39 K , 23 Na , and 31 P NMR studies of ion transport in Saccharomyces cerevisiae ( stoichiometry / cation exchange / proton pump / intracellular pH 1 / dysprosium tripolyphosphate )

The relationship between efflux and influx of K+, Na', and intracellular pH (pH' ) in yeast cells upon energizing by oxygenation was studied by using the noninvasive technique of -9K, DNa, and 31P NMR.spectroscopy. By introducing an anionic paramagnetic shift reagent, Dy3+(P3O)2i, into the medium, NMR'signals of intraand extracellular K+ and Na+ could be resolved, enabling us to study ion transport processes by NMR. Measurements showed that 40% of the intracellular K+ and Na+ in yeast cells contributed to the NMR intensities. By applying this correction factor, the intracellular ion concentrations were determined to be 130-170mM K+ and 2.5 mM Na+ for fresh yeast cells. With the aid of a home-built solenoidal coil probe for mK and a double-tuned probe for 'Na and 31p, we could follow time courses of K+ and Na+ transport and of pHin with a time resolution of 1 min. It was shown that H+ extrusion is correlated with K+ uptake and not with Na+ uptake upon energizing yeast cells by oxygenation. When the cells were deenergized after the aerobic period, K+ efflux, H+ influx, and Na+ influx were calculated to be 1.6, 1.5, and 0.15 jimol/min per ml of cell water, respectively. Therefore, under the present conditions, K+ efflux is balanced by exchange for H+ with an approximate stoichiometry of 1:1. 31p, 13C, and 1H NMR have been applied to the study of bioenergetics and of metabolism in intact cells, organelles, and perfused organs and to whole animals (1, 2). In particular, 31P NMR has been used to measure intracellular pH (pHin) together with levels of phosphate metabolites. Therefore, 31P NMR gives information about transport of protons across the cell membrane (refs. 3 and 4; A. Ballarin-Denti, personal communication). Because transport of protons is intimately connected with the transport of cations, NMR studies of transport of ions such as K+ and Na+ would complement the 31P NMR studies. Until recently, however, direct observation by NMR of physiologically relevant ion transport was not possible because separate resonances from intraand extracellular ions could not be observed. Balschi et aL (5) and Gupta and Gupta (6) have introduced anionic paramagnetic shift reagents, which allowed separation of resonances from intraand extracellular 2'Na' (Naj+ and Naut) in human erythrocytes, frog skeletal muscle, and yeast. This approach has opened the possibility of applying 39K and 23Na NMR to the study of ion transport in intact cells. When yeast cells oxidize ethanol in the presence of K+, K' is taken up by the cells and H+ is excreted into the medium (7). The kinetics of influx and efflux pathways for cations in yeast cells have been extensively studied,but relationships and mechanisms of cation transports have not yet been clearly established (ref. 7; ref. 8 and references cited therein). For example, Conway et aL (9) and Ryan and Ryan (10) suggested that separate carriers were involved in the influx of K' and in the efflux of Na'. However, Rothstein (11) has proposed a single cation transport system with different cation specificities and kinetics on the inside and outside of the membrane. More recently, Rodrigues-Navarro and Sancho (12) have shown that Mg2" in the medium inhibits cation exchanges in yeast and that, in the absence of Mg2+, a two-transport-system hypothesis gives a better explanation of their observed data. Yeast cells possess a proton translocating ATPase in the plasma membrane; however, the precise role of K+ in proton pumping is still the subject of controversy (ref. 13 and references cited therein; ref. 14). In this work we studied by the noninvasive technique of NMR spectroscopy the relationship between efflux and influx of Na', K+, and pHin when energizing yeast cells by oxygenation. With the aid of a home-built solenoidal coil probe and an anionic shift reagent, we now can follow time courses of K+ with a time resolution of 1 min in yeast suspensions. In separate experiments under the same conditions, Na+ transport and pHin could be followed-on one sample by using a probe double-tuned for 23Na and 31P and switching back and forth every 1 min. EXPERIMENTAL The Saccharomyces cerevisiae strain NCYC 239 was grown at 30°C for 24 hr in 2% Bacto-peptone, 1% yeast extract, and 2% glucose. Before harvest, the cultures were chilled to 4°C, after which the cells were collected by low-speed centrifugation. The cells were washed twice andresuspended to a pellet/medium density of .1:2 in a resuspension medium consisting of 0.85 g of KH2PO4, 0.15 g of K2HPO4, 0.5 g of MgSO4, and 0.1 g of NaCl per ml in 50mM 2-(N-morpholino)ethanesulfonic acid/25 mM Tris buffer. The extracellular pH was about 6.0. The cells were kept at ice temperature until used. A BrukerWH 360 WB NMR spectrometer was used at 25°C, operating at 145.78 MHz for 31p, 95.26 MHz for 23Na, and 16.81 MHz for 39K. For 31p, 450 pulses were used with pulse intervals of 0.3 s; for 2'Na and for 39K, 900 pulses were used with intervals of 0.05 s. The Bruker 20-mm 13C probe was tuned without modification to 23Na. In addition, we used the Bruker 13C/ 31P double-tuned probe (15-mm OD sample tubes), which could be double-tuned to 23Na and 31p. 39K experiments were done in a separate experiment with our home-built solenoidal coil probe and a sample volume of 25 ml. The home built coil was a 12-turn solenoid tuned to 16.81 MHz with a sample diameter of 28 mm. We obtained with this probe a signal-to-noise ratio of 63:1 for a 150 mM KC1 solution in one pulse, which is >5 times better than with the Bruker 20-mm broad-band probe. In all experiments, gas was bubbled through the suspension. In aerobic experiments, a 95% 02/5% CO2 was used; in anAbbreviations: pHm, intracellular pH; Na:,t and -Kout, extracellular Na+ and K+; Na+ and K,+, intracellular Na+ and K+. 5185 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisenent" in accordance with 18 U.S.C. §1734 solely to indicate this fact. D ow nl oa de d by g ue st o n N ov em be r 18 , 2 02 1 Proc. Natl. Acad. Sci. USA 80 (1983) aerobic experiments, 95% N2/5% CO2 was used (3). Calibration of intracellular concentrations was done by assuming that 1.67 g of wet yeast cells contain 1 ml of cell water. The shift reagent used was Dy3(P3A05 )2 (6). It was prepared by adding a stoichiometric amount of Dy203 to a solution of Na5P3O10. In most experiments, 2.5 mM shift reagent was used, leading to the addition of 25 mM Na' to the suspension. RESULTS Fig. 1 shows the 16.81-MHz 39K NMR spectra of aerobic and anaerobic yeast suspensions at 25°C in the presence of the shift reagent. Two well-resolved peaks were observed, separated by about 10 ppm. The peak shifted upfield corresponds to extracellular K+ (K' t), whereas the unshifted position corresponds to intracellular K+ (Kj+). The peak assignments were based upon the observation that the K,+ chemical shift was virtually unaffected by the presence of the paramagnetic shift reagent. The linewidth of the Kj+ signal was about 10 ppm, and deconvolution helped to separate the two signals (Fig. 1, spectrum C). Relative peak intensities of K, and K+ t signals were quite constant for at least 3 hr, indicating that net movement of K+ was small under these conditions (anaerobic, without exogenous substrate). The observation of separate signals for K'I and 'K't can be utilized to determine the fraction of K, that is NMR visible.