INTRODUCTION

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Yeast cells maintain a stable K⁺ content and growth rate in a wide range of extracellular K⁺ concentrations (18, 23). Consequently, the mechanisms responsible for K⁺ uptake must be capable of adapting to a wide range of external K⁺ concentrations. This is reflected in the broad range of apparent K_m values measured for K⁺ uptake in Saccharomyces cerevisiae growing in different nutritional conditions. The K_m values for K⁺ uptake vary from 15 µM during K⁺ starvation to 5 mM in cells growing at low millimolar K⁺ concentrations (18, 23) to values as high as 50 mM in cells growing in K⁺ concentrations exceeding 100 mM (20).

High- and moderate-affinity K⁺ uptake in S. cerevisiae is mediated by the products of two genes, TRK1 and TRK2 (11, 20). Consequently, trk1 trk2 cells possess only low-affinity K⁺ uptake and exhibit a growth rate much lower than that observed for wild-type yeast in media containing less than 10 mM extracellular K⁺ (11, 20).

The identity of the low-affinity K⁺ uptake pathway in yeasts is unknown. In the few electrophysiological studies performed on yeasts, only two currents have been reported at the plasma membrane; (i) a small instantaneous inward current in Schizosaccharomyces pombe (13) and S. cerevisiae (4) which is probably mediated by the TRK gene products and (ii) a time-dependent outward current mediated by a K⁺-selective outward rectifier (3, 6) which is the product of TOK1 (10, 12, 29). In plants, low-affinity K⁺ uptake is thought to be mediated by K⁺-selective influx channels (14, 21). However, the characteristics of the pathway which mediates low-affinity K⁺ uptake in yeast have not been reported.

In previous electrophysiological experiments on yeast, plasma membrane currents were measured in high (5 mM) extracellular Ca²⁺ and Mg²⁺ (e.g., references 3, 5, and 13). In the present study, we applied the patch clamp technique to spheroplasts isolated from a trk1 trk2 strain of S. cerevisiae and observed whole-cell cation-selective inward currents which are revealed by reducing extracellular divalent cation concentrations to 0.1 mM. K⁺ uptake and growth experiments suggest that the inward current mediates low-affinity K⁺ uptake in intact yeast cells. Inward currents with characteristics very similar to those reported here were also described for S. cerevisiae at the 42nd American Biophysical Society Annual Meeting (7).

	MATERIALS AND METHODS

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All chemicals were supplied by Sigma (Poole, United Kingdom) unless otherwise stated.

Yeast culture and spheroplast isolation. A trk1 trk2 double-deletion strain of S. cerevisiae (W3; MATa ura3 his3 trp1 ade2 leu2 trk1::LEU2 trk2::HIS3) was used unless otherwise stated. Cells were grown overnight at 30°C, with shaking at 100 rpm in 30 ml of liquid medium (LM; 2% [wt/vol] glucose, yeast nitrogen broth [Difco Laboratories], 100 mM KCl) supplemented with the appropriate amino acids. A method based on that described by Bertl and Slayman (2) was used for spheroplast isolation. Cells were harvested from 10 ml of suspension culture by centrifugation (188 × g for 5 min). The cell pellet was resuspended in 10 ml of buffer A (50 mM KH₂PO₄, 40 mM 2-mercaptoethanol; adjusted to pH 7.0 with KOH), pelleted again, resuspended in 4 ml of buffer B (1.2 M sorbitol, 50 mM KH₂PO₄, 40 mM 2-mercaptoethanol, 400 U of lyticase, 1 U of chitinase, and 2,000 U of -glucuronidase per ml; adjusted to pH 7.0 with KOH), and incubated at 30°C, with shaking at 100 rpm. After 90 min, the digest was centrifuged at 188 × g for 5 min, and the pellet was resuspended in 2 ml of ice-cold buffer C (2 M sorbitol, 1 mM CaCl₂, 50 mM KH₂PO₄, 40 mM 2-mercaptoethanol; adjusted to pH 7.0 with KOH). Below this was layered 2 ml of ice-cold buffer D (buffer C, with 2 M sorbitol replaced with 1.5 M sucrose and 0.5 M sorbitol), followed by 2 ml of ice cold buffer E (buffer C, with 2 M sorbitol replaced with 2 M sucrose). This sucrose step gradient was centrifuged at 488 × g for 3 min, and clean spheroplasts were collected from the top layer. Spheroplasts were washed in 5 ml of buffer F (1 M sorbitol, 10 mM HEPES, 1 mM CaCl₂; adjusted to pH 7.0 with KOH) and centrifuged at 188 × g for 5 min. The pellet was resuspended in 1 ml of buffer F and stored on ice. Spheroplasts with diameters of 4 to 5 µm were used.

Electrophysiology. All recordings were made in a continuously perfused chamber in which a glass coverslip formed the base to which the spheroplasts adhered loosely. Patch pipettes were fabricated on a two-stage puller (Kopf Instruments, Tujunga, Calif.) from borosilicate glass (Kimax-51; Kimax Products, Vineland, N.J.). To reduce pipette capacitance, electrodes were coated with a 50% (wt/wt) mixture of mineral oil and Parafilm (American National Can Co., Chicago, Ill.). Pipette resistances varied between 5 and 50 M, depending on the solution used in the pipette. An Ag-AgCl reference electrode was connected to the bath via a 2 M KCl agar bridge. Whole-cell currents were recorded at room temperature (approximately 20°C) with an RK-400 amplifier (Biologique, Claix, France) connected to an A/D converter (Digidata 1200; Axon Instruments, Foster City, Calif.). Recording and storage of data was controlled by the software package pClamp 6.03 (Axon Instruments) and a 486 personal computer. Data were filtered at 1 kHz. Results are presented as means ± standard errors of the means (SEM).

Solutions used in electrophysiology. All solutions were filtered (0.2-µm pore diameter; Millipore) before use and were adjusted to 600 mosmol kg¹ with sorbitol. Seals in excess of 12 G were formed in sealing solution that contained 10 mM KCl, 10 mM CaCl₂, 5 mM MgCl₂, and 5 mM HEPES-Tris base (pH 7.4). After the whole-cell configuration (indicated by an increase in capacitance of between 0.5 to 0.7 pF) was obtained, the solution was replaced by a standard bath solution (SBS; 0.1 mM CaCl₂, 5 mM morpholineethanesulfonic acid [MES]-choline base, pH 5.5) containing various concentrations of K₂SO₄ or KCl. Pipettes were filled with either K⁺-depleted medium (LKM; 10 mM KCl, 2 mM MgCl₂, 3 mM MgATP, 10 mM HEPES, 4 mM EGTA, 22 mM choline base [pH 7.4]) or K⁺-replete medium (HKM; 100 mM KCl, 5 mM MgCl₂, 3 mM K₂ATP, 10 mM HEPES, 4 mM EGTA, 23 mM KOH [pH 7.4]). Ionic equilibrium potentials were calculated after correction for ionic activity with GEOCHEM-PC (17a). K⁺ activities in LKM and HKM were 8.6 and 97.6 mM, respectively.

⁸⁶Rb⁺ uptake experiments. W3 cells were grown overnight at 30°C, with shaking at 100 rpm, in 60 ml of LM, harvested by centrifugation (188 × g for 5 min), and washed with deionized water. Cells were repelleted and resuspended in 10 ml of reaction buffer (2% [wt/vol] glucose, 50 mM KCl, 5 mM HEPES-choline base [pH 7.0]) supplemented with 0.1 mM EGTA or CaCl₂ ranging from 0.1 to 20 mM. The assay was started by the addition of ⁸⁶Rb⁺ (0.1 µCi/ml; Amersham International, Amersham, United Kingdom) and shaking at 100 rpm at room temperature (ca. 20°C). ⁸⁶Rb⁺ was used as a tracer for K⁺ and assumed to simulate K⁺ uptake. The reaction was stopped after 10 min by filtering 1 ml of cell suspension onto nitrocellulose filters (0.45-µm pore diameter; Millipore) and washing with 30 ml of wash solution (50 mM RbCl, 5 mM HEPES-choline base [pH 7.0]). Radioactivity on the filters was then counted.

Growth experiments. W3 cells were grown overnight at 30°C in 30 ml of liquid SD, prepared as described by Sherman et al. (24) but containing 200 µM Mg²⁺ and 100 µM Ca²⁺, and supplemented with the appropriate amino acids and 100 mM KCl. One milliliter of liquid culture of cells was added to 7 ml of SD or SD supplemented with 20 mM Ca²⁺ and incubated at 30°C, with shaking at 100 rpm. Optical density of cell cultures was measured at 600 nm to determine cell density.

	RESULTS

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Inward current revealed in low extracellular Ca²⁺. Using 129 mM cytosolic K⁺ (HKM in the pipette) and 10 mM extracellular Ca²⁺, we always observed time-dependent (TD) spiky outward currents and very little inward current in W3 cells (n = 81) (Fig. 1A and C). The outward current has been characterized previously (3, 6) and shown to be mediated by an outwardly rectifying K⁺ channel encoded by the TOK1 gene (10, 12, 29). In contrast, the inward current represents a resistance of approximately 14 G, and hence a significant portion of this current might result from the seal conductance between the glass pipette and plasma membrane (see below).

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Whole-cell currents in yeast spheroplasts are revealed at low extracellular Ca2+ concentrations. Pipette medium was HKM. (A) Current traces in response to voltage pulses ranging from +80 to 160 mV in 20-mV steps. Holding potential was 30 mV. Bath solution was SBS supplemented with 10 mM CaCl2 and 25 mM K2SO4. (B) Like panel A except that bath solution was SBS supplemented with 25 mM K2SO4. (C) Steady-state current-voltage relationship of currents in the presence of 0.1 () and 10 mM () extracellular Ca2+ derived from panels A and B.

Selectivity. The reversal potential (E_rev) of the inward current closely followed changes in the equilibrium potential for K⁺ (E_K). With 129 mM cytosolic K⁺ and 50 mM extracellular K⁺, the E_rev was approximately 12 mV (Fig. 1), much closer to E_K (22 mV) than the reversal potential for any other ion in the bath or pipette medium. Furthermore, with 10 mM cytosolic K⁺ and 50 mM extracellular K⁺, E_rev was approximately equal to E_K (+40 mV; Fig. 2C). This is consistent with the instantaneous current being carried predominantly by K⁺.

View larger version (26K): [in this window] [in a new window]   FIG. 2.   Whole-cell cation-selective inward currents in yeast spheroplasts. Pipette solution was LKM. (A) Current traces in response to voltage pulses ranging from +100 to 140 mV in 20-mV steps. Holding potential was +40 mV. Bath solution was SBS supplemented with 25 mM K2SO4. (B) As panel A except that bath solution was SBS supplemented with 50 mM KCl. (C) Steady-state current-voltage (I-V) relationship of currents in the presence of either 25 mM K2SO4 () or 50 mM KCl () in the bath. Data are taken from traces shown in panels A and B. (D) I-V relationship of currents in the presence of a range of extracellular K+ concentrations from 1 to 200 mM. Data are the mean values (±SEM) of five separate cells. Bath solution was SBS supplemented with appropriate concentrations of K2SO4. (E) Effects of various cations on inward current at different voltages. Data are the mean values (±SEM) from four separate cells. Current values are shown for 140 (open bars), 100 (hatched bars), 60 (double hatched bars), and 20 (solid bars) mV. Ch, choline.

Block by divalent cations. The inward current carried by K⁺ was insensitive to extracellular TEA⁺ up to 50 mM (data not shown) but was sensitive to extracellular Ca²⁺ and Mg²⁺. Typically, up to 70% of the inward current was inhibited by increasing extracellular Ca²⁺ concentrations from 0 to 20 mM, indicating that although the majority of the current is Ca²⁺ sensitive, a Ca²⁺-insensitive component is also evident (Fig. 3). The apparent K_i for the Ca²⁺-sensitive component was derived from equation 1: I = I_Ca · (1/(1 + ([Ca]/K_i)))+I_ins where I is the total current, I_Ca is the Ca²⁺-sensitive current, I_ins is the Ca²⁺-insensitive current, K_i is the constant for inhibition of the Ca²⁺-sensitive current, and [Ca] is extracellular Ca²⁺ activity. We obtained K_i values at various voltages (Fig. 3B) and found that they did not vary significantly with voltage (i.e., K_i varied between 653 ± 335 µM at 60 mV to 363 ± 78 µM at 140 mV [Fig. 3B, inset]), indicating that the Ca²⁺ inhibition was voltage independent. With respect to the Ca²⁺-insensitive component, it was not possible to distinguish between currents resulting from the seal between the plasma membrane and glass pipette and specific cation transporters in the plasma membrane. However, radiotracer experiments (see below and Fig. 7) indicated that at least part of the Ca²⁺-insensitive current is physiologically relevant.

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Inhibition of the inward current by extracellular Ca2+. Pipette medium was LKM, and bath solution was SBS supplemented with 25 mM K2SO4, 0.1 mM MgCl2, and the appropriate concentration of CaCl2. For 0 mM Ca2+, bath solution was as above except that CaCl2 was omitted from SBS. Data are the mean values (±SEM) of four separate cells. (A) Current-voltage relationship for currents in the presence of 0, 1, and 10 mM extracellular Ca2+. (B) Inhibition of inward current at 160 (), 140 (), 120 (), 100 (), 80 (), and 60 () mV plotted as a function of extracellular Ca2+ activity. Data are fitted with equation 1. Error bars have been omitted for clarity but are of the order of 15 to 25% of the mean. Inset, Ki values plotted as a function of voltage. Data are taken from fits with equation 1.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Inhibition of the inward current by extracellular Mg2+. Pipette medium was LKM, and bath solution was SBS supplemented with 25 mM K2SO4 and the appropriate concentration of MgCl2. Data are the mean values of four separate cells ± SEM. (A) Current-voltage relationship for currents in the presence of 0, 1, and 10 mM extracellular Mg2+. (B) Inhibition of inward current at 140 (), 120 (), 100 (), 80 (), and 60 () mV plotted as a function of extracellular Mg2+ activity. Data are fitted with equation 1. Error bars have been omitted for clarity but are of the order of 10 to 15% of the mean. Inset; Ki values plotted as a function of voltage. Data are taken from fits with equation 1.

Regulation of the inward current by cytosolic K⁺. Figures 1 and 2 indicate that decreasing cytosolic K⁺ increases the magnitude of the inward current. Figure 5 shows the results of an analysis in which the plasma membrane conductance was calculated for extracellular K⁺ concentrations ranging from 1 to 200 mM (with a permissive extracellular Ca²⁺ concentration of 0.1 mM) with pipette solutions either of LKM (10 mM cytosolic K⁺) or HKM (129 mM cytosolic K⁺). The chord conductance was calculated after steady-state currents were normalized with respect to the electrochemical potential for K⁺ (see the legend to Fig. 5). The conductance saturates as a function of extracellular K⁺ activity. Fitting the data with Michaelis-Menten functions yielded maximal conductances of 0.44 ± 0.10 and 1.25 ± 0.31 nS for HKM and LKM, respectively. This finding indicates that the increased inward current observed with low cytosolic K⁺ (compare Fig. 1 and 2) was not solely the result of increasing the electrochemical potential gradient for K⁺ influx but rather the result of the inward current regulation by cytosolic K⁺.

View larger version (17K): [in this window] [in a new window]   FIG. 5.   Control of the inward current by cytosolic K+. Plasma membrane conductance at 140 mV plotted as a function of extracellular K+ activity. Pipette solutions were LKM and HKM for 10 () and 129 () mM cytosolic K+, respectively. Conductance was calculated as Iss/(140  EK), where Iss is the steady-state current and EK is the equilibrium potential for K+. Data are fitted with Michaelis-Menten functions. For 10 mM cytosolic K+, Km was 90 ± 50 mM and Vmax was 1.25 ± 0.31 nS; for 129 mM cytosolic K+, Km was 62 ± 55 mM and Vmax was 0.44 ± 0.10 nS. Data for 10 mM cytosolic K+ were taken from Fig. 2C.

Does TOK1 mediate the inward current? Since W3 is a trk1 trk2 strain, the inward current cannot be mediated by TRK1 or TRK2. However, the possibility remains that the inward current is mediated by the TOK1 gene product. This possibility was tested with a strain of W3 in which the TOK1 gene was additionally deleted (W3-tok1; as W3 except tok::TRP; manufactured by J. Rosamond, Zeneca Pharmaceuticals). Using HKM as the pipette solution, significant outward current was never observed in W3-tok1 (n = 15), confirming earlier reports showing that TOK1 exhibited K⁺-selective outward rectifier activity (10, 12, 29). However, Fig. 6 shows that the inward current was apparent in W3-tok1 cells; indeed, all W3-tok1 cells tested exhibited this current (n = 15). There was no significant difference in the membrane conductance measured at 140 mV between W3-tok1 (0.19 ± 0.11 nS; n = 5) and W3 (0.15 ± 0.06 nS; n = 5) cells when tested with HKM in the pipette and SBS supplemented with 25 mM K₂SO₄ in the bath. Thus, it was concluded that the K⁺-selective outward rectifier does not mediate the Ca²⁺-sensitive inward current.

View larger version (11K): [in this window] [in a new window]   FIG. 6.   Whole-cell currents in W3-tok1 yeast spheroplasts. Pipette medium was HKM, and bath solution was SBS supplemented with 25 mM K2SO4. (A) Current traces in response to voltage pulses ranging from +80 to 160 mV in 20-mV steps. Holding potential was 10 mV. (B) Current-voltage relationship of plasma membrane currents shown in panel A.

Ca²⁺-inhibitable K⁺ uptake and growth in intact yeast. If the inward currents that we measure are responsible for low-affinity K⁺ uptake, then it should be possible to demonstrate that K⁺ influx is Ca²⁺ inhibitable in intact cells. We have used ⁸⁶Rb⁺ as a convenient tracer for K⁺ since the patch clamp experiments indicate that Rb⁺ has essentially the same permeability properties as K⁺ (Fig. 2D). Influx of ⁸⁶Rb⁺ into intact cells was measured in a background of 50 mM K⁺ to simulate low-affinity K⁺ uptake and, as shown in Fig. 7A, was strongly inhibited by Ca²⁺. The resultant K_i of 48 ± 1 µM (Fig. 7A), which, although lower than that observed for the inward current, is consistent with the patch clamp data. It is also noteworthy that approximately 87% of the low-affinity K⁺ uptake is Ca²⁺ sensitive. This indicates that at least some of the Ca²⁺-sensitive inward current measured with the patch clamp technique is physiologically relevant and not solely the result of leak between the glass pipette and plasma membrane.

View larger version (10K): [in this window] [in a new window]   FIG. 7.   Effects of extracellular Ca2+ on K+ uptake and growth in intact cells. (A) K+ uptake plotted as a function of extracellular Ca2+ activity. Data are fitted as Z = ZCa · (1/(1 + ([Ca]/Ki))) + Zins, were Z is the total K+ uptake, ZCa is the Ca2+-sensitive K+ uptake, Zins is the Ca2+-insensitive K+ uptake, Ki is the constant for inhibition of the Ca2+-sensitive K+ uptake and [Ca] is extracellular Ca2+ activity. Ki was 48 ± 1 µM. Results are the means (±SEM) for three separate experiments. (B) Growth of cells in SD medium containing 100 µM () and 20 mM () extracellular Ca2+. Solid lines represent linear regressions from which doubling times of 203 () and 283 () min were calculated. Results are the means (±SEM) of eight separate experiments.

trk1 trk2

	DISCUSSION

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Cation-selective current in the plasma membrane of yeast cells. Reducing extracellular Ca²⁺ and Mg²⁺ revealed a large inward monovalent cation current in the plasma membrane of S. cerevisiae. This current is independent of the previously characterized K⁺ transporters in yeast, namely, TRK1, TRK2, and TOK1. It is known that extracellular Ca²⁺ aids formation of a seal between the glass pipette and membrane in the patch clamp technique (15). Thus, it is necessary to address the potential criticism that the inward current in the present study may represent the loss of seal. The following characteristics of the inward current in yeast illustrate that the current is not an artifact of the patch clamp technique but rather is mediated by specific transporters in the plasma membrane: (i) strong rectification favoring inward current, (ii) strong selectivity for monovalent cations over anions or H⁺, (iii) differential permeability among monovalent cations, and (iv) membrane conductance regulated by cytosolic K⁺. Furthermore, growth and ⁸⁶Rb⁺ uptake experiments suggest that the inward current represents the principal pathway for low-affinity K⁺ uptake in intact trk1 trk2 cells, thus supporting the conclusion that the inward current has physiological significance.

trk1 trk2

Physiological roles. The magnitude of the inward current suggests that it represents a pathway for rapid uptake of K⁺ in the low-affinity range. The inward current is enhanced when cytosolic K⁺ is reduced, which may suggest that it functions to rapidly restore cytosolic K⁺ levels when they become depleted. The regulation by cytosolic K⁺ might also aid growth at high Ca²⁺ levels, where uptake tends to be blocked. Consistent with this function, Ramos and Rodriguez-Navarro (19) showed that low-affinity K⁺ uptake is enhanced in yeast cells which have been treated with azide to reduce their internal K⁺.

Molecular identity of the transporter underlying the inward current. The large magnitude of the inward current suggests that it might be mediated by channel-type molecules which have high turnover rates. Ideally, it should be possible to identify channel-type activity which may underly whole-cell currents in excised patches. However, excised patches from yeast spheroplasts (which are only 4 to 5 µm in diameter) are difficult to obtain and consequently were isolated from only a few cells (n = 5). No single inward channel activity was observed in these patches (data not shown). However, carrier-type mechanisms (for which turnover rates are lower than for channels) could also mediate the inward current if sufficiently large numbers of the carrier-type transporters are present in the plasma membrane. Indeed, TRK-mediated inward currents can be measured in the whole-cell configuration of the patch clamp technique (13), yet it is not known whether these currents are mediated by channel-type or carrier-type molecules. In addition, there are no reports of yeast genes which may encode for a K⁺ uptake channel. However, recent studies suggest that low-affinity K⁺ uptake may be mediated by a number of non-K⁺ transporters which have intrinsic K⁺ transport capabilities, for example, permeases involved in amino acid, sugar, inositol, or choline uptake (16, 27, 28). K⁺ uptake has also been reported for the H⁺/tetracycline antiport in bacteria (8). It is possible that permease-type molecules can mediate the inward current even though the turnover rates of these transporters at first sight seem unlikely to be sufficient to generate the large currents observed in this study.