cotransport expressed in Xenopus oocytes

Transcription

1 JBC Papers in Press. Published on April 27, 2004 as Manuscript M Voltage-dependent H+ buffering Voltage dependence of H+ buffering mediated by sodium-bicarbonate cotransport expressed in Xenopus oocytes Holger M. Becker & Joachim W. Deitmer Abteilung für Allgemeine Zoologie, FB Biologie, TU, Postfach 3049, D Kaiserslautern, Germany Running title:voltage-dependent H + buffering Key words: ph, intracellular sodium, membrane potential, membrane current, lactate transport, monocarboxylate transporter (MCT1) Correspondence to: Dr. J.W. Deitmer (at above address) Tel Fax deitmer@rhrk.uni-kl.de Acknowledgments. We thank Christian Lohr for his comments on an earlier version of this manuscript. This study has been supported by a stipend of the state Rheinland-Pfalz to H.B., 1 Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

2 and a grant from the Deutsche Forschungsgemeinschaft to J.W.D. (De 231/16-1, 16-2). Abbeviations: NBCe1 sodium-bicarbonate cotransporter (electrogenic, isoform 1) MCT1 monocarboxylate transporter (isoform 1) 2

3 Summary The electrogenic sodium bicarbonate cotransporter (NBCe1) is expressed in many epithelial cells and, in the brain, in glial cells. Little is known about the physiological significance of the NBCe1 for proton homeostasis and for other acid/base-coupled transporters in these cells. We have measured the voltage-dependent transport activity of a NBC from human kidney, type hknbce1, expressed in oocytes of the frog Xenopus laevis, by recording membrane current, and the changes of intracellular ph and sodium at different membrane potentials between 20 and 100 mv. The apparent intracellular buffer capacity was increased and became dependent on membrane voltage, when the NBCe1 was expressed; the measured buffer capacity increased by up to 7 mm/10 mv membrane depolarization. Lactate transport by the electroneutral monocarboxylate transporter (MCT1) became enhanced and dependent on membrane potential, when MCT1 was co-expressed with NBCe1 in oocytes. Our results indicate that the electrogenic NBCe1 renders the cell membrane potential an effective regulator of intracellular H+ buffering and acid/base-coupled metabolite transport. 3

4 Introduction Many processes in living cells are ph-dependent, in particular enzymatic reactions and acid/base-coupled transport; therefore cytosolic proton buffering and proton regulation are essential properties of all cells. In fact, no ion is as well buffered as protons; for each free cytosolic H +, 10 4 to 10 5 H + are bound. This is reflected in a nanomolar concentration of free H + in cells (~40-80 nm), although the total amount of protons is in the ten-millimolar range. Regulation of intracellular H+ is mainly due to Na+- and/or HCO 3 --dependent carriers, which can recover intracellular ph from an acidification or alkalinization. These transporters can be so effective in muffling H + that they can contribute apparent buffer capacity intraand extracellularly (1). The electrogenic sodium-bicarbonate cotransport NBCe1, which carries Na + with HCO - 3 across cell membranes with a stoichiometry of 1:2 or 1:3 (2,3,4), has been reported to be highly active in some cell types such as epithelial and glial cells (5,6). Due to the electrogenic nature of the NBCe1, the activity of this co-transporter is dependent on membrane potential. We have recently shown that the NBCe1, when heterologously expressed in Xenopus oocytes, increased the total apparent buffering power (7). In co-expression with another acid/basecoupled transporter, the monocarboxylate transporter (MCT1), which co-transports one monocarboxylate anion with one H+, the NBCe1 considerably increased the metabolite transport capacity of the MCT1. This functional transport cooperation was presumably due to 4

5 the NBCe1 counteracting the dissipation of the H + gradient following H + -lactate flux across the cell membrane. We have now studied, whether the apparent buffering follows the voltage-dependent activity of the NBCe1. Therefore, we have expressed the NBCe1 in Xenopus frog oocytes, and determined the total buffer capacity of the cytosol as dependent on the activity of the NBCe1, in comparison to H2O-injected control oocytes, and to oocytes, in which the MCT1 was expressed alone or together with the NBCe1. Our results suggest that, due to the NBCe1 activity, H + buffering becomes voltage-dependent, which also renders the transport activity of the electroneutral MCT1 membrane potential-dependent. Experimental procedures Oocytes and Injection The constructs and procedures have been described recently (7). The human kidney NBCe1 cdna (hknbce1) cloned in the oocyte expression vector pgh19 was kindly provided by Dr. Walter Boron (8), and subcloned into the vector pgemhejuel. Rat MCT1 cloned in the vector pgemhejuel, which contains the 5 and the 3 untranscribed regions of the Xenopus ß-globulin flanking the multiple cloning site, were used (9). Briefly, plasmid DNA was linearized with NotI and transcribed in vitro with T7 RNA-Polymerase in the presence of the 5

6 cap analogon m 7 G(5 )ppp(5 )G (mmessage mmachine, Ambion Inc., USA) to produce a capped RNA transcript. The crna was purified and stored at 70 C in DEPC-H2O. Integrity of the crna was checked by formaldehyde-gel electrophoresis. Xenopus laevis females were purchased from Horst Köhler, Hamburg, Germany. Oocytes were isolated and singularized by collagenase (Collagenase A, Roche, Mannheim, Germany) treatment in Ca 2+ -free oocyte Ringer solution at 26 o C for 1.5 h. The singularized oocytes were left overnight in Ca 2+ - containing oocyte Ringer solution to recover. The oocyte saline (OR2+) had the following composition (in mm): NaCl, 82.5; KCl, 2.5; CaCl 2, 1; MgCl 2, 1, Na 2 HPO 4 ; 1, HEPES, 5, titrated with NMDG to ph 7.0 or 7.4. The bicarbonate-containing saline contained (in mm): NaCl, 58.5; KCl, 2.5; CaCl 2, 1; MgCl 2, 1; Na 2 HPO 4, 1; NaHCO 3, 24, gassed with 5% CO 2 (ph 7.4) and HEPES, 5, to stabilize the ph. Experiments with MCT1-expressing oocytes were done in saline buffered to 7.0, containing 72.5 mm NaCl and 10 mm NaHCO3. Lactate (3 mm and 10 mm) was added as Na-L-lactate and exchanged for equimolar amounts of NaCl. In Na + -free saline, NaCl was exchanged by N-methyl-D-glucamine (NMDG) and titrated with HCl to ph 7.0; lactate was added to Na + -free salines as free acid. Oocytes of the stages V and VI were selected and injected either with 14 ng of NBCe1- crna, 13 ng of MCT1-cRNA, or with 7 ng of MCT1- and 14 ng of NBCe1-cRNA using glass micropipettes and a microinjection device (Nanoliter 2000, World Precision Instruments, Berlin, Germany). (Injection of 7 ng MCT1-cRNA and more gave similar expression of MCT1, but injection of more than 7 ng MCT1-cRNA sometimes interfered 6

7 with the expression of NBCe1.) Control oocytes were injected with an equivalent volume of DEPC-H 2 O. Intracellular ph and Na + measurements For measurement of intracellular ph (ph i ) and membrane potential double-, and for intracellular Na+ (Na+ i ) single-barrelled microelectrodes were used; the manufacture and application have been described in detail previously (10). Briefly, for double-barrelled microelectrodes two borosilicate glass capillaries of 1.0 and 1.5 mm in diameter were twisted together and pulled to a micropipette. The ion-selective barrel was silanized with a drop of 5% tri-n-butylchlorsilane in 99.9% pure carbon tetrachloride, backfilled into the tip. The micropipette was baked for 4.5 min at 450 C on a hot plate. H+-sensitive cocktail (Fluka 95291, Fluka, Buchs, Switzerland) was backfilled into the tip of the silanized ion-selective barrel and filled up with 0.1 M Na-citrate, ph 6.0. The reference barrel was filled with 3 M KCl. To increase the opening of the electrode-tip, it was bevelled with a jet stream of aluminium powder suspended in H 2 O. Calibration of the electrodes was carried out in oocyte salines with a ph of 7.0 and 6.4. The recording arrangement was the same as described previously (10;11). The central and the reference barrel of the electrodes were connected by chlorided silver wires to the headstages of an electrometer amplifier. Electrodes were accepted for use in the experiments, when their response exceeded 50 mv per unit change in ph; on average, they responded with 54 mv for unit change in ph. In the experimental 7

8 chamber they responded faster to a change in saline ph than the fastest reaction expected to occur in the oocyte cytosol. For single-barrelled Na + -sensitive microelectrodes, a 1.5 mm borosilicate glass capillary was silanized as described above and backfilled with Na + -sensitive cocktail, made of 10 wt% sodium ionophore VI (Fluka 71739), 89.5 wt% 2-nitrophenylether (o-npoe) and 0.5 wt% sodium-tetraphenylborate. The pipette was filled up with 100 mm NaCl+10 mm MOPS buffer, ph 7.0. Calibration of the electrodes was carried out in oocyte saline with Na + concentration of 5, and 84.5 mm; on average, the electrodes responded with 52 mv and 50 mv for a tenfold change in the Na+ concentration with NMDG or K+ as Na+ substitutes, respectively. As described previously (12) optimal ph changes were detected when the electrode was located near the inner surface of the plasma membrane. This was achieved by carefully rotating the oocyte with the impaled electrode. All experiments were carried out at room temperature (22-25 o C). Only oocytes with a membrane potential negative to -30 mv were used for experiments. Buffering power and proton fluxes The measurements of ph i were stored digitally using homemade PC software (13), and could be converted into intracellular H+ concentration, [H+] i. This should provide changes in the 8

9 [H+] i, which take into account the different ph baseline, as e.g. measured in HEPES- and CO 2 /HCO 3 --buffered salines. Amplitude and rate of change of the measured phi or of the [H + ]i were continuously recorded. The intrinsic buffering power, ßi, was calculated from the maximal instantaneous ph i changes recorded when changing from HEPES- to 5% CO 2 /24 mm or 10 mm HCO buffered saline (ph 7.4 or 7.0, respectively). The CO 2 -dependent buffering power, ß CO2, was calculated from the intracellular bicarbonate concentration in the oocytes (ß CO2 = 2.3 x [HCO - 3 ]i ), and the bicarbonate concentration was obtained from the Henderson-Hasselbalch equation. The total buffer capacity, ß T, was defined as the sum of ß i and ß CO2. Net acid/base flux rate, J A/B (mm/min), defined as the net transport of acid and/or base equivalents across the cell membrane, was calculated as the product of the rate of ph i change, ph i /t, and the buffering power ß. For calculation of the flux rates in HEPES-buffered saline, ph i /t was multiplied with the intrinsic buffer capacity ß i of the oocyte. For calculation of the flux rate in CO 2 /HCO 3 - -buffered saline, phi /t was multiplied with the total buffering capacity ß T. Voltage-Clamp Recording 9

10 A borosilicate glass capillary, 1.5 mm in diameter, was pulled to a micropipette and backfilled with 3 M KCl. The resistance of the electrode measured in oocyte saline was around 1 MΩ. For voltage-clamp, both electrodes were connected to the head-stages of an Axoclamp 2B amplifier (Axon Instruments, USA). The experimental bath was grounded with a chlorided silver wire coated by agar dissolved in oocyte saline. For calculation of significance in differences Student s t-test or, if possible, a paired t-test was used. In the figures shown a significance level of p < 0.05 is marked with *, p < 0.01 with ** and p < with ***. Results Transport activity of the NBCe1 We determined the transport activity of the NBCe1 expressed in Xenopus oocytes by simultaneously measuring the membrane current, the intracellular sodium activity, Na + i, and the intracellular proton activity, H+ i of voltage-clamped oocytes (Fig. 1 A). When a HEPESbuffered saline was replaced by a saline containing 5% CO2/24 mm HCO3 - at a constant ph of 7.4, an outward current was activated, which slowly declined, the Na + i and the H+ i increased. The rate and extent of these changes were dependent on the membrane potential. 10

11 At more depolarized potential (at 20 mv), the current and the amplitude and rate of Na+ i change were enhanced, while the amplitude and rate of H+ i change were reduced, as compared to more negative membrane potentials (-60 mv, -100 mv). In contrast, H 2 O- injected control oocytes acidified similarly at all membrane potentials (Fig. 1 B), and neither current nor Na+ i changes were observed in these control oocytes (not shown). The changes in membrane current and Na + i were taken to be due to NBCe1 transport activity, challenged by the intracellular acidification following diffusion of CO2 into the oocytes: The outward current and the rise in Na + i, indicate inwardly directed NBCe1, the co-transported HCO3 - counteracting the CO2-induced acidification. Consistent with the electrogenic nature of the NBCe1 (stoichiometry 1 Na + : 2 HCO - 3 ; 3), the inwardly directed transport was larger at more positive potentials, and smaller, as the membrane potential was held at more negative values. The relationships between the measured parameters and the membrane potential are shown in Fig. 2. A non-linear change in membrane current, H + i and Na+ i was observed in NBCe1- expressing oocytes. The CO 2 -induced rise in H+ i decreased from 42.4 ±4.8 nm at 100 mv to 17.6 ±1.7 nm at 20 mv (n=11; Fig. 2 A), while the rate of H + i rise decreased from 16.2 ±2.6 nm/min at 100 mv to 8.5 ±1.0 nm/min at 20 mv (n=11; Fig. 2 B). The values for both, 11

12 H+ i rise and rate of H+ i rise at 40 mv and 80 mv, respectively, were significantly different (p<0.01). The mean change in H+ i as induced by CO 2 in control oocytes (H 2 O-injected; Fig. 2 F) was between 74 and 88 nm at membrane potentials between 20 and 100 mv, showing no significant voltage dependence (n=6). The rise in Na + i following the addition of CO 2 increased from 3.1 ±0.3 mm at 100 mv to 5.0 ±0.7 mm at 20 mv (Fig. 2 C), and the rate of Na+ i rise increased from 0.43 ±0.07 mm/min at 100 mv to 0.63 ±0.09 mm/min at 20 mv (n=10; Fig. 2 D). While the values for the rise in Na + i at 40 mv and 80 mv were significantly different (p<0.05), the values for the rate of Na+ i rise were not significantly different at these potentials, respectively. The membrane current challenged by CO 2 was highly voltage-dependent; it increased from 278 ±22 na at 100 mv to 547 ±49 na at 20 mv, and was significantly different at 40 mv and 80 mv (p<0.001; n=11; Fig. 2 E). Consistent with electrogenic NBCe1 activity, the membrane current and the Na + i rise increased, and the rise in H+ i and the rate of H+ i rise decreased, with membrane depolarisation. Taken the values for membrane current, and the rate of H + i changes, the activity of the NBCe1 increased by a factor of 2 and 1.9 between 100 and 20 mv, respectively. Membrane potential-dependent buffer capacity 12

13 From the intracellular ph and HCO 3 - changes induced by CO 2 (see Fig. 1), the intrinsic and CO 2 -dependent buffer capacity was calculated and summed up to the total buffer capacity (ß T, see Methods). The plot of ß T against the membrane potential for NBCe1-expressing oocytes showed a marked voltage dependence; ß T increased from 63 ±6 mm to 119 ±14 mm, when the membrane potential changed from 100 mv to 20 mv (n=11; Fig. 3 A). The ß T of H 2 O-injected control oocytes, in contrast, remained unchanged near 40 mm for all membrane voltages, of which 22 mm was due to HCO 3 - dependent buffer capacity at an intracellular HCO 3 - concentration of 9.5 ±0.4 mm in saline buffered with 5% CO 2 /24 mm HCO 3 - at ph 7.4. When the values for ß T of NBCe1-expressing oocytes and control oocytes were subtracted, the buffering contributed by the NBCe1 was obtained (ß NBCe1 ; Fig. 3 A), increasing from 24 mm at -100 mv to 79 mm at -20 mv. A voltage dependence of nearly 0.7 mm/mv was indicated for ß NBCe1 by the regression line through the points, suggesting that the NBCe1 renders the buffer capacity voltage-dependent, and adds 7 mm apparent buffer capacity for a 10 mv membrane depolarization. The total buffer capacity of NBCe1- expressing oocytes displayed a similar voltage dependence as the CO 2 -induced membrane current and Na+ i rise, which are indicative for NBCe1 activity; the slope being 0.59 for the buffer capacity, 0.56 for the current, and 0.60 for the change in Na + i (Fig. 3 B). 13

14 Transport activity by monocarboxylate transporter (MCT1) co-expressed with NBCe1 In order to evaluate the functional significance of the voltage dependence of buffering imposed by the NBCe1, we co-expressed a second acid/base transporter together with the NBCe1. The monocarboxylate transporter 1 (MCT1) cotransports an organic anion, such as lactate or pyruvate, with one proton in an electroneutral 1:1 mode (14). If buffering by NBCe1 affected the transport activity of the MCT1, lactate/pyruvate transport might be expected to become voltage-dependent. We measured membrane current and H + i changes in voltageclamped oocytes expressing NBCe1+MCT1 in the absence and presence of the substrates of NBCe1, Na + and HCO3 - (Fig. 4 A). Lactate, as a substrate for MCT1, was applied in two concentrations (3 and 10 mm; the Km of MCT1 in oocytes had been determined to be 3-5 mm; (12)) in saline buffered to 7.0 with either HEPES or 5% CO 2 /10 mm HCO - 3 (the lower ph value of the saline was chosen to ensure prominent MCT1 activity, which depends on external H+ as co-substrate). The intracellular acidifications during lactate application, indicative for the activity of MCT1, were neither Na + - nor HCO3 - -dependent, as they were unchanged in nominally CO 2 / HCO 3 - -free, HEPES-buffered, as well as in Na + -free saline. Upon addition of 5% CO 2 /10 mm HCO 3 -, there was a small intracellular acidification and a prominent outward current, indicative for the diffusion of CO 2 into the cell and activation of NBCe1 in the inwardly directed mode, respectively. In the presence of CO2/HCO3 -, lactate 14

15 now produced smaller intracellular acidifications, but large outward currents, due to NBCe1 activity (7). Following removal of external Na +, the cytosol acidified by more than 100 nm H +, accompanied by a transient inward current, indicative for the reversed, outwardly directed, transport mode of the NBCe1. The lactate-induced acidifications became larger as compared to those in the presence of external Na +, while the currents were suppressed. The results show that lactate transport via MCT1 is accompanied by a larger change in H+ i in the absence of Na + in NBCe1+MCT1-expressing oocytes by a factor of 3.5 and 3.0 for 3 and 10 mm lactate, respectively, but not in oocytes expressing MCT1 alone (Fig. 4 B). The membrane current induced by the addition and removal of 3 and 10 mm lactate was reduced to 28% and 23% for the outward current (inwardly directed mode of NBCe1), and to 13% and 9% for the inward current (outwardly directed mode of NBCe1). This Na+-dependence in CO 2 /HCO 3 - -buffered saline indicates that the current is largely due to NBCe1 activity (Fig. 4 C; see also (7)). Lactate produced small membrane currents in the nominal absence of CO2/HCO3 -, which were suppressed after Na+ removal, suggesting that these currents were due to NBCe1 activity present in the nominal absence of CO 2 /HCO 3 -, which still contain a few hundred µm HCO 3 - (see also (7)). The rise of H+ i induced by lactate due to H+-lactate co-transport via MCT1 was presumably reduced due to the intracellular acidification, and due to the increased 15

16 buffering capacity in the presence of CO 2 /HCO 3 -. The H+ i rise as evoked by 3 mm lactate was compared when holding the oocyte membrane potential at 40 mv and at 80 mv for NBCe1+MCT1-expressing oocytes, and for oocytes expressing MCT1 alone (Fig. 5 A). While the rise in H + was similar for MCT1-expressing oocytes at the two voltages, it was significantly smaller at 80 mv for the NBCe1+MCT1- expressing oocytes. Similarly, the HCO - 3 concentration, as calculated from the Henderson- Hasselbalch equation, was also significantly larger in NBCe1+MCT1-expressing oocytes at 40 mv, being 15 ±0.7 mm, than at 80 mv, being 9.9 ±0.5 mm (p<0.001; n=7-9; Fig. 5 B), reflecting the voltage dependence of the NBCe1 activity. In contrast, similar HCO 3 - concentrations were determined in MCT1-expressing oocytes at 40 and 80 mv, being 8.2 ±0.7 mm and 8.6 ±0.2 mm (n=5, 6), respectively. When the total buffer capacity was determined for the three different types of oocytes, only the buffer capacity of oocytes (co-)expressing the NBCe1 displayed a voltage-dependence, which was 42% larger at 40 mv than at 80 mv in NBCe1+MCT1-expressing oocytes (Fig. 5 C). This corresponds to the increase of ß T by 35% in oocytes expressing NBCe1 alone at these potentials (see Fig. 3 A). Oocytes expressing MCT1 alone or H 2 O-injected control oocytes had a buffer capacity of between 27 and 36 mm (Fig. 5 C), and were similar at 40 and 80 mv, respectively. Using the total buffer capacity to calculate the rate of acid/base flux, JA/B, as induced by 3 mm lactate, revealed that the transport activity of the MCT1 was 16

17 more than doubled at -40 mv when the NBCe1 was co-expressed (Fig. 5 D), rising from 0.8 ±0.1 mm/min in MCT1-expressing oocytes to 1.9 ±0.2 mm/min in NBCe1+MCT1-expressing oocytes. In contrast, lactate transport was significantly smaller in NBCe1+MCT1-expressing oocytes at -80 mv, being 0.3 ±0.1 mm/min as compared to 0.8 ±0.1 mm in oocytes expressing MCT1 alone. At -80 mv, where the inwardly directed NBCe1 is reversed by the steep electrical gradient, the NBCe1 appears to counteract uptake of lactate, and would rather support efflux of lactate via MCT1. However, the values for the flux rate as determined in NBCe1+MCT1-expressing oocytes may be underestimated due to some conversion of HCO - 3 transported into the cell by the NBCe1 to CO 2, which could leave the cell silently, and would hence be missed by the ph sensor (see also Discussion). Discussion The results of the present study clearly show that expression of NBCe1 adds apparent buffer capacity to the cytosol, which is voltage-dependent, presumably due to electrogenic nature of the NBCe1. The NBCe1 co-transports 1 Na + and 2 HCO3 - (8, 3, 15). In oocytes held at -40 mv, the reversal potential of the NBCe1 was -56 mv (7). Thus, at a potential more positive 17

18 to the reversal potential, the NBCe1 transports Na+ and HCO 3 - into the cell, and hence adds apparent buffer capacity, while at a potential more negative to the reversal potential, the NBCe1 extrudes Na + and HCO 3 -, and removes base equivalents, and hence cytosolic buffer capacity. This is concluded from comparing the NBCe1 activity and buffer capacity at -40 mv and -80 mv. In most physiological experiments using intracellular ph sensors, the total cell capacity for muffling H + is measured (1), which includes the instantaneous, chemical, H + buffering, due to binding and release of H+ by intracellular molecules, and the dampening of ph changes contributed by fast acid/base transport across the cell membrane. Among other factors, the determination of the buffer capacity therefore depends on the speed and sensitivity of the method sensing H+ i, and on the capacity and velocity of the acid/base transport. The voltage dependence of the total buffer capacity indicated that a 10 mv depolarisation activated the NBCe1 to increase the buffer capacity by up to 7 mm. The relationship between buffer capacity and membrane potential was linear over the potential range between 100 mv and 20 mv. The HCO 3 - concentration in NBCe1+MCT1-expressing oocytes was 52% larger at 40 mv than at 80 mv, which also results from the activity of the NBCe1. If the CO 2 - dependent buffer capacity, as derived from the HCO3 - concentration (see Methods), is compared with the measured total buffer capacity, a discrepancy becomes evident: while the total buffer capacity increased by about 27 mm between 80 and 40 mv, the increase in 18

19 HCO 3 - concentration by 5 mm indicates an increase of 11.5 mm ß CO2. Since it must be assumed that apparent buffer capacity contributed by NBCe1 activity is CO 2 -dependent buffer capacity, there is a deficit of 15.5 mm buffer capacity (compare Figs. 5 B and C), or, 57% of the calculated buffer capacity is not reflected by a change in intracellular HCO 3 - concentration. This deficit can be explained by the fact that we can only measure net H + changes with limited velocity, during addition of CO 2 or lactate, hence miss fluxes of H+ and HCO3 -, which are rapidly converted into CO2, and as such return silently across the cell membrane in the opposite direction. Hence, some short-circuit of acid/base-coupled fluxes occurs within the open CO 2 /HCO 3 - buffer system, which would lead to an underestimation of the buffer capacity and acid/base-coupled fluxes in our measurements. The electroneutral lactate transport via MCT1 became membrane potential-dependent, when the MCT1 was co-expressed with NBCe1. We have shown in a previous study that this coexpression facilitates lactate transport, presumably due to the increased buffer capacity in the presence of NBCe1 expression (7). Our present results support this conclusion, and show that lactate transport was enhanced at a more depolarized membrane, when NBCe1 adds apparent buffer capacity, and was reduced at a more negative membrane potential, where the activity of the NBCe1 is directed opposite to that of the MCT1 during lactate uptake. It is the NBCe1 activity that changes as a function of membrane voltage, while the MCT1 activity follows that of NBCe1; we have no evidence that MCT1 itself becomes electrogenic following co- 19

20 expression with NBCe1. Nevertheless, in a cell expressing NBCe1, the membrane potential becomes an important regulator of acid/base-coupled metabolite transport. In epithelial cells and glial cells, where the NBCe1 is expressed (5, 6), an electroneutral acid/base-coupled transport, such as the MCT, may become membrane potential-dependent due to the interaction via the acid/base gradient and the buffer capacity, which is rendered voltage-dependent by the electrogenic NBCe1. This might also lead to some confusion, when trying to determine the stoichiometry of transporters, which interact with electrogenic transporters in a way as described here; the apparent voltage dependence would suggest an electrogenic nature of even electoneutral transporters. There are several types of cells, in which functional expression of NBCe1 and MCT has been reported, including epithelial cells, muscle and glial cells (14, 16). Considering co-expression of NBCe1 and MCT1, our present results suggest that factors activating the outward mode of NBCe1, which results in an intracellular acidification, would stimulate efflux of lactate and H + via MCT1. Such factors could be a membrane hyperpolarization, due to activation of a K + conductance or electrogenic transporters, such as e.g. the Na + -K + -ATPase, or an increase in the intracellular Na + concentration due to Na + -dependent uptake of neurotransmitters, such as glutamate or GABA (17, 18). Activity of glutamatergic neurons, which could initiate these processes, might thus lead to a net efflux of Na+ and lactate from glial cells (19), linking the uptake of glutamate by glial cells with the transfer of energetic compounds, such as lactate or pyruvate from glial cells to neurons (20, 21). 20

21 References 1. Thomas, R. C., Coles, J. A., and Deitmer, J. W. (1991) Nature 350, Boron, W. F., and Boulpaep, E. L. (1983) J. Gen. Physiol. 81, Heyer, M., Müller-Berger, S. Romero, M. F., Boron, W. F., and Frömter, E. (1999) Pflügers Arch. Eur. J. Physiol. 438, Gross, E., Hawkins, K. Abuladze, N., Pushkin, A., Cotton, C. U., Hopfer, U., and Kurz, I. (2001) J. Physiol. 531, Romero, M. F., and Boron, W. F. (1999) Annu. Rev. Physiol. 61, Chesler, M. (2003) Physiol. Rev. 83, Becker, H., Bröer, S., and Deitmer, J.W. (2004) Biophys. J. 86, Choi, I, Romero, M.F., Khandoudi, N., Bril, A., and Boron, W. E. (1999) Am. J. Physiol. 276, C576-C Bröer, S., Rahman, B., Pellegri, G., Pellerin, L., Martin, J.-L., Verelysdonk, S., Hamprecht, B., and Magistretti, P. J. (1997) J. Biol. Chem. 272, Deitmer, J.W. (1991) J. Gen. Physiol. 98, Munsch, T., and Deitmer, J. W. (1994) J. Physiol. (Lond) 474, Bröer, S., Schneider, H.-P., Bröer, A., Rahman, B., Hamprecht, B., and Deitmer, J. W. (1998) Biochem. J. 333, Deitmer, J. W., and Schneider, H.-P. (1995) J. Physiol. 485, Halestrap, A. P., and Price, N. T. (1999) Biochem. J. 343,

22 15. Sciortino, C. M., and Romero, M. F. (1999) Am. J. Physiol. 277, F611-F Soleimani, M., and Burnham, C. E. (2001) J. Membr. Biol. 183, Schousboe, A. (2000) Neurochem. Res. 25, Amara, S. G., and Fontana, A. C. (2002) Neurochem. Int. 41, Deitmer, J.W. (2002) J. Neurochem. 80, Magistretti, P. J., Pellerin, L., Rothman, D. L., and Shulman, R. G. (1999) Science 283, Deitmer, J.W. (2000) BioEssays 22,

23 Figure legends Fig. 1: (A) Intracellular recordings of membrane current (I m, upper trace), of sodium concentration (Na + i, middle traces), and of proton concentration (H + i, lower traces) in an oocyte expressing the sodium-bicarbonate cotransporter hknbce1 (15 ng crna injected) at three different membrane potentials, when 5% CO2/24 mm HCO3 - (at ph 7.4) was applied, replacing a HEPES-buffered, nominal CO 2 /HCO 3 - -free saline, as compared to the changes in H + i in a H 2O-injected control oocyte (B). Fig. 2: Changes in intracellular proton ( H + i, A, F), in the rate of intracellular proton rise ( H+ i /t, B), in membrane current ( I m, E), in intracellular sodium ( Na+ i, C) and in the rate of intracellular sodium rise ( Na + i, D), upon adding 5% CO2/24 mm HCO3 - (ph 7.4) in oocytes expressing NBCe1 (A-E), and changes in H + i in a H 2O-injected control oocyte (F). A significance test was included for the values at 40 and 80 mv in each graph, respectively. Fig. 3: (A) Voltage dependence of the total buffer capacity (ßT) of oocytes expressing NBCe1 23

24 (filled circles) and in H 2 O-injected oocytes (open circles), and the difference of the buffer capacity of NBCe1-expressing and H2O-injected control oocytes (filled diamonds), indicating the voltage-dependent apparent buffer capacity contributed by NBCe1 expression ß NBC of 0.7 mm/mv membrane depolarization. (B) Comparison of the voltage dependence of the buffer capacity ß NBC (filled circles) with the voltage dependence of the change in membrane current ( Im; open diamonds) and change in intracellular sodium ( Na + i; open squares) upon addition of 5% CO 2 /24 mm HCO - 3. Fig. 4: (A) Membrane current (Im, upper trace) and intracellular H + i (lower trace) in an oocyte co-expressing NBCe1 and MCT1, during application of 3 and 10 mm lactate in HEPES, nominally CO2/HCO3 - -free saline (left part), and in saline containing 5% CO2/10 mm HCO - 3 (ph 7.0; right part), respectively, both in the presence and absence of external Na+. (B) Summary of changes in intracellular proton concentration ( H+ i ) induced by 3 and 10 mm lactate in CO 2 / HCO 3 --buffered saline in the presence and absence of external Na+ in oocytes expressing either NBCe1+MCT1 or MCT1 alone. (C) Summary of lactate-induced membrane current in NBCe1+MCT1-expressing oocytes in CO 2 / HCO 3 --buffered saline in the presence (84.5 mm) and absence of external Na +. 24

25 Fig. 5: The changes in intracellular H+, H+ i, as induced by 3 mm lactate (A), the HCO 3 - concentration (B) in NBCe1+MCT1-expressing oocytes, and in oocytes expressing MCT1 alone at 40 mv and 80 mv. (C) The total cytosolic buffer capacity was compared for NBCe1+MCT1-expressing oocytes, for MCT1-expressing oocytes, and for H 2 O-injected control oocytes at 40 and 80 mv. (D) The rate of acid/base flux, J A/B, as induced by 3 mm lactate in NBCe1+MCT1- and in MCT1-expressing oocytes at 40 mv and 80 mv. The HCO 3 - concentration (B), the buffer capacity (C) and the acid/base flux rate, indicative for lactate transport by MCT1 (D), were significantly larger at 40 mv only in NBCe1+MCT1- expressing oocytes. 25

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29 Fig. 4 + m A I m 0.5 µa 0Na 0Na H nm 10 mm Lac - 10 min 3 mm Lac - - 5% CO 2/ 10 mm HCO3 B H (nm) % CO 2 / 10 mm HCO3 MCT1 + NBC MCT1 ** ** 3 mm lactate 10 mm lactate C I (na) n=6-3mm lactate +3mM lactate - 5% CO 2 / 10 mm HCO3-10mM lactate +10mM lactate + Na -free * * ** * Na -Na +Na -Na mm lactate 10 mm lactate

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31 Voltage dependence of H+ buffering mediated by sodium-bicarbonate cotransport expressed in xenopus oocytes Holger M. Becker and Joachim W. Deitmer J. Biol. Chem. published online April 27, 2004 Access the most updated version of this article at doi: /jbc.M Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts