaying long flickery openings of several hundred milliseconds, separated by long gaps (G). Elevating cytoplasmic calcium shortens the gaps but also introduces brief blocks (B, closures of 2-3 msec duration). On the assumption that the flickery open intervals constitute bursts of very brief openings and closings, below the time resolution of the recording system, analysis via the beta distribution revealed typical closed durations (interrupts, I) near 0.3 msec, and similar open durations. Overall behavior of the channel is most simply described by a kinetic model with a single open state (O), and three parallel closed states with significantly different lifetimes: long (G), short (B) and very short (I). Detailed kinetic analysis of the three open/closed transitions, particularly with varied membrane voltage and cytoplasmic calcium concentration, yielded the following stability constants for channel closure: K1 = 3.3 x e-zu in which u = eVm/kT is the reduced membrane voltage, and z is the charge number; KG = 1.9 x 10(-4) ([Ca2+].ezu)-1; and KB = 2.7 x 10(3)([Ca2+].ezu)2. Because of the antagonistic effects of both membrane voltage (Vm) and cytoplasmic calcium concentration ([Ca2+]cyt) on channel opening from the B state, compared with openings from the G state, plots of net open probability (Po) vs. either Vm or [Ca2+] are bell-shaped, approaching unity at low calcium (microM) and high voltage (+150 mV), and approaching 0.25 at high calcium (10 mM) and zero voltage. Current-voltage curves of the open channel are sigmoid vs. membrane voltage, saturating at large positive or large negative voltages; but time-averaged currents, along the rising limb of Po (in the range 0 to +150 mV, for 10 microM [Ca2+]) make this channel a strong outward rectifier. The overall properties of the channel suggest that it functions in balancing charge movements during secondary active transport in Saccharomyces.

Complex modulation of cation channels in the tonoplast and plasma membrane of Saccharomyces cerevisiae: single-channel studies.

Detailed patch-clamp studies have been made of ion channels in the plasma membrane and tonoplast of the yeast Saccharomyces cerevisiae. The predominant tonoplast channel is a high-conductance cation-selective inward rectifier (passing ions easily into the cytoplasm from the vacuole), with its open probability (Po) peaking at about -80 mV (cytoplasm negative) and falling to near zero at +80 mV. It has a maximal slope conductance of approximately 150 pS in 100 mmol l-1 KCl, and conducts Na+, K+ and Ca2+. Elevated cytoplasmic Ca2+ concentration, alkaline pH and reducing agents can activate the channel, its likely physiological function being to adjust cytoplasmic Ca2+ concentration from the vacuolar reservoir. The predominant plasma-membrane channel is a strongly outward rectifying K+ channel (passing K+ easily out of the cytoplasm to the extracellular medium), which is activated by positive-going membrane voltages as well as by elevated cytoplasmic Ca2+ concentration and alkaline pH. Interaction between membrane voltage and [Ca2+]cyt is complex and defines three parallel closed states for the channel: a Ca(2+)-independent brief closure (I), a calcium-inhibited long closure (G) and, at large positive voltages, a calcium-induced brief blockade (B). This channel is likely to function in steady-state turgor regulation and in charge balancing during proton-coupled substrate uptake.