31P and 35Cl Nuclear Magnetic Resonance Measurements

Transcription

1 THE JOURNAL OF BOLOGCAL CHEMSTRY by The American Society of Bioloocal Chemists, nc. Vol. 260, No. 21, ssue of September 25, PP ,1385 Printed in U. S. A. 31P and 35Cl Nuclear Magnetic Resonance Measurements Transport in Human Erythrocytes* of Anion (Received for publication, February 6, 1985) Manfred BrauerS, Carole Y. Spread#, Reinhart A. F. Reithmeierq, and Brian D. SykesS From the Department of Biochemistry and $Medical Research Council Group on Protein Structure and Function, Uniuersity of Alberta, Edmonton, Alberta, Canada T6G 2H7 The exchange of anions across the erythrocyte membranehasbeenstudiedusing P nuclearmagnetic resonance (NMR) to monitor inorganic phosphate influx and C1 NMR to monitor chloride ion efflux. The slp NMR resonances for intracellular Pi and extracellular Pi could be observed separately by adjusting the initial extracellular ph to 6.4, while the intracellular ph was 7.3. The C1 NMR resonance for intracellular C1- was so broad as to be virtually undetectable (line width >200 Hz), while thatof extracellular C1- is relatively narrow (line width of about 30 Hz). The transports of Pi and C1- were both totally inhibited by 4,4 -diisothiocyanostilbene-2,2 -disulfonate, a potent inhibitor of the band 3 protein. Since the resonance of Pi varies with ph, intra- and extracellular ph changes could also be determined during anion transport. The extracellular ph rose and intracellular ph fell during anion transport, consistent with the protonated monoanionic HzPOZ form of Pi being transported into the erythrocyte rather than the deprotonated dianionic HPOZ- form. The rates of C1- efflux and Pi influx were determined quantitatively and were found to be in close agreement with values determined by isotope measurements. The C1- efflux was foundto coincide with the influx ofthemonoanionic H,PO; form of Pi. The transport of anions, particularly C1- and HCO:, across the erythrocyte membrane plays a crucial role in the removal of COz from the body and in acid-base balance. Band 3 protein is an integral membrane protein in human erythrocytes which catalyzes the one-for-one exchange of anions across the membrane (1-4). The anions bind to specific anion binding sites on the band 3 protein for which saturation kinetics are observed at high substrate concentrations (1-4). Various specific inhibitors of band 3, which can bind covalently or noncovalently to the anion binding site, are known, such as 4,4 - diisothiocyanostilbene-2,2 -disulfonate (DDS ) (4, 5). The * The Medical Research Council of Canada and the Alberta Heritage Foundation for Medical Research provided generous financial support. A preliminary report of this work has been published (Brauer, M., Spread, C. Y., Reithmeier, R. A. F., and Sykes, B. D. (1985) Biophys. J. 47, 158a). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 8 Alberta Heritage Foundation for Medical Research Summer Student. ll Scholar of the Medical Research Council of Canada. The abbreviations used are: DDS, 4,4 -diisothiocyanostilbene- 2,2 -disulfonate; Pi, inorganic phosphate; Pi, intracellular inorganic phosphate; PiE, extracellular inorganic phosphate; ph, intracellular ph; phe, extracellular ph; CE, extracellular chloride ion; NMR, nuclear magnetic resonance; PPES, 1,4-piperazinediethanesulfonic acid; 1, liter normal mechanism of action involves facilitative transport of anions in which electroneutrality is preserved. While C1- and HCO: exchange is the most important process physiologically, other anions such as sulfate (6) and inorganic phosphate (4, 7) can also be transported by band 3. Nuclear magnetic resonance spectroscopy provides an ideal direct probe for the study of anion transport in erythrocytes but has not been widely utilized. 35Cl NMR has been used to study the binding of C1- with a wide variety of proteins (8). The binding of Cl- to band 3 was first studied by Rothstein and his co-workers (9), who showed that the broadening of the 35Cl resonance ofc1- in the presence of erythrocytes ghosts or their Triton X-100 extracts was found to decrease in the presence of the specific inhibitor DDS. These studies were expanded upon by Chan and co-workers (10, 11) who found two types ofc1- binding sites on erythrocyte membranes, a high affinity transporter site and a low affinity site, possibly involved in transport inhibition. The high affinity transporter sites could be further resolved into sites facing the intra- or extracellular solution, and the 35Cl NMR studies indicated an alternating site transport mechanism for the band 3 protein (11). The actual transport ofc1- across the erythrocyte membrane was not studied in any of these reports, however. 31P NMR spectroscopy has enjoyed considerable usage as a noninvasive tool to study the phosphorus metabolism of intact cells and tissues (12-14). The change in the chemical shift of the Pi resonance with ph has allowed investigators to determine intracellular ph in the intact erythrocyte (15) as well as many other cellular and organ systems (12-14). 31P NMR has been used to demonstrate the accumulation of Pi within resealed erythrocyte ghosts containing high levels of intracellular Tris buffer (16). Pi transport in anaerobic mitochondria has been monitored by 31P NMR (17). The ph gradient across the mitochondrial membrane could also be measured from the chemical shifts of the Pi resonances inside and outside the organelle. n this study, 31P and 35Cl NMR spectroscopy were used to study the transport of Pi into erythrocytes and C1- out of erythrocytes. n the 35Cl NMR experiments, the line width of the intracellular C1- was very broad so that only extracellular C1- was observed under the spectral conditions used. Both intra- and extracellular Pi could be observed with 31P NMR. Since the chemical shift of the 31P resonance of Pi changes appreciably with ph, the resonance for extracellular Pi was shifted away from that of intracellular Pi by varying the extracellular ph. With 31P NMR, we could also determine intracellular and extracellular ph changes with time. Thus, we were able to follow intra- and extracellular H2PO;, HPOi-, and H levels as well as extracellular C1- levels during the course of the transport process. This study demonstrates the power of 31P and 35Cl NMR in monitoring anion transport

2 P and 35Cl NMR of Anion Transport in Erythrocytes in erythrocytes and may also be applied to anion transport in other cells, organelles, and tissues. MATERALS AND METHODS Bloodwas freshly drawn from the antecubital vein of normal, young adults or was obtained as outdated blood from the Red Cross blood bank. Erythrocytes for 31P NMR experiments were washed three times and incubated overnight at 4 "C in 5 mm Pi, 150 mm NaCl, ph 7.4. For %C1 NMR experiments, where initial extracellular chloride ion levels were to be kept low, three washes were done with 28.5 mm citrate, 205 mm sucrose, ph 6.4. (Citrate does not penetrate the erythrocyte membrane.) n both cases, the erythrocytes were then collected by centrifugation and mixed in most experiments with an equal volume of 130 mm Pi, ph 6.4, to start the transport process. An external ph of 6.4 was chosen since it is optimal for Pi transport (7), and a -1 ph unit difference across the erythrocyte membrane allowed us to clearly distinguish 31P resonances for intra- and extracellular Pi. For experiments designed to follow anion transport as a function of initial extracellular Pi concentration, the solution of 130 mm Pi, ph 6.4, was mixed in varying proportions with a solution of 28.5 mm sodium citrate, 205 mm sucrose, ph 6.4 (to maintain constant ionic strength and osmolarity), prior to addition to the packed erythrocytes to give a range of external phosphate concentration (0-130 mm). All buffers were prepared fresh to minimize the accumulation of dissolved COz. For inhibition studies with DDS (obtained from Pierce Chemical Co.), solutions of 2.5 mm DDS were prepared fresh in 28.5 mm citrate, 205 mm sucrose, ph 6.4 buffer, aliquots were added to the erythrocytes to a final concentration of 25 pm, and the erythrocytes were allowed to incubate for at least 20 min at room temperature before the transport process was initiated. Calibration curves relating the intensity of the 35Cl NMR signal corresponding to extracellular C1- to the concentration of extracellular C1- were obtained in the following manner. A fresh 2.5 mm DDS solution was prepared, and 100 pl was added to 5.0 mlof washed, packed erythrocytes and allowed to incubate for at least 20 min at room temperature. The erythrocytes were then mixed with an equal volume of 130 mm Pi, ph 6.4, and various amounts of a standard 2.5 M KC1 solution were added to the extracellular environment (20 pl of2.5 M KC1 per 10 mm final extracellular c1- concentration). Calibration curves for 31P NMR experiments were determined in a similar manner. 15 ~1 of a fresh 2.5 mm DDS solution was added to 0.75 ml of washed, packed erythrocytes and allowed to incubate for 2 min. Solutions of various concentrations of Pi ranging from 0 to 130 mm were prepared by mixing in various proportions a solution of 130 mm Pi, ph 6.4, with a solution of 28.5 mm citrate, 205 mm sucrose, ph ml of these solutions was then added to the erythrocytes, and 31P NMR spectra were run. To follow transport, both 31P and 35C1 NMR spectra were taken at discrete time intervals immediately after the 130 mm Pi, ph 6.4, solution was mixed with the packed erythrocytes. The NMR samples were run nonspinning to minimize centrifugation of the erythrocytes. Even 20-Hz spinning of the NMR samples, while enhancing the homogeneity of the magnetic field, caused the erythrocytes to pack to the sides and bottom of the NMR tube in min, while nonspinning samples exhibited only minimal settling after 12 h. 31P NMR spectra were taken at MHz on a Bruker HXS-270 NMR spectrometer, using a 55' excitation pulse, repetition time, and a spectral width of f2500 Hz. Typically, 176 scans were accumulated per spectrum (requiring 6.0 min), and a line broadening of 5 Hz was used to decrease spectral noise. O-mm round-bottom NMR tubes were used, and the sample volume was 1.5 ml. All 31P chemical shifts are relative to 85% H3P04 external standard, with upfield shifts negative. 35Cl NMR spectra were obtained at MHz on a Nicolet NT-BOOWB NMR spectrometer using a sweep width of f2000 Hz, a 90" excitation pulse, and a s repetition time scans were accumulated per spectrum (requiring 2.14 min), and a 2-Hz line broadening was generally used to decrease spectral noise. 20-mm flatbottom NMR tubes were used for 36C1 NMR experiments with a total sample volume of 10.0 ml. All %C1 NMR experiments were run at 22 "C. 31P NMR experiments were run at 27 "C, or at 22 "C for those experiments run concurrently with 35Cl NMR experiments. RESULTS nitially, the study of the transport of chloride ions across the erythrocyte membrane using 35Cl NMR was approached similarly to experiments involving cation transport and phosphorus metabolism (18). The line width of the 35Cl resonance of 100 mm KC1 in 50 mm PPES buffer, ph 6.5, is 15 Hz; in the presence of 50% erythrocytes in 28.5 mm citrate, 205 mm sucrose, ph 6.4, the line width observed was 35 f 3 Hz. Attempts were made to separate the resonances corresponding to extracellular and intracellular chloride ions using paramagnetic shift reagents. Cobalt, copper, nickel, and dysprosium were added to the extracellular environment, and the resulting spectra showed significant shifting and broadening of the extracellular C1- resonance. However, the intracellular C1- resonance, unaffected by the extracellular reagents, was found to be a very broad peak (>200 Hz) and, therefore, difficult to see. The increase in the line width of the intracellular C1- resonance is the result of interaction of the C1- ion with the hemoglobin in the cell. (Since the 35Cl nucleus has an appreciable quadrapolar moment, a minor distortion of the spheri- cal symmetry of the C1- ion electron cloud due to protein binding will result in greatly enhanced spin-spin and spinlattice relaxation rates for the observed resonance, given conditions of fast exchange between free and bound C1-.) From this point on, the spectral conditions used were such that only the extracellular C1- resonance was observed. n order to correlate the height of a 35Cl NMR resonance corresponding to extracellular C1- with a concentration of C1- ions in the extracellular environment, calibration curves were done (Fig. 1). Samples were prepared by adding equal volumes of fresh blood (pretreated with 25 PM DDS) with 130 mm Pi, ph 6.4. Known amounts of C1- were added to the extracellular environment, and 35Cl NMR spectra were taken. n order to determine if the small amount of extracellular C1- seen in Fig. 1A at 0 mm added C1- was due to leakage (even though DDS was present), the final sample of 68 mm was left for 16 h, and then the spectrum was retaken. The amount of leakage of C1- across the membrane in 16 h could not significantly have contributed to the increase in extracellular C1- over just a few minutes. The average of two curves obtained in an B t- X P W X a W 0, [a-] - FG. 1. A, %C1 NMR spectra of various extracellplar C1- concentrations. Each spectrum represents 1000 scans (2.14 min) with a line broadening of 5 Hz. Fresh washed blood with 25 pm DDS was mixed with an equal volume of 130 mm Pi, ph 6.4, and 0 --.) 136 pl of 2.5 M KC1 was added to the extracellular environment to give extracellular concentrations in the range from 0 to 68 mm. The final 68 mm sample was left for 16 h at 22 "C, and then the spectrum was retaken. B, plot of the average of two calibration curves obtained as shown in A. Height of the 35C1 resonance is plotted as a function of the extracellular concentration of chloride ion. Error bars and the best fit line are shown.

3 31P and 35Cl NMR of Anion Transport in Erythrocytes %1 NMR of C' Efflux 2.14 mins TME - FG. 2. s6c1 NMR spectra of erythrocytes undergoing anion transport (Cl- efflux and P influx). An equal volume of 130 mm Pi, ph 6.4, was added to fresh washed erythrocytes and acquisition of spectra was begun immediately. 50 sequential spectra were taken of 1000 scans each, requiring 2.14 min per spectrum. identical fashion to Fig. 1A is shown in Fig. 1B. This graph correlates the height of the 35Cl resonance with the concentration of chloride ion in the extracellular environment. Resonance heights rather than areas could be used because the 35Cl resonance line widths were constant over the full range of C1- concentrations used. To study C1- efflux fresh blood was suspended in an equal volume of 130 mm inorganic phosphate, ph 6.4. %C1 NMR spectra were then taken at regular intervals for a period of about 100 min (Fig. 2). This was done using blood from various individuals and each 100-min series resulted in a horizontal stacked plot similar to Fig. 2. This plot shows the height of the extracellular C1- resonance as a function of the elapsed time since addition of the 130 mm Pi solution to the washed erythrocytes. The average calibration curve (shown in Fig. 1A) was then used to convert the resonance heights in the kinetics experiments to the actual amount of C1- ion present in the extracellular environment. The result was a graph of the increase in [C1-lE (concentration of extracellular C1-) during the experiment, for the blood of each individual (Fig. 3). The kinetics of C1- transport across the erythrocyte membranes for the different individuals was found to bevery similar-an early quick phase of transport which levels off as the experiment nears completion. Considerable individual variability in the final extracellular C1- concentration may reflect variation in the initial intracellular C1- levels. The intracellular C1- concentration is known to vary considerably with the ph within the erythrocyte and to be subject to individual variability (19). When a solution of Pi, ph 6.4, was added to an equal volume of packed erythrocytes and 31P NMR spectra were taken with time, twowell separated resonances were clearly observed (Fig. 4A). The upfield resonance at -1.7 ppm represents Pi outside the erythrocyte (P?), and the downfield resonance at -2.1 ppm represents intracellular Pi (P!). With time, the PiE resonance decreased and the Pi' resonance increased, consistent with the net influx of Pi (Fig. 4, B-G). The total area of the Pi' and PiE resonances varied by less than 10% over the course of the transport experiment, indicating that any T differences between the Pi' and PF resonances did not signif- lo TME frnin) FG. 3. Graphs obtained when the average calibration curve from Fig. 1B were used to determine the extracellular C1- concentration from the %C1 resonance heights (e.g. Fig. 2). The results are graphs of the increase in extracellular C1- as transport occurs for 60 min across the erythrocyte membrane after the addition of 130 mm Pi, ph 6.4. The symbols e, a,., and A represent results from the blood of various individuals. icantly affect area measurements. The two resonances moved towards one another with time as the ph gradient between the intra- and extracellular solution decreased, i.e. as the Pi was transported into the erythrocyte, the extracellular ph increased and the intracellular ph decreased. When the same experiment was done, except for a preincubation of the erythrocytes with 25 p~ DDS, a negligible change in the intensities

4 P and 35Cl NMR of Anion Transport in Erythrocytes a P P ~ FG. 4. "P NMR spectra of erythrocytes undergoing anion transport (Pi influx and C1- efflux). 25 mm Pi, ph 6.4, was added to an equal volume of packed erythrocytes equilibrated with 150 mm C- and 5 mm Pi, and spectra were run nonspinning at 27 "C. Each spectrum was acquired over 6.0 min and 5-Hz line broadening was used to decrease spectral noise. The erythrocytes used were from aged blood with very little &phosphoglycerate, so the Pi resonance1 3 would not be obscured. Spectra were taken after 30 s (A). 2.0 min ( B), 9.5 and chemical shift of the Pi' and PiE resonances occurred over 12 h. Addition of 1% octaethylene glycol mono-n-dodecyl ether, a monoanionic detergent which lyses red cells, collapsed the ph to intermediate values, with only one Pi resonance observable. n order to quantitate the changes in intensity of the Pi resonances and the changes in ph, calibration curves relating 31P resonance intensity to concentration of Pi and relating 31P chemical shift to ph were obtained (Figs. 5, A and B, respectively). n Fig. 5A, erythrocytes were preincubated with 25 MM DDS to inhibit anion transport, and equal volumes of Pi of known concentration were added to the packed erythrocytes. The areas of the PiE resonance were found to be linearly dependent upon the concentration of added Pi. The areas of the Pi' resonances increased slightly with high concentrations of added extracellular Pi, indicating some Pi influx in spite of the DDS. n Fig. 5B, spectra of Pi taken under identical conditions as those of the anion transport experiments were titrated from ph 4.5 to 9.0. The variation in 31P chemical shift for Pi over this ph range corresponded to the second ionization constant for Pi (H2PO; c, H' + HPO;') (20). The pk, for Pi determined from Fig. 5B was 6.80, and the chemical shifts of the mono- and dianionic form were +0.7 and +3.4 ppm, respectively, in good agreement with literature values (20,21). A potential problem in determining intra- and extracellular ph for a solution of erythrocytes by NMR methods is that the magnetic susceptibility changes as a function of the oxygenation of the hemoglobin (22, 23). Oxyhemoglobin is diamagnetic, while deoxyhemoglobin is paramagnetic. The effects of magnetic susceptibility changes on the chemical shifts of 31P resonances were assessed by monitoring the chemical shift of the a-phosphate of intracellular ATP, a resonance which does not change appreciably with ph, metal ion (24), or hemoglobin concentration (25). The chemical shift of this resonance was ppm in all experiments for which ph.values were reported. Since the chemical shift of the a-phosphate of ATP did not change O 9.0 PH FG. 5. A, calibration curve of "P resonance area relative to the concentration of Pi. Erythrocytes were preincubated with 25 PM DDS for min, followed by the addition of au equal volume of various concentrations of Pi (prepared by mixing 130 mm Pi, ph 6.4, with 28.5 mm citrate, 205 mm sucrose, ph 6.4, to maintain constant ionic strength and osmolarity). Spectra were taken under standard conditions, as in Fig. 1A. 0, PE 0, Pi'. B, calibration curve of 31P resonance chemical shift relative to ph. Pi (10 mm) in 100 mm KClwas titrated from ph 4.5 to 9.0 and spectra were run under standard conditions TME (rnin) FG. 6. nflux of Pi into erythrocytes obtained from various blood samples. The initial Pi concentration for all the transport experiments was 130 mm. 0, erythrocytes obtained from freshly drawn blood; 0, A, and V, erythrocytes obtained from outdated blood (stored 30 days); 0, erythrocytes obtained from old outdated blood (stored >60 days).

5 31P and 35Cl NMR of Anion Transport in Erythrocytes TME (rnin) i E LO} // - / FG. 7. A, influx of Pi into erythrocytes with time at various initial PiE concentrations. Concentrations of PiE from 0 to 130 mm., (under conditions of constant osmolarity and ionic strength) were added to an equal volume of packed erythrocytes. The increase in Pi' with time was determined from the area of the Pi' resonance. A, 0 mm initial concentration PiE; A, 10 mm initial concentration PF; 25 mm initial concentration PiE; 0, 50 mm initial concentration PiE; 0, 100 mm initial concentration PiE; 0, 130 mm initial concentration PiE. B, initial velocity of Pi influx into erythrocytes as a function of initial concentration of PF. The initial rates of Pi influx were determined from A. appreciably from its normal chemical shift in the absence of erythrocytes of ppm (26) or in erythrocyte hemolysates of ppm (results not shown), the ph values determined from the chemical shifts of the Pi resonances were judged to be reliable. The transport properties of different blood samples were compared at constant initial P? concentrations of 130 mm (Fig. 6). The rates of influx of Pi for fresh blood, outdated blood (stored 30 days), and old outdated blood (>SO days storage were comparable. Erythrocytes that have been severely depleted of ATP have been reported to have a decreased rate of anion transport (27,28). This effect, however, was not as pronounced at ph' > 7.0, in agreement with our results. The 31P NMR spectra of fresh blood showed appreciable levels of diphosphoglycerate and adenosine 5'-triphosphate, while the levels of these metabolites in outdated blood were very low, and the levels in old outdated blood were undetectable (data not shown). The fact that the rates of Pi influx are TME (min) FG. 8. Concentration of CE, Pi', and PE and ph* and phe during the course of anion transport. Transport was initiated by the addition of 130 mm P? to an equal volume of packed erythrocytes. The same blood sample was used for both 31P and %C NMR results. Concentrations of Pi' and P? were determined from the areas of the 31P resonances of Pi' and PiE (Fig. 5A), while ph' and phe were determined from the chemical shifts of the Pi' and PF resonances (Fig. 58). The concentration of extracellular C1- was determined from the area of the observed %C1 resonance (Fig. 18). Open circles symbolize intracellular Pi concentration or ph while closed circles symbolize extracellular Pi concentration or ph. Open triangles symbolize extracellular C1-. The high initial Cl- concentration was due to incomplete washing of the erythrocytes. The lines through the CE, PiE, and Pi' data were generated by a computerized theoretical calculation using a mobile carrier model involving 1:l anion exchange in which the carrier cannot traverse the membrane without a bound anion (see "Results"). independent of the concentration of intracellular adenosine 5"triphosphate indicates that Pi influx is facilitative rather than active transport. Since the kinetic properties of the erythrocyte samples under our experimental conditions were found to be independent of the age of the blood (Fig. 6) or the intracellular ATP levels, data can be compared between batches of outdated blood and with literature values based on fresh blood. The transport of Pi into erythrocytes from outdated blood was studied as a function of initial extracellular Pi concentration. The increase in intracellular Pi concentration with time, determined from the area of the Pi' resonance, is shown in Fig. 7A at various starting concentrations of P?. The rate of influx of Pi increased linearly with increasing P? concentrations up to about 130 mm Pi (Fig. 7B), in agreement with similar kinetic data determined by classical radiotracer studies (4, 7). At 130 mm PiE concentration and extracellular ph of 6.4, Pi was transported into the cell at a velocity of 2.6 mmol of Pi 1" min" erythrocyte, compared to literature rates of 2.8 (4) and about 2.6 mmol of Pi 1" min" erythrocyte (7) under similar experimental conditions. A detailed analysis of the concentration of CE, Pi', and PiE and of ph' and phe over the course of a transport experiment (130 mm PiE initial concentration) is shown in Fig. 8 and

6 ~ ~~~ P and 35Cl NMR of Anion Transport in Erythrocytes r TABLE Values for kinetic analysis of Pi influx and C1- efflwl 'lp' ph' P,' [HsPO;]' phe Pi" [H2PO;E time min mm mm mm mm "C1, time Time [C1-E Time [Cl-]' min mm min mm min mm brane movement of anion-free carrier C' c, CE is negligible. A computer program in Basic was written to calculate PiE, Pi', and CE as a function of time using equation 38a of Ref. 29 (program available upon request). Literature values (1) for the binding constants of C1- (66 mm) and Pi (50 mm) to the anion binding site of band 3 protein were used. The initial intracellular C- concentration was estimated to be 110 mm, based on the work of Dalmark (19) and of Gunn and Frohlich (30). Taking initial CE of 20 mm, Pi' of 10 mm, and PiE of 110 mm by extrapolating to zero time, and using maximum rates of 3.3 mmol of Pi 1" min" and 10,000 mmol of C1 1" min-', close agreement between the experimentally determined CE, Pi', and PiE levels and the theoretical curves was obtained (Fig. 8).' Varying the maximum velocity of C1- transfer from 50 to 100,000 mmol of C11" min" did not alter the theoretical curves, indicating that C1- transfer was not rate-limiting. ndeed, the rate of C1- transport is known to be >lo4 faster than the rate of Pi transport (1-3). The maximum rate for Pi transport of 3.3 mmol of Pi 1" min-' is in reasonable agreement with literature values of 2.8 (4) and 2.6 mmol of Pi 1" min" (7). From the experimentally determined concentrations of Pi and from the ph during the course of the anion transport, the concentration of the monoanionic (H'PO;) and the dianionic (HPO$-) forms of Pi could be calculated, using the pk,, of 6.80 for Pi (Fig. 5B) and the Henderson-Hasselbalch equation ph = pka + log [HPOi-]/[H,PO;]. From the PiE and phe values in Fig. 8, concentrations of extracellular H,PO; and HP0:- were calculated. The net influx of H2PO; and HPOi-, determined from the loss of extracellular H2PO; and HPOi- during transport, was plotted as a function of time (Fig. 9). These curves were compared to a curve for the net efflux ofc1- determined from the increase in CE. The results show that the C1- efflux coincides stoichiometrically with the influx of total Pi (within experimental error) and that it is mainly the monoanionic form of extracellular Pi which is transported. n O D Y o. u n o DSCUSSON TME (rnin).. n this report, -. FG. 9. Net influx of &POZ and HPOi- and net efflux of C1- during anion transport. The results of Fig. 8 for CE, PiE, and phe were analyzed in terms of net loss of PiE and net increase in CE with time. The concentrations of HzPOT and HPOf were determined from PiE and phe, using pk, of , C1- efflux; 0, HzPO: influx; 0, HP0:- influx. Table. 36Cl and 31P NMR spectra were taken of the same blood sample under identical conditions. The increase in Pi' is readily reflected in the decrease in P? and increase in CE in Fig. 8. Also, the ph' decreases concurrently with an increase in phe. The convergence of ph' and phe is consistent with selective transport of the protonated monoanionic form of Pi relative to the deprotonated dianionic form. Both initial ph' and phe values of 7.25,and 6.39 were in close agreement with expected values of (13, 14) and 6.40, respectively. The change in CE, Pi', and PiE concentrations during anion transport were analyzed in terms of a mobile carrier model: P? + CE u PiE.CE Y Pi'.C' u C' + Pi' CE + CE u CE.CE u C11.C' u C' + C1' where CE and C' represent forms of the carrier having their anion binding site exposed to the extra- or intracellular solvent, respectively (1, 29). t is assumed that the transmem- we have been able to accurately monitor anion transport across the red cell membrane using NMR. This represents the first study of chloride and phosphate fluxes in intact human erythrocytes using NMR. Sodium and potassium ion flux have been measured using NMR (18). We have chosen a simple, well-characterized cellular anion transport system to determine the utility of NMR as a technique to measure anion transport. NMR has a number of advantages. Transport can be monitored continuously using a single sample. The time required for each NMR measurement during the course of an experiment is limited only by the data acquisition time plus the delay time for T1 relaxation, providing adequate signal-to-noise can be obtained with one scan. n these experiments, one scan could be taken in "1 s for the resonances and ~ 0.1 s for the 35Cl resonances. The volume of sample required (-1-10 ml) is minimal and depends upon specific probe design within an NMR spectrometer. NMR experiments can be done over a wide temperature range and are limited only by the requirements of the biological sample. There is also considerable flexibility in other experimental conditions such as ionic strength, ph, concentration of cells, etc. NMR allows the monitoring of external chloride and extra- After 60 min, the experimentally determined Pi' concentrations increase significantly more than the theoretical curve, perhaps indicating HC05 efflux after C1- levels have reached equilibrium.

7 cellular and intracellular phosphate, as well as internal and external ph. nternal chloride was barely discernible above base-line under the spectrometer conditions used due to its binding to hemoglobin and the sensitivity of the quadrapolar 31P and 35Cl NMR of Anion Transport in Erythrocytes than 80% of the net Pi influx was due to transport of the nucleus to this binding. n contrast, both internal and external monoanionic form of Pi, indicating that the band 3 protein phosphate concentrations could be accurately measured. This preferentially transports H2PO; rather than HPOi-. method does not use unphysiological probes. The ability to The transport of only the monoanionic form of Pi is commeasure Pi and C1- allowed us to confirm the stoichiometry patible with kinetic data in the literature. Schnell et al. (7) of transport as one Pi for each C1- (1-5, 30). The initial transport rates andependence of transport on external phosphate also agreed with literature values (4, 7). No difference in transport was noted between fresh blood samples and outdated samples that were depleted of intracellular ATP. The 31P and 35Cl NMR methods described in this paper allow us to determine whether Donnan equilibrium is achieved for Pi and OH- without perturbing the erythrocyte system in any way. The values for Pi' and P? after the anion transport experiment has reached equilibrium (extrapolating to infinite time in Fig. 8) were -74 and -57 mm, respectively, giving a ratio for P,'/PiE of At infinite time, ph' and phe were and 6.65 giving a ratio of OH"/OH-E of The value for CE at infinite time was -70 mm. The Donnan equilibrium for C1- could not be tested, as C1' could not be detected in these experiments. Dalmark (19) determined a ratio of intracellular to extracellular anion concentration of about 1.25 for erythrocytes (nystatin-free) in 150 mm KC1, phe Schnell et al. (7) reported values of P;/P? = 1.36 and Cl'/CE = 1.24 for amphotericin B-treated erythrocytes in 60 mm PF, phe 6.5. Thus, the final distributions of Pi and OH- in our transport experiments appear to follow Donnan equilibrium and are in reasonable agreement with literature values. The complementarity of techniques is illustrated by the fact that Schnell et al. (7) used radioisotopic methods to determine [Pi]'/[PilE,[Cl]'/[Cl]", [PiE, and phe and assumed a Donnan equilibrium for [OH]'/[0HlE to calculate ph', [Pi]', and [H2PO;]'. By NMR, we could directly measure ph' and Pi' and confirm that Donnan equilibrium applies for [OH]'/ [OHE. While the human erythrocyte anion transport system is known to be involved in the movement of monoanions such as C1-, HCOF, F-, Br-, and -, dianions such as SO:- are also exchanged by the anion transport system (1). The transport of Pi was presumed to involve the dianionic form (1) or both the mono- and dianionic forms equally (7). More recently, the rate of Pi influx over a range of phe from 6.45 to 8.0 was found to obey simple Michaelis-Menten kinetics when substrate concentrations were plotted as H2P06, implying that the monoanionic form is the actively transported form of Pi (31). t was also found that only the monoanionic form of Pi appears to inhibit C1- exchange (32). This investigation of Pi transport across the erythrocyte membrane by 31P and 35Cl NMR points to the monoanionic form of Pi as the ionic form preferentially accepted by the anion transport system. The decrease in ph' and increase in phe during anion transport indicate the loss of the acidic H2PO; form (with its proton) from the extracellular solution and the accumulation of the H2PO; form and release of its proton within the erythrocyte. The preferential transport of the dianionic HP0:- form would result in ph changes opposite to those observed. The possibility that the increase in phe and decrease in ph' observed represent merely the passive collapse of the ph gradient is unlikely because permeability of the membrane to cations such as protons is extremely low (33), and the transport of OH- in carbonic anhydrase-inhibited systems is very slow (34). n our own control experiments, the presence of DDS prevented the anion transport and the ph changes observed, since the resonance areas and chemical shifts of the Pi' and PiE resonances did not change with time. The efflux of C1- is approximately equimolar with total Pi influx (Fig. 9). More studied the unidirectional flux of Pi as a function of ph and obtained a bell-shaped curve with optimum flux at ph 6.7 and ph values for half-maximal activation and inhibition of 6.0 and 7.4, respectively. Using a pk, for activation of 6.2, based on the rate of C1- transport as a function of ph (1,351, and a pk, of 6.8 for the inhibition of flux based on the pk, of Pi and the premise that only the monoanionic form of Pi is transported, a similar bell-shaped curve could be generated? This curve had a ph of optimum flux of 6.7, with ph values for half-maximum fluxes of 5.9 and 7.7. Thus, activation of Pi flux may involve the same group on the band 3 carrier protein as is involved in C1- flux. C1- flux does not undergo inactivation as the ph is increased from ph 7 to 11 (1). The inactivation of Pi influx as the ph is increased above ph 6.8 can be adequately accounted for by the simple titration of the active substrate HzPO; to inactive substrate HPOi-, or the presence of a titratable group on the carrier protein (pk., -6.8), the basic form of which does not catalyze Pi transport. Even SO*, which is generally accepted to be transported as a dianion (pk, -1.9), moves across the erythrocyte membrane in conjunction with a proton (6) and could be transported as a protein-stabilized HSO; monoanion. The experimental results of this paper are compatible with the model of transport of HzPO; or of cotransport of HPOi- and H+. The difference between these two models may be very subtle on a molecular level, in that the proton transported may be bound to substrate HzPO;, bound to a residue on the carrier protein or hydrogen-bonded to both. Acknowledgments-We would like to thank D. Lieberman for drawing fresh blood and preparing the blood samples for these studies and G. A. McQuaidfor maintaining the Bruker HXS-270 and Nicolet NTJOOWB NMR spectrometers. REFERENCES 1. Knauf, P. A. (1979) Curr. Top. Membr. Tramp. 12, Passow, H., Kampmann, L., Fasold, H., Jennings, M., and Lepke, S. (1980) Membr. Tramp. Erythrocytes 14, Macara,. G., and Cantley, L. C. (1983) Cell Membr. 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