Cation and voltage dependence of rat kidney electrogenic Na+-HCOminus 3 cotransporter, rkNBC, expressed in oocytes

Christopher M. Sciortino1 and Michael F. Romero1,2

Departments of 1 Physiology and Biophysics and 2  Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Recently, we reported the cloning and expression of the rat renal electrogenic Na+-HCO-3 cotransporter (rkNBC) in Xenopus oocytes [M. F. Romero, P. Fong, U. V. Berger, M. A. Hediger, and W. F. Boron. Am. J. Physiol. 274 (Renal Physiol. 43): F425-F432, 1998]. Thus far, all NBC cDNAs are at least 95% homologous. Additionally, when expressed in oocytes the NBCs are 1) electrogenic, 2) Na+ dependent, 3) HCO-3 dependent, and 4) inhibited by stilbenes such as DIDS. The apparent HCO-3:Na+ coupling ratio ranges from 3:1 in kidney to 2:1 in pancreas and brain to 1:1 in the heart. This study investigates the cation and voltage dependence of rkNBC expressed in Xenopus oocytes to better understand NBC's apparent tissue-specific physiology. Using two-electrode voltage clamp, we studied the cation specificity, Na+ dependence, and the current-voltage (I-V) profile of rkNBC. These experiments indicate that K+ and choline do not stimulate HCO-3-sensitive currents via rkNBC, and Li+ elicits only 3 ± 2% of the total Na+ current. The Na+ dose response studies show that the apparent affinity of rkNBC for extracellular Na+ (~30 mM [Na+]o) is voltage and HCO-3 independent, whereas the rkNBC I-V relationship is Na+ dependent. At [Na+]o vmax (96 mM), the I-V response is approximately linear; both inward and outward Na+-HCO-3 cotransport are observed. In contrast, only outward cotransport occurs at low [Na+]o (<1 mM [Na+]o). All rkNBC currents are inhibited by extracellular application of DIDS, independent of voltage and [Na+]o. Using ion-selective microelectrodes, we monitored intracellular pH and Na+ activity. We then calculated intracellular [HCO-3] and, with the observed reversal potentials, calculated the stoichiometry of rkNBC over a range of [Na+]o values from 10 to 96 mM at 10 and 33 mM [HCO-3]o. rkNBC stoichiometry is 2 HCO-3:1 Na+ over this entire Na+ range at both HCO-3 concentrations. Our results indicate that rkNBC is highly selective for Na+, with transport direction and magnitude sensitive to [Na+]o as well as membrane potential. Since the rkNBC protein alone in oocytes exhibits a stoichiometry of less than the 3 HCO-3:1 Na+ thought necessary for HCO-3 reabsorption by the renal proximal tubule, a control mechanism or signal that alters its in vivo function is hypothesized.

sodium/bicarbonate cotransport; NBC; Xenopus oocyte expression; intracellular pH; sodium transport; bicarbonate transport; kinetics; voltage clamp


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE ELECTROGENIC Na+-HCO-3 cotransporter was first described in the renal proximal tubule (8) and later cloned from the salamander kidney by functional expression using Xenopus oocytes (34). By homology, several Na+-HCO-3 cotransporter (NBC) cDNA isoforms have been cloned from different mammalian tissues including rat kidney (33), human kidney (9), human heart (13), human pancreas (2, 29), and brain (5). These proteins are either 1,035 (kidney) or 1,079 (other organs) amino acids in length and are at least 95% identical to one another. Variation in sequence occurs predominantly in the NH2-terminal 45-85 amino acids. Expression studies in oocytes (13, 33, 34) show that the basic functions of these NBC isoforms are similar: they are 1) electrogenic, 2) Na+ dependent, 3) HCO-3 dependent, and 4) inhibited by stilbenes such as DIDS.

Although Na+-HCO-3 cotransport systems are physiologically implicated in many tissues, these cotransporters appear to function differently depending on the tissue. NBC is located at the basolateral membrane of renal proximal tubule cells (37) and electrogenically moves Na+/HCO-3 out of the cell into the blood. This mechanism is responsible for 80-90% of HCO-3 reabsorbed in the kidney. However, in pancreatic ductal cells, Na+/HCO-3 influx is thought to occur (19). In cultured hippocampal glial cells (6, 36) and in the eye (21, 23), both HCO-3 influx and efflux have been measured and are electrogenic (6, 26, 27). Yet recovery from an acid load elicited by increasing heart rate in cat papillary muscle is also attributed to electrogenic Na+/HCO-3 influx (3, 10, 11), whereas experiments in guinea pig ventricular myocytes indicate that the Na+-HCO-3 cotransport is electroneutral (24).

Electrogenic NBC transport moves a net negative charge in the direction of transport (i.e., a ratio of HCO-3:Na+ > 1:1). Both electrochemical gradients and cell membrane potential dictate the direction of ion flux. The HCO-3:Na+ coupling ratio has been used as a predictor of transport direction. The larger this ratio, the more effective the cell potential acts as a driving force to move Na+/HCO-3 out of the cell against the Na+ gradient. Studies of the electrogenic Na+/HCO-3 cotransporter in renal tubules (48), proximal tubule cell lines (16), and vesicles made from rabbit basolateral membranes (39) predict a HCO-3:Na+ coupling of 3:1. It is thought that this ratio is necessary to move HCO-3 out of the proximal tubule cell across the basolateral membrane. However, the Na+-HCO-3 cotransporter in the pancreas and brain is thought to have a coupling ratio of 2:1 (6, 15, 19) and the heart may either be 2:1 (11) or 1:1 (14, 24).

Although different physiological characteristics have been attributed to these different tissues, the NBC clones are greater than 95% homologous. Thus the purpose of the present study was to elucidate NBC transport characteristics to understand how and if NBC can fulfill all of these roles. Specifically, we expressed rkNBC in Xenopus oocytes to 1) determine the monovalent cation specificity, 2) define the voltage dependence, and 3) determine apparent affinity for extracellular Na+ ([Na+]o). For rkNBC specificity, all ionic and current changes attributed to rkNBC are inhibitable by the stilbene, DIDS. From measurements of the [Na+]o dose response of rkNBC-stimulated HCO-3 current, and with measurements of intracellular pH (pHi) and sodium activity (aNai), we directly calculate the stoichiometry of rkNBC-mediated Na+-HCO-3 cotransport over a range of [Na+]o levels. Interestingly, our studies indicate that the stoichiometry of rkNBC is 2 HCO-3:1 Na+, rather than 3:1 as previously reported for in vitro tissue studies, and is independent of the Na+ gradient and [HCO-3]o.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Oocyte Experiments

Oocyte isolation and injection. Xenopus laevis were purchased from Xenopus Express (Beverly Hills, FL). Oocytes were removed and collagenase dissociated as previously described (32, 33). To optimize rkNBC expression, we used a rkNBC-cDNA construct in the Xenopus expression vector pTLN2 (33). Capped cRNA was synthesized using a linearized cDNA template and the SP6 mMessage mMachine kit (Ambion, Austin, TX). Oocytes were injected with 50 nl of rkNBC cRNA (0.2 µg/µl) or water and incubated at 18°C in OR3 media (32). Oocytes were studied 3-10 days after injection. Each experimental procedure was studied on at least two batches of oocytes from different Xenopus to account for possible biologic variations between animals.

Electrophysiology

Solutions. Experimental solutions are detailed in Table 1.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Experimental solutions

Two-electrode voltage clamp. Oocyte currents were recorded with OC-720C voltage clamp (Warner Instruments, Hamden, CT). Electrodes were fashioned from borosilicate glass using a model P-97 puller (Sutter, Novato, CA). Electrode tips were filled with 1% agarose/3 M KCl and backfilled with 3 M KCl. Current and voltage electrodes had resistances of 0.5-1 MOmega . Current signals were filtered with an eight-pole Bessel filter (-3-dB cutoff, frequency of 2-5 kHz) and digitized at 10 kHz. Current and voltage signals were acquired via an EPC-16 I/O interface using Pulse software, and data were analyzed using the PulseFit program (HEKA). Oocytes were clamped at a holding potential (Vh) of -60 mV; and current was constantly monitored and recorded at 1 Hz. Initial experiments determined that currents elicited by voltage steps saturated in 50 ms to steady-state levels. Thus current-voltage (I-V) protocols consisted of 70-ms steps from Vh to potentials from -160 mV to 60 mV in 20-mV steps. The mean steady-state current is plotted against voltage (Fig. 2, A-E).

Ion-selective microelectrode. Ion-selective microelectrodes were used to monitor pHi and aNai of rkNBC and water-injected oocytes as previously described (33). Intracellular ion activity was measured as the difference between the ion-selective electrode (pH or Na+) and a KCl voltage electrode impaled into the oocyte; membrane potential (Vm) was measured as the potential difference between the KCl microelectrode and an extracellular calomel (33, 34). Briefly, ion-selective microelectrodes were fabricated using filamented borosilicate glass pulled to 0.5-µm tips and silanized at 210°C with bis-(dimethylamino)dimethylsilane (Fluka, Ronkonkoma, NY), and the shanks were coated with Sylgard (Dow Corning, Midland, MI). Micropipettes were cooled under vacuum, and the tips were filled with either Na+ ionophore cocktail A or H+ ionophore I-cocktail B ion-selective resin (Fluka Chemical). Na+ electrodes were backfilled with 150 mM NaCl. H+ electrodes were backfilled with (in mM) 40 KH2PO4, 23 NaOH, and 15 NaCl, pH 7.0. pH electrodes were calibrated using pH 6.0 and 8.0 (traceable National Bureau Standards; Fisher Scientific, Pittsburgh, PA) followed by point calibration in ND96 (pH 7.50, solution 1, Table 1). Na+ electrodes were calibrated with 10 mM and 100 mM NaCl, and the specificity was checked using 100 mM KCl, followed by point calibration in ND96 (96 mM Na+). Na+ electrodes had a selectivity1 of 52 ± 5 for Na+ over K+ (calculated as described by Abdulnour-Nakhoul and coworkers Ref. 1). Both types of ion-selective electrodes had slopes of at least -56 mV/decade change.

Buffering power was calculated as previously reported (35). Briefly, the total apparent buffering power (beta T, see Table 2) is defined as the change in [HCO-3] before and after application of CO2/HCO-3 (once steady-state is reached) divided by the change in pHi elicited from the same solution changes, i.e., beta T = Delta [HCO-3]steady-state/Delta pHi.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Intracellular ion activity measurements

Cation selectivity and apparent extracellular Na+ vmax. The cation selectivity of rkNBC-mediated cotransport and the apparent maximal extracellular Na+-stimulated HCO-3 current (vmax) were measured using the bath solution protocol shown in Fig. 4A. The ability of K+, Li+, and choline to stimulate HCO-3-dependent current via rkNBC is tested by measuring cation current responses ± HCO-3. Briefly, an oocyte was perfused with a test cation (solution 3, 4, or 5, Table 1) non-HCO-3 solution and an I-V relation was recorded. The bath solution was switched to the respective HCO-3/test cation solution (1.5% CO2/10 mM HCO-3/pH 7.5, solutions 8-12 and 5, Table 1), i.e., Li+/ND96 (solution 3, Table 1) to Li+/1.5% CO2/HCO-3 (solution 10, Table 1). An I-V relation was measured at the peak HCO-3-stimulated current. The HCO-3-dependent response was calculated as the difference between the two I-V relationships (Fig. 4). The [Na+]o at which rkNBC-mediated HCO-3 current saturates (i.e., attains an apparent current vmax) was determined using the solution protocol in Fig. 4A. The HCO-3-mediated I-V responses for [Na+]o ranging from 84 to 120 mM were tested. Solution osmolalities were matched with choline (solutions 2, 6, 9, 13, Table 1).

[Na+]o concentration dependence. Figure 1A shows a schematic of the experimental protocol used to study the extracellular Na+ dependence of rkNBC. Our protocol was designed to monitor Na+-induced currents while maintaining pHi and aNai at steady state. An oocyte was put into an ~100-µl perfusion chamber and perfused at 8-10 ml/min. Dye exchange experiments showed that the entire chamber volume was exchanged in <1 s. Therefore, the initial current response of rkNBC cotransport can be measured without cell rundown due to long mixing times. The Vh (-60 mV) current of rkNBC-expressing oocytes was recorded for the duration of the experiment at 1 Hz. A baseline non-HCO-3 I-V relation was recorded once current stabilized after initial electrode impalements. The bath solution was changed to 1.5% CO2/10 mM HCO-3/96 mM Na+ (solution 8, Table 1) or 5% CO2/33 mM HCO-3/96 mM Na+ (solution 14, Table 1) for 10 min to allow a steady-state current and pHi to be reached (compare Figs. 1B and 3B). Unless otherwise specified, Na+ replacement was with choline. The bath solution was changed to 0 Na+ (solution 12, Table 1) for 24 s, pulsed for 8 s to a test Na+ (in mM 0, 1, 10, 24, 36, 48, 72, 84, 96), then to 96 mM Na+ for 24 s. An I-V relation was recorded at the peak current induced by each Na+ change. This quick pulse protocol maintains a steady-state pHi and aNai, and baseline 0 Na+ current, allowing three to six randomly ordered test measurements of [Na+]o per oocyte to be taken and ensures that test Na+ responses are measured from the same "fully outward" transport state of rkNBC (Figs. 1B and 2B).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1.   Current-voltage (I-V) response protocol. A: model of Na+ dose protocol. Rat renal electrogenic Na+-HCO-3 cotransporter (rkNBC) cRNA or water-injected (control) oocytes were voltage clamped to a holding potential (Vh) of -60 mV in ND96 (solution 1, Table 1). Bath solution was then switched to 1.5% CO2/10 mM HCO-3 or 5% CO2/33 mM HCO-3 at pH 7.5/96 mM Na+ (solutions 8 and 14, respectively, Table 1) for 10 min. Extracellular Na+ ([Na+]o) was then removed (0 Na+, choline replacement solutions 9 and 15, respectively, Table 1) for 24 s, followed by a 12-s perfusion in test Na+ (in mM: 1, 10, 24, 36, 48, 72, 84, 96). An I-V response was recorded at the peak current induced by the Na+ solution (x). Na+ was reapplied for 24 s. The 0-test-96 mM Na+ pulse sequence was used to maintain a stable baseline current and was repeated for 3-6 test Na+ concentrations per oocyte. B: Vh recorded during a Na+ dose response experiment. Vh current response of an rkNBC-expressing oocyte resulting from the solution protocol in A was monitored at 1 Hz. A steady-state current was reached in 8-10 min after 1.5% CO2/10 mM HCO-3 administration. Test pulses are to 96, 48, and 24 mM Na+. Peak current stimulated by these test pulses was Na+ concentration dependent.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 2.   I-V response of rkNBC. A: steady-state current sweeps ± [Na+]o ± DIDS. Unsubtracted current sweep data recorded from of a rkNBC-expressing oocyte are shown in presence of extracellular 1.5% CO2/10 mM HCO-3 with either 96 mM Na+ (black squares, solution 8) or Na+ replaced with choline (green diamonds, solution 12). Addition of 200 µM DIDS to the CO2/HCO-3-containing bath solution irreversibly inhibited current in presence (red square) or absence (red diamond) of extracellular Na+. B: rkNBC I-V relationship ± [Na+]o ± DIDS. ND96 (non-HCO-3) I-V relationship is subtracted from I-V relations measured from current sweep data in A to yield the HCO-3-stimulated current. Current response in presence of extracellular Na+ (black squares) is approximately linear, whereas in absence of Na+ (green diamonds) only negative currents are observed. After a 5-min exposure to 200 µM DIDS (red), while still in CO2/HCO-3, all current was blocked. C: control oocyte I-V relationships. A water-injected control oocyte (H2O) was voltage clamped, and an I-V response was recorded in ND96 (blue triangles, solution 1), 1.5% CO2/10 mM HCO-3/pH 7.5 with 96 mM Na+ (black squares, solution 8), 0 Na+ ND96 (green diamonds, solution 5), and in presence of 200 µM DIDS (red solutions 1 and 8 plus DIDS, respectively). The five I-V responses of the water-injected oocyte are superimposable, indicating absence of an endogenous HCO-3-stimulated current. On the same axis, a rkNBC-expressing oocyte (rkNBC) was voltage clamped, and an I-V relationship was recorded in non-HCO-3 Ringer solution of ND96 (black squares, solution 1), ND120 (blue diamonds, solution 2), 0 Na+ ND120 (green diamonds, solution 6), and in presence of 200 µM DIDS (red circle, solution 2 plus DIDS). In the absence of HCO-3, the four I-V responses of the rkNBC-expressing oocyte to these test solutions (solutions 1, 2 ± DIDS, and 6) are also superimposable. No current was recorded by either increasing the Na+ concentration or by complete Na+ removal. In addition, there is not a DIDS-inhibitable current present in absence of HCO-3. D: HCO-3-dependent steady-state current sweeps. Unsubtracted current sweep data recorded from a rkNBC-expressing oocyte are shown in presence of extracellular 1.5% CO2/10 mM HCO-3 with 96 mM Na+ (black squares, solid line) or Na+ replaced with choline (green diamonds, solid line). The same oocyte was then exposed to 5% CO2/33 mM HCO-3 with 96 mM Na+ (black squares, dotted line) or Na+ replaced with choline (green diamonds, dotted line). E: [HCO-3]o- and [Na+]o-dependent I-V relationships. A rkNBC-expressing oocyte was voltage clamped and equilibrated in 1.5% CO2/10 mM HCO-3/96 mM Na+ for 10 min (see Fig. 1). An I-V relation was then recorded in presence of 96 mM Na+ (black squares, solid line) and 0 Na+ (choline; green diamonds, solid line). This protocol was then repeated using 5% CO2/33 mM HCO-3. I-V relations were again recorded with the oocyte exposed to 96 mM (black square, dotted line) and 0 mM (green diamonds, dotted line) extracellular Na+. F: transport convention. From the I-V relationships of rkNBC-expressing oocytes, both the direction and magnitude of cotransport were measured. Positive current indicates a net negative charge movement into the cell. This is the unbalanced inward movement of HCO-3/Na+ via rkNBC. Negative currents indicate the outward movement of Na+/HCO-3 as net negative charge exits the cell.

DIDS inhibition. The stilbene sulfonate DIDS (Sigma, St. Louis, MO), a known inhibitor of rkNBC (33), was used to determine 1) the non-HCO-3 current via rkNBC, 2) the voltage dependence of DIDS inhibition, and 3) the [Na+]o dependence of DIDS block. Preliminary experiments showed that 100 µM DIDS was sufficient to block rkNBC-mediated cotransport (Fig. 5, bottom). To determine the non-HCO-3 current through rkNBC, oocytes were bathed in ND96 or 0 Na+ ND96 (solutions 1 and 5, Table 1) and I-V relationships were recorded. DIDS, 200 µM, was then added to the bath solution, and the oocyte was perfused for 5 min. I-V relations were recorded in ND96 and 0 Na+ ND96 in the presence of DIDS. Voltage sensitivity of DIDS inhibition was tested by first recording an I-V relation in the presence of CO2/HCO-3. DIDS, 200 µM, was then added to the CO2/HCO-3 solution, and the oocyte was incubated for 5 min. An I-V relationship was then recorded in the presence of DIDS. The extracellular Na+ dependence of DIDS inhibition was measured by first recording a Na+ dose response experiment as described above, perfusing the oocyte for 5 min in 200 µM DIDS/CO2/HCO-3, and repeating the Na+ dose solution protocol in the continued presence of 200 µM DIDS.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Voltage Dependence of rkNBC

In the presence of extracellular CO2/HCO-3, the electrogenic Na+-HCO-3 cotransporter expressed in Xenopus oocytes moves a net negative charge into the cell, and this movement is blocked by extracellular DIDS (13, 33, 34). Using the two-electrode voltage clamp, we studied the voltage dependence of rkNBC expressed in Xenopus oocytes. First we identified the contribution of rkNBC expression on the basal currents of oocytes (Fig. 2C). The I-V relationships in ND96 (non-HCO-3, solution 1, Table 1) are similar in the presence (black) and absence (green) of extracellular Na+. Addition of 200 µM DIDS (red) to the bath solution does not change this basal current. Therefore the oocyte current in the absence of HCO-3 is not due to charge movement via rkNBC. Moreover, Fig. 2C illustrates that control oocytes show no endogenous electrogenic Na+-HCO-3 cotransporter, i.e., no Na+-dependent, DIDS-inhibitable current with or without HCO-3. Consequently, the ND96 (non-HCO-3) I-V response can be subtracted from the HCO-3 elicited I-V relations to yield the rkNBC-dependent current.

Figure 2, A and B, illustrates that addition of extracellular CO2/HCO-3 stimulates a DIDS-inhibitable current in rkNBC oocytes not observed in water-injected oocytes (Fig. 2C). In the presence of 96 mM extracellular Na+, the I-V response of rkNBC oocytes is almost linear, i.e., the direction and magnitude of current (Na+/HCO-3 transport) depends on Vm. Positive currents represent the net inward movement of Na+/HCO-3 (Fig. 2F), whereas negative currents represent the outward movement of Na+/HCO-3. At the reversal potentials, Erev, there is no net current (transport). Erev values for 10 and 33 mM extracellular HCO-3 and [Na+]o from 10 to 96 mM are listed in Table 3. These data show that decreasing extracellular Na+ shifts Erev more positive. The positive shift of Erev does not mirror the calculated shift in the Nernst potential for Na+ (Table 3), indicating that transport direction is not dependent on the Na+ gradient alone. Furthermore, for our conditions (Fig. 1), pHi and thus [HCO-3]i do not change (Fig. 3B), indicating that for the brief solution changes, the HCO-3 gradient is approximately static.

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Erev and stoichiometry



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3.   Ion-selective microelectrode experiment. A: water, intracellular pH (pHi) experiment. An experiment monitoring pHi and membrane potential (Vm) of a control oocyte is shown. Addition of 1.5% CO2/10 mM HCO-3 to bath elicited no change in Vm and resulted in cell acidification of 0.195 ± 0.016. This is due to the diffusion of CO2 across the membrane, formation of H2CO3, and subsequent generation of H+ and HCO-3 from the fast dissociation. B: rkNBC, pHi experiment. pHi and Vm of a rkNBC-expressing oocyte is shown. Initial pH is ~0.2 pH units higher than the water control. Addition of 1.5% CO2/10 mM HCO-3 resulted in a large, transient hyperpolarization and fall of pHi (0.145 ± 0.010 pH units). Vm and pHi reached a steady state in 8-10 min; pHi was 7.28 ± 0.02 (n = 9, 4 frogs) and intracellular [HCO-3] was 4.8 ± 0.2 mM (calculated from the Henderson-Hasselbach equation). After 10-min perfusion in CO2/HCO-3, bath Na+ is removed (replaced with choline at bars) for 30-s intervals. The oocyte quickly depolarizes and then repolarizes upon readdition of Na+ without change in pHi. This experiment mimics the Na+ dose response protocol in Fig. 1, illustrating that brief changes in extracellular Na+ do not significantly alter steady-state pHi, and therefore HCO-3, of the oocyte. C: water, aNai experiment. Intracellular Na+ activity (aNai) and Vm were monitored in control oocyte. CO2/HCO-3 addition resulted in no changes in either Vm or aNai. D: rkNBC, aNai experiment. As seen in B, 1.5% CO2/10 mM HCO-3 addition results in a hyperpolarization of an rkNBC oocyte. aNai increases and is ~9 mM at steady state. Extracellular Na+ removal decreases aNai and depolarizes the cell. Chloride removal does not effect aNai.

When Na+ is removed from the bath solution, only negative current is observed (Fig. 2, B and E). These data indicate that NBC-mediated influx of negative charge does not occur without extracellular Na+. Additionally, without extracellular Na+, the negative currents are augmented, indicating increased outward Na+-HCO-3 cotransport. Taken together, these data indicate that neither Na+ nor HCO-3 alone is capable of inducing rkNBC currents. Moreover, all of these currents are inhibited by 200 µM bath DIDS (Fig. 2, A and B), independent of extracellular Na+ and Vm.

Intracellular Ion Effects of rkNBC Expression

CO2/HCO-3 addition to the bath solution results in the acidification of oocytes due to CO2 diffusion across the plasma membrane, intracellular hydration, and subsequent hydrolysis to form H+ and HCO-3. pHi and aNai changes are quantified in Table 2 for rkNBC and water-injected oocytes perfused with 1.5% CO2/10 mM HCO-3 (pH 7.5) or 5% CO2/33 mM HCO-3 (pH 7.5). Water-injected oocytes exposed to 1.5% CO2/10 mM HCO-3/pH 7.5, acidify by 0.195 ± 0.016 (n = 5, 3 frogs, means ± SE) (Fig. 3A). In contrast, the initial pHi of rkNBC oocytes is ~0.2 pH units more alkaline than water-injected controls (Fig. 3, A vs. B, and Table 2), and the addition of 1.5% CO2/10 mM HCO-3 elicits an immediate hyperpolarization of -72 ± 1.8 mV (n = 9, 4 frogs) concomitant with the fall in pHi (0.145 ± 0.010 pH units, Fig. 3B). The apparent buffering power (beta T) (35) of rkNBC-expressing oocytes at 1.5% CO2/33 mM HCO-3 is 35.7 ± 3.1 mM/pH unit (n = 9), whereas that of water-injected controls is 16.7 ± 1.8 mM/pH unit (n = 5) (Table 2). The increased beta  of rkNBC-expressing oocytes (beta rkNBC) indicates that an additional buffering system (i.e., acid-base transporter) is present. The increased movement of HCO-3 across the plasma membrane increases [HCO-3]i and thus buffers more of the cytosolic H+ generated by the hydration of CO2 entering the oocyte.

CO2/HCO-3 addition to rkNBC-oocytes also results in an aNai increase in concert with the hyperpolarization (Fig. 3D) but not in water-injected controls (Fig. 3C). Nominal air CO2 over several days (days 0-3) provides sufficient solution HCO-3 to raise initial aNai by ~2 mM in rkNBC oocytes over water controls. rkNBC oocytes exhibit a pHi recovery (HCO-3 influx) over 5-10 min in the continued presence of CO2/HCO-3. Removal of extracellular Na+ causes an immediate and reversible depolarization (Na+/HCO-3 efflux), followed by a delayed fall in pHi (33) and aNai (Fig. 3D). Both pHi and aNai responses of rkNBC-expressing oocytes are blocked by 200 µM DIDS (not shown).

Figure 3B also illustrates that pHi can remain at steady state upon short and repeated removals and replacement of extracellular Na+ (every 30 s). Thus we can measure changes in whole cell current on the second time scale without significantly disturbing the steady-state [HCO-3]i or aNai.

Cation Dependence of rkNBC

Li+ appears to substitute for Na+ for transport via the Na+HCO-3 cotransporter when assayed in basolateral membrane vesicles from rabbit kidney (40). Studies using human kidney NBC (hkNBC) expressed in HEK-293 cells also indicate that Li+ induces a 25% DIDS-sensitive HCO-3 pHi recovery from an acid load (4). We used two-electrode voltage clamp to determine whether cations other than Na+ (K+, Li+, and choline) could be transported in Xenopus oocytes expressing rkNBC. No inward transport (positive current) was observed when Na+ is replaced by choline in the bath solution. To ensure that any current measured was due to the test cation, we pulsed solutions from non-HCO-3 to HCO-3 solution with constant 96 mM cation present (Fig. 4A). The HCO-3-stimulated current is the difference between the I-V relationships in these two solutions. As illustrated in Fig. 4, B and C, K+ and choline do not facilitate HCO-3 transport via rkNBC. Li+ is only capable of 3 ± 2% of the Na+ response (n = 5, 2 frogs) over a voltage range from -160 to +60 mV.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4.   Cation dependence. A: solution pulse protocol. This solution protocol is used to test whether K+, Li+, or choline are capable of stimulating a HCO-3-dependent current in rkNBC oocytes. Oocytes were voltage clamped and bathed in ND96 (solution 1, Table 1) for 5 min before switching to test cation/non-HCO-3 Ringer (solutions 3 to 5, Table 1) for 5 min. Solution was then switched to the corresponding 1.5% CO2/10 mM HCO-3 solution for 2 min (solutions 10 to 12, Table 1, i.e., Li+-ND96 to 1.5% CO2/10 mM HCO-3/96 mM Li+) and returned to non-HCO-3 Ringer for 2 min. An I-V relation was recorded before and after each solution change. HCO-3-stimulated current for each cation was taken as the difference between the non-HCO-3 and HCO-3 I-V responses. Cation solutions were tested in random order. B: current sweeps of cation replacement. The unsubtracted current sweep data from a rkNBC-expressing oocyte exposed to extracellular K+-HCO-3 (red), Li+-HCO-3 (blue), Na+-HCO-3 (black), and choline-HCO-3 (green) using the protocol described in A. C: rkNBC I-V response of cations ± HCO-3. Subtracted I-V response curves from the current sweeps in B show that only Na+ (black) stimulates a strong HCO-3-dependent current. Extracellular K+ (red) and choline (green) have I-V relations that lie on the voltage axis, indicating no transport. Li+ (blue) shows only a slight current response of a maximal 3 ± 2% of the Na+ response over the voltage range tested.

Extracellular Na+ Dependence of rkNBC

Determination of experimental vmax. We next determined the maximal Na+ response (vmax) of rkNBC-expressing oocytes in constant to CO2/HCO-3. Recording the I-V responses after varying [Na+]o between 84 and 120 mM (osmolalities matched with choline), we found no difference between the 96 and 120 mM Na+ I-V response (Fig. 5, top). Thus we conclude that the [Na+]o at which vmax current is observed is ~96 mM. Even at supermaximal Na+ concentrations, DIDS inhibits all Na+/HCO-3 current responses (Fig. 5, top).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 5.   Experimental determination of apparent rkNBC vmax (maximal Na+ and HCO-3 stimulated current). Top: vmax ± DIDS. To avoid changes in solution osmolality that could activate endogenous acid-base transporters or Cl- channels, experiments depicted at top used derivatives of solutions 2, 9, and 13 (Table 1). Shown are eight I-V relationships. The I-V profile of a rkNBC-expressing oocyte does not change between ND96 (yellow diamonds, solution 1) and ND120 (yellow squares, solution 2). Subsequently, all solutions in top were based on the 120 mM cation formulation, i.e., made by mixing solutions 9 and 13 in appropriate ratios. The vmax for extracellular Na+ was determined using the solution protocol described in Fig. 4A. CO2/HCO-3 I-V relations for 96 mM (black squares) and 120 mM (blue triangles) extracellular Na+ overlap (<4% divergence at +60 mV, n = 5, 2 frogs). Therefore, vmax occurs at ~96 mM Na+. In absence of extracellular Na+, no outward current was observed (120 mM choline, green diamonds). DIDS, 200 µM, inhibited rkNBC current for all Na+ concentrations (120 mM Na+, red circles; 96 mM Na+ red squares; and 0 Na+, red diamonds). Middle: rkNBC I-V response, extracellular Na+ dependence. Experimental protocol outlined in Fig. 1 was used to determine the extracellular Na+ dependence of a rkNBC-expressing oocyte. I-V responses for 96 mM (black squares), 72 mM (blue diamonds), 24 mM (blue open circles), 10 mM (blue open diamonds), 1 mM (inverted blue open triangles), and 0 mM (green diamonds) extracellular Na+ are shown (all solutions are 96 mM cation based). Bottom: rkNBC extracellular Na+ dependence ± DIDS. Na+ concentration dependence I-V relationships of a rkNBC-expressing oocyte were measured at 96 mM (black squares), 48 mM (blue triangles), 24 mM (blue open circles), and 0 mM (green diamonds) extracellular Na+. Oocyte was then incubated for 5 min in 1.5% CO2/10 mM HCO-3 with 100 µM DIDS, and experiment was repeated (extracellular Na+: 96 mM, red squares; 48 mM, red triangles; 24 mM, red circles; and 0 mM, red diamonds). DIDS, 100 µM, inhibits inward and outward Na+/HCO-3 transport at all Na+ concentrations and voltages.

Extracellular Na+ concentration dependence. We developed the solution protocol in Fig. 1A to study the extracellular Na+ dose response profile of rkNBC. As described above, a rkNBC oocyte reaches a pHi and voltage steady state within 8-10 min perfusion with CO2/HCO-3 at saturating [Na+]o. Figure 1B illustrates that the resulting current, at Vh = -60 mV, also plateaus within the 10-min initial perfusion. Figure 5, middle, shows a set of I-V relations measured for a dose response experiment. The magnitude of outward current (NaHCO3 influx) is decreased, and inward current (NaHCO3 efflux) is augmented with decreasing extracellular Na+. As noted above, Erev shifts in the positive direction, but is not equated with the Na+ reversal potential (Table 3). We also found that 100 µM DIDS inhibits rkNBC current at all Na+ concentrations tested, 0 to 120 mM (Fig. 5, top and bottom), independent of Na+ and voltage.

Calculation of apparent K0.5. Figures 5, middle, and Fig. 6A illustrate that rkNBC transport direction is voltage and extracellular Na+ dependent. From these experiments, we calculated the apparent affinity coefficient of rkNBC for extracellular Na+ (K0.5) of Na+-HCO-3 cotransport at each voltage measured. To plot the rkNBC-specific currents, we subtracted the current at a given [Na+]o and Vm from the current elicited at that same Vm by the 0 Na+/CO2/HCO-3 solution, i.e., I - I0 Na+ (Fig. 6B). This subtraction yields the full range of rkNBC activity (maximal outward I to maximal inward I). We fit these data at each pulse Vm with a right rectangular hyperbolic function2 (Michaelis-Menten) and calculated the apparent K0.5 as ~30 mM (Fig. 6). Remarkably, these data indicate that at every test Vm (-160 to +60 mV) that the apparent K0.5 for extracellular Na+ is ~30 mM. Since all curves were similar, after normalizing currents, we grouped all voltages for each test [Na+] to generate a composite graph (Fig. 6C).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 6.   Apparent extracellular Na+ affinity for rkNBC. A: rkNBC I-V response. I-V responses elicited using the solution protocol outline in Fig. 1 are shown for 1.5% CO2/10 mM HCO-3. As previously, current responses are subtracted from the non-HCO-3 current. Subtracted currents at 96 mM (, solid line), 84 mM (, broken line), 72 mM (black-diamond , broken line), 48 mM (black-triangle, broken line), 36 mM (, broken line), and 0 mM (black-diamond , solid line) extracellular Na+ are shown. B: Na+-stimulated current measurement. Na+-stimulated current at each voltage tested (-160 to 60 mV in 20-mV steps) in A was determined by subtracting the 0 Na+/CO2/HCO-3 current (I0 Na+) from the test Na+/CO2/HCO-3 current (I). The "I - I0 Na+" current is plotted vs. extracellular [Na+] for each voltage. These points were fit with a two-parameter right rectangular hyperbolic function of generalized form v = a(b[Na+]o)/(1 + b[Na+]o) (Michaelis-Menten). All lines fit with an R2 value >0.99. C: apparent K0.5 of rkNBC for extracellular Na+. Na+-stimulated currents (Delta I = I - I0 Na+) were determined as illustrated in B, normalized to the 96 mM Na+ response (experimental vmax, i.e., Imax) and plotted vs. [Na+]o and fit as described above. Apparent K0.5 was calculated from the resulting equation as the [Na+]o that elicits the half-maximal current (0.5). Apparent K0.5 values were similar for the 12 test voltages (not shown). Thus we grouped the voltage data together and calculated the overall apparent K0.5 of rkNBC for extracellular Na+ as 30 mM (solid points, n = 180, 6 frogs). Open circles represent data from 5% CO2/33 mM HCO-3.

HCO-3:Na+ stoichiometry of rkNBC. Cotransport of Na+ and HCO-3 through rkNBC can be described as a coupled transport process by the chemical equation
[Na<SUP>+</SUP>]<SUB>i</SUB> + <IT>b</IT> · [HCO<SUP>−</SUP><SUB>3</SUB>]<SUB>i</SUB> ↔ [Na<SUP>+</SUP>]<SUB>o</SUB> + <IT>b</IT> · [HCO<SUP>−</SUP><SUB>3</SUB>]<SUB>o</SUB> (1)
The stoichiometry of transport (b) can be determined from the concentration gradients across the cell membrane and Erev, i.e., when
&Dgr;&mgr;<SUP>i-o</SUP><SUB>Na</SUB> = <IT>b</IT>&Dgr;&mgr;<SUP>o-i</SUP><SUB>HCO<SUB>3</SUB></SUB> (2)
where Delta µi-oNa is the in-to-out electrochemical potential difference for Na+, Delta µo-iHCO3 is the out-to-in electrochemical potential difference for HCO-3, and b is the cotransport ratio of HCO-3:Na+. By substituting measured values, the expression becomes
[Na<SUP>+</SUP>]<SUB>i</SUB>/[Na<SUP>+</SUP>]<SUB>o</SUB> = ([HCO<SUP>−</SUP><SUB>3</SUB>]<SUB>o</SUB>/[HCO<SUP>−</SUP><SUB>3</SUB>]<SUB>i</SUB>)<SUP><IT>b</IT></SUP> (3)
× exp [(<IT>b</IT> − 1)<IT>FV</IT><SUB>m</SUB>/<IT>RT</IT>]
where R, T, and F have their usual meaning (gas constant, absolute temperature, and Faraday constant, respectively). The Vm at which there is no net ionic flux across the cell membrane is the reversal potential, or Erev. The values of Erev, [HCO-3]i, and aNai were measured or determined from the previously described experiments (Tables 2 and 3). Solving for b in Eq. 4, we calculate the transport stoichiometry of rkNBC for our conditions to be 2 HCO-3:1 Na+ at 10 and 33 mM [HCO-3]o (Fig. 7). These results are obtained between 10 and 96 mM extracellular Na+. Thus rkNBC stoichiometry does not appear sensitive to the cellular Na+ gradient.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 7.   Stoichiometry of rkNBC. HCO-3:Na+ stoichiometry was calculated from experimentally determined reversal potentials (Table 3) and the measured values of intracellular HCO-3 and aNai as outlined in the text (Equation 3). Stoichiometry for [Na+]o ranging from 10 to 96 mM is 2 HCO-3:1 Na+. Solid bars represent data from 1.5% CO2/10 mM HCO-3, and open bars represent data from 5% CO2/33 mM HCO-3.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The process of electrogenic Na+-HCO-3 cotransport is functionally described in a variety of tissues, e.g., kidney, brain, heart, eye, and pancreas. Cloning of these NBC isoforms reveals that they are 95% homologous at the amino acid level (30). However, function in these tissues ranges from strict HCO-3 excretion (kidney), to mainly HCO-3 influx (pancreas),3 and both modes of transport (eye and heart). To begin to define the determinants of NBC function, we studied the voltage- and cation-dependent properties of Na+-HCO-3 cotransport expressed in Xenopus oocytes. Our studies indicate that rkNBC is specific for Na+ over K+ and choline. These results are consistent with studies of the human kidney isoform, indicating that K+ is not a substrate for NBC (4). However, our Li+ results differ, i.e., Li+ minimally stimulates rkNBC-mediated HCO-3 transport. Yet the hkNBC and rkNBC isoforms are 97% homologous. Of the 22 amino acid difference between the two clones, 10 alter charge. H502 in rat is switched to N502 in human and has the predicted location at the putative extracellular portion of transmembrane span 3 (TM-3, L482 to F498). Such charge switches may sufficiently alter a Na+ selectivity filter of NBC in the human clone to allow Li+ but not K+ to be transported. This amino acid is conserved between the human kidney (9, 13, 29), human heart (13), and human pancreas (2) NBC isoforms. The ability of human NBC to cotransport Li+ could have therapeutic consequences resulting from Li+ administration. Such Li+ cotransport capacity might not be predicted to alter NBC renal function, but might affect both the brain and the heart where NBC-mediated cotransport is bidirectional.

Since extracellular Na+ is high and cytosolic Na+ is low, the normal, physiological, electrochemical gradient for Na+ is inward. Under this condition, we find the I-V response of rkNBC-expressing oocytes is roughly linear. Extracellular Na+ of 96 mM is the experimentally determined saturation point of rkNBC current with bath CO2/HCO-3, or the functional vmax for our conditions. At this apparent vmax, the Erev is -94 mV and -100 mV for 10 and 33 mM extracellular HCO-3, respectively. Only at voltages more negative than Erev is outward cotransport observed. At -73 mV (49), the approximate basolateral membrane potential of mammalian, renal proximal tubule cells, rkNBC-mediated cotransport in oocytes is inward (raising both pHi and aNai compared with controls), not outward as expected for electrogenic Na+-HCO-3 cotransporter function of the proximal tubule basolateral membrane. The observed rkNBC function in oocytes is more comparable to that described in tissues where NBC functions as an acid extruder, such as the brain and pancreas. Our results suggest that a mechanism of directional control for the cotransporter is present in the kidney and not found in these other tissues. Additionally, our data predict that variable modes of cotransport would occur with changes in cell membrane potential, physiological or pathological. For example, Camilion de Hurtado and coworkers (10, 11) reported that cat papillary muscle could recover from an acid load caused by increased heart rate. These authors argued that depolarization during cardiac contraction increased the activity of the electrogenic Na+-HCO-3 cotransporter causing HCO-3 influx. This finding is consistent with our data which show a depolarization could move the membrane potential through Erev of rkNBC reversing the cotransport mode from outward to inward and thus increase pHi.

Our data indicate that addition of the stilbene DIDS to the bath solution inhibits both the inward and outward Na+-HCO-3 cotransport modes of rkNBC. Blockade is not overcome by increasing extracellular Na+ or by altering Vm, and with time the blockade is irreversible (not shown). In the murine band 3 protein (AE1), H2-DIDS covalently binds to Lys539 and Lys851 (28). The NBCs and the anion exchangers (AE1-3) are the initial members of a HCO-3 superfamily of transporters (30, 33) for which a predicted DIDS binding motif [K-(Y)-(X)-K for Y = M, L, and X = I, V, Y] seems to exist (30). rkNBC has two putative motifs: 1) KMIK at 558-561 (near H502N described above) and 2) KLKK at 768-771. Perhaps these sites are close to a substrate binding site or permeant path. Upon DIDS binding, this path may be occluded or the regional structure altered, leading to complete abolition of transport.

Using BSC1 cells to examine the native electrogenic Na+-HCO-3 cotransporter, Jentsch and associates (22) found that for 10 mM HCO-3, the apparent K0.5 for extracellular Na+ was between 35 and 45 mM. To study the native electrogenic Na+-HCO-3 cotransporter, presumably NBC, Gross and Hopfer (17) used a rat proximal tubular cell line (SKPT-0193). Employing a "zero-trans" condition (15 mM intracellular HCO-3 with no basolateral Na+ present), these investigators calculated the apparent K0.5 for intracellular Na+ binding as ~18 mM. Nevertheless, it is unclear whether intra- and extracellular Na+ affinities of NBC are similar. For rkNBC expressed in Xenopus oocytes, we calculate the apparent K0.5 for extracellular Na+ to be ~30 mM at a "normal" Vm (-60 mV). The Na+ sensitivity of rkNBC cotransport appears unaffected by membrane potential. That is, over the entire voltage, range the apparent K0.5 for extracellular Na+ is ~30 mM (Fig. 6). However, although the apparent K0.5 is stable over the entire voltage range tested (-160 to +60 mV), the current magnitude increases as Vm becomes more positive. Thus either more rkNBC protein is inserted into the plasma membrane or the rate of cotransport is increasing. Since insertion and retrieval of protein is unlikely to occur in the millisecond-to-second time scale, the cotransport-mediated current increase likely indicates that the overall transport rate is voltage sensitive, yet extracellular Na+ binding is not. Moreover, with physiological Na+ concentration greater than 30 mM, it is unlikely that extracellular Na+ binding is a rate-limiting step of cotransport.

This study was designed to elucidate some of the fundamental properties of rkNBC in an isolated system. A HCO-3:Na+ stoichiometry of 3:1 is thought to be necessary for HCO-3 efflux from renal proximal tubule cells. This stoichiometry is necessary to overcome the Na+ gradient and use the favorable movement of charge down the voltage gradient. However, here we have determined the stoichiometry of rkNBC expressed in Xenopus oocytes as 2:1. This stoichiometry is [Na+]o independent (Fig. 7), as evidenced by a stoichiometry of 2:1 between 10 and 96 mM extracellular Na+. For normal physiological conditions (high extracellular Na+) and Vm more positive than -80 mV, we find only inward Na+-HCO-3 cotransport (outward current). This same stoichiometry is found at both 10 and 33 mM HCO-3 (Fig. 7). Taken together, these results indicate that rkNBC alone can mediate HCO-3 influx as described for some tissues (e.g., brain, heart, liver, and pancreas). However, these data alone cannot explain the HCO-3 reabsorption by the renal proximal tubule cell. A recent report examining rkNBC function in giant patches has also found a 2 HCO-3:1 Na+ stoichiometry (18). Additionally, several groups have measured or calculated a stoichiometry of 3 HCO-3:1 Na+ for native membranes of basolateral membrane vesicles (39), intact proximal tubules (48), or proximal tubular cell monolayers (16). Although formally one could hypothesize another NBC isoform in the kidney, Schmitt and associates (37) in a recent immunolocalization study have found that two different antibodies, specific for NBC, recognize a major protein at the basolateral membrane of mammalian proximal tubules. The same study explicitly demonstrated that these antibodies recognize the functional and recombinant rkNBC expressed in Xenopus oocytes. Moreover, in the several years since NBC was cloned by expression (31, 34), no other electrogenic Na+-HCO-3 cotransporter has been reported for the kidney nor have similar renal clones appeared in the Expressed Sequence Tag (EST) databases. Consequently, although formally possible, it seems unlikely that another (novel and major protein) electrogenic Na+-HCO-3 cotransporter mediating HCO-3 reabsorption in the mammalian proximal tubule, will be found. It is possible that a factor(s) is absent in the Xenopus oocyte but present endogenously in HEK-293 cells or native proximal tubule membranes. Such a factor might be regulated such that it, in turn, could shift the activity of NBC from an acid extruder to that of an acid loader, i.e., HCO-3 reabsorption. Alternatively, an as yet unknown endogenous protein of the Xenopus oocyte could interact with rkNBC to mask the "true" stoichiometry of rkNBC transport. Additional studies using other heterologous expression systems or planar lipid bilayers will be required to entirely rule out this latter possibility.

Our data suggest that in the intact proximal tubule or vesicles there are likely other factors, such as binding partners or cellular factors, which modify the rkNBC function allowing NaHCO3 efflux (HCO-3 reabsorption). Regulation of rkNBC activity or coupling by protein kinase A (PKA) and/or PKC might be important, since there is a predicted PKA phosphorylation site and seven PKC consensus phosphorylation sites (30, 33). Such activation could shift the cotransporter voltage dependence such that Na+/HCO-3 efflux occurs. Currently, we have no information indicating that other proteins associate with rkNBC. However, accessory proteins, such as those recently illustrated with the Na/H exchangers (25, 46, 47), could modify rkNBC function. Moreover, AE1 has long been known to form dimers (7, 20, 43-45), associate with cytoskeletal proteins (12), and has recently been shown to associate with carbonic anhydrase II (42). Although it is unclear whether such modifications or protein associations alter the transport function of either the AEs or the NBCs, it is attractive speculation. By extension, tissue-specific expression of a modulator could allow the same NBC protein to be used for both HCO-3 influx and efflux in different locations. The specific regulator(s) of rkNBC function that allow HCO-3 efflux in the renal proximal tubule is (are) yet unknown. Further studies that explore the isolated protein properties will help to determine whether factors or binding partners can alter NBC function.


    ACKNOWLEDGEMENTS

We thank Drs. Eberhard Frömter, Ulrich Hopfer, and Suzanne Müller-Berger for helpful suggestions on the manuscript. We thank Dr. Stephen Jones for suggesting how to present the Na+ current response data.


    FOOTNOTES

Portions of this work have been presented in preliminary form (38).

This work was supported by a grant from the American Heart Association (to M. F. Romero) and a HHMI-institutional grant (to Case Western Reserve University). C. M. Sciortino was supported by National Institute of Diabetes and Digestive and Kidney Diseases Predoctoral Fellowship DK-07678.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

1 Selectivity for the ion of interest (i) over the interfering ion (j) is calculated as Kij = ai/[ajexp(zi/zj)] × 10exp[(Ej - Ei)/m], where a is activity an m is the slope of the electrode. Na+ electrodes maintain their calibrated slope even at 5 mM NaCl.

2 Note that similar results are obtained if the data are fit by a second order polynomial.

3 A preliminary report from our laboratory (41) indicates that models of pancreatic HCO-3 absorption and secretion may not include all of the relevant transporters. In rat pancreatic ductal epithelia, NBC protein is mainly basolateral but is also present apically. Moreover, the basolateral membranes of rat pancreatic acinar cells also contain NBC protein.

Address for reprint requests and other correspondence: M. F. Romero, Dept. of Physiology & Biophysics, Case Western Reserve Univ. School of Medicine, 2119 Abington Rd., Cleveland, OH 44106-4970 (E-mail: mfr2{at}po.cwru.edu).

Received 17 December 1998; accepted in final form 18 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abdulnour-Nakhoul, S., R. N. Khuri, and N. L. Nakhoul. Effect of norepinephrine on cellular sodium transport in Ambystoma kidney proximal tubule. Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36): F725-F736, 1994[Abstract/Free Full Text].

2.   Abuladze, N., I. Lee, D. Newman, J. Hwang, K. Boorer, A. Pushkin, and I. Kurtz. Molecular cloning, chromosomal localization, tissue distribution, and functional expression of the human pancreatic sodium bicarbonate cotransporter. J. Biol. Chem. 273: 17689-17695, 1998[Abstract/Free Full Text].

3.   Aiello, E. A., M. G. Petroff, A. R. Mattiazzi, and H. E. Cingolani. Evidence for an electrogenic Na+-HCO-3 symport in rat cardiac myocytes. J. Physiol. (Lond.) 512: 137-148, 1998[Abstract/Free Full Text].

4.   Amlal, H., Z. Wang, C. Burnham, and M. Soleimani. Functional characterization of a cloned human kidney Na+:HCO-3 cotransporter. J. Biol. Chem. 273: 16810-16815, 1998[Abstract/Free Full Text].

5.   Bevensee, M. O., B. M. Schmitt, M. F. Romero, and W. F. Boron. Cloning of a putative Na/HCO3 cotransporter (NBC) from rat brain (Abstract). FASEB J. 12: 1031, 1998.

6.   Bevensee, M. O., M. Apkon, and W. F. Boron. Intracellular pH regulation in cultured astrocytes from rat hippocampus. II. Electrogenic Na/HCO3 cotransport. J. Gen. Physiol. 110: 467-483, 1997[Abstract/Free Full Text].

7.   Boodhoo, A., and R. A. Reithmeier. Characterization of matrix-bound Band 3, the anion transport protein from human erythrocyte membranes. J. Biol. Chem. 259: 785-790, 1984[Abstract/Free Full Text].

8.   Boron, W. F., and E. L. Boulpaep. Intracellular pH regulation in the renal proximal tubule of the salamander. Basolateral HCO-3 transport. J. Gen. Physiol. 81: 53-94, 1983[Abstract].

9.   Burnham, C. E., H. Amlal, Z. Wang, G. E. Shull, and M. Soleimani. Cloning and functional expression of a human kidney Na+:HCO-3 cotransporter. J. Biol. Chem. 272: 19111-19114, 1997[Abstract/Free Full Text].

10.   Camilion de Hurtado, M. C., B. V. Alvarez, N. G. Perez, and H. E. Cingolani. Role of an electrogenic Na+-HCO-3 cotransport in determining myocardial pHi after an increase in heart rate. Circ. Res. 79: 698-704, 1996[Abstract/Free Full Text].

11.   Camilion de Hurtado, M. C., N. G. Perez, and H. E. Cingolani. An electrogenic sodium-bicarbonate cotransport in the regulation of myocardial intracellular pH. J. Mol. Cell. Cardiol. 27: 231-242, 1995[Medline].

12.   Casey, J. R., Y. Ding, and R. R. Kopito. The role of cysteine residues in the erythrocyte plasma membrane anion exchange protein, AE1. J. Biol. Chem. 270: 8521-8527, 1995[Abstract/Free Full Text].

13.   Choi, I., M. F. Romero, N. Khandoudi, A. Bril, and W. F. Boron. Cloning and characterization of an electrogenic Na-HCO3 cotransporter from human heart. Am. J. Physiol. 274 (Cell Physiol. 43): C576-C584, 1998.

14.   Dart, C., and R. D. Vaughan-Jones. Na+-HCO-3 symport in the sheep cardiac Purkinje fibre. J. Physiol. (Lond.) 451: 365-385, 1992[Abstract].

15.   Deitmer, J. W., and W. R. Schlue. An inwardly directed electrogenic sodium-bicarbonate co-transport in leech glial cells. J. Physiol. (Lond.) 411: 179-194, 1989[Abstract].

16.   Gross, E., and U. Hopfer. Activity and stoichiometry of Na+:HCO-3 cotransport in immortalized renal proximal tubule cells. J. Membr. Biol. 152: 245-252, 1996[Medline].

17.   Gross, E., and U. Hopfer. Voltage and cosubstrate dependence of the Na+-HCO-3 cotransporter kinetics in renal proximal tubule cells. Biophys. J. 75: 810-824, 1998[Abstract/Free Full Text].

18.   Heyer, M., S. Müller-Berger, M. F. Romero, W. F. Boron, and E. Frömter. Stoichiometry of rat kidney Na-HCO3 cotransporter (rkNBC) expressed in Xenopus laevis oocytes. Pflügers Arch. 438: 322-329, 1999[Medline].

19.   Ishiguro, H., M. C. Steward, A. R. Lindsay, and R. M. Case. Accumulation of intracellular HCO-3 by Na+-HCO-3 cotransport in interlobular ducts from guinea-pig pancreas. J. Physiol. (Lond.) 495: 169-178, 1996[Abstract].

20.   Jennings, M. L., and J. S. Nicknish. Localization of a site of intermolecular cross-linking in human red blood cell band 3 protein. J. Biol. Chem. 260: 5472-5479, 1985[Abstract].

21.   Jentsch, T. J., S. K. Keller, M. Koch, and M. Wiederholt. Evidence for coupled transport of bicarbonate and sodium in cultured bovine corneal endothelial cells. J. Membr. Biol. 81: 189-204, 1984[Medline].

22.   Jentsch, T. J., P. Schwartz, B. S. Schill, B. Langner, A. P. Lepple, S. K. Keller, and M. Wiederholt. Kinetic properties of the sodium bicarbonate (carbonate) symport in monkey kidney epithelial cells (BSC-1). Interactions between Na+, HCO-3, and pH. J. Biol. Chem. 261: 10673-10679, 1986[Abstract/Free Full Text].

23.   Kenyon, E., A. Maminishkis, D. P. Joseph, and S. S. Miller. Apical and basolateral membrane mechanisms that regulate pHi in bovine retinal pigment epithelium. Am. J. Physiol. 273 (Cell Physiol. 42): C456-C472, 1997[Abstract/Free Full Text].

24.   Lagadic-Gossmann, D., K. J. Buckler, and R. D. Vaughan-Jones. Role of bicarbonate in pH recovery from intracellular acidosis in the guinea-pig ventricular myocyte. J. Physiol. (Lond.) 458: 361-384, 1992[Abstract].

25.   Murthy, A., C. Gonzalez-Agosti, E. Cordero, D. Pinney, C. Candia, F. Solomon, J. Gusella, and V. Ramesh. NHE-RF, a regulatory cofactor for Na+-H+ exchange, is a common interactor for merlin and ERM (MERM) proteins. J. Biol. Chem. 273: 1273-1276, 1998[Abstract/Free Full Text].

26.   Newman, E. A. Sodium-bicarbonate cotransport in retinal Muller (glial) cells of the salamander. J. Neurosci. 11: 3972-3983, 1991[Abstract].

27.   Newman, E. A., and M. L. Astion. Localization and stoichiometry of electrogenic sodium bicarbonate cotransport in retinal glial cells. Glia 4: 424-428, 1991[Medline].

28.   Okubo, K., D. Kang, N. Hamasaki, and M. L. Jennings. Red blood cell band 3. Lysine 539 and lysine 851 react with the same H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonic acid) molecule. J. Biol. Chem. 269: 1918-1926, 1994[Abstract/Free Full Text].

29.   Romero, M. F., C. R. Sussman, I. Choi, M. A. Hediger, and W. F. Boron. Cloning of an electrogenic Na/HCO3 cotransporter (NBC) isoform from human kidney and pancreas (Abstract). J. Am. Soc. Nephrol. 9: 11, 1998.

30.   Romero, M. F., and W. F. Boron. Electrogenic Na/HCO3 cotransporters: expression cloning and physiology. Annu. Rev. Physiol. 61: 699-723, 1999[Medline].

31.   Romero, M. F., M. A. Hediger, E. L. Boulpaep, and W. F. Boron. Expression cloning of the renal electrogenic Na/HCO3 cotransporter (NBC-1) from Ambystoma tigrinum (Abstract). FASEB J. 10: 89, 1996.

32.   Romero, M. F., Y. Kanai, H. Gunshin, and M. A. Hediger. Expression cloning using Xenopus laevis oocytes. Methods Enzymol. 296: 17-52, 1998[Medline].

33.   Romero, M. F., P. Fong, U. V. Berger, M. A. Hediger, and W. F. Boron. Cloning and functional expression of rNBC, an electrogenic Na+-HCO-3 cotransporter from rat kidney. Am. J. Physiol. 274 (Renal Physiol. 43): F425-F432, 1998[Abstract/Free Full Text].

34.   Romero, M. F., M. A. Hediger, E. L. Boulpaep, and W. F. Boron. Expression cloning and characterization of a renal electrogenic Na+-HCO-3 cotransporter. Nature 387: 409-413, 1997[Medline].

35.   Roos, A., and W. F. Boron. Intracellular pH. Physiol. Rev. 61: 296-434, 1981[Free Full Text].

36.   Rose, C. R., and B. R. Ransom. Regulation of intracellular sodium in cultured rat hippocampal neurones. J. Physiol. (Lond.) 499: 573-587, 1997[Abstract].

37.   Schmitt, B. M., D. Biemesderfer, E. L. Boulpaep, M. F. Romero, and W. F. Boron. Immunolocalization of the electrogenic Na+/HCO-3 cotransporter in mammalian and amphibian kidney. Am. J. Physiol. 276 (Renal Physiol. 45): F27-F36, 1999[Abstract/Free Full Text].

38.   Sciortino, C. M., and M. F. Romero. Na+ and voltage dependence of the rat kidney electrogenic Na/HCO3 cotransporter (rkNBC) expressed in Xenopus oocytes (Abstract). J. Am. Soc. Nephrol. 9: 12, 1998.

39.   Soleimani, M., S. M. Grassi, and P. S. Aronson. Stoichiometry of Na+-HCO-3 cotransport in basolateral membrane vesicles isolated from rabbit renal cortex. J. Clin. Invest. 79: 1276-1280, 1987[Medline].

40.   Soleimani, M., G. A. Lesoine, J. A. Bergman, and P. S. Aronson. Cation specificity and modes of the Na+:CO2-3:HCO-3 cotransporter in renal basolateral membrane vesicles. J. Biol. Chem. 266: 8706-8710, 1991[Abstract/Free Full Text].

41.   Thévenod, F., E. Roussa, B. M. Schmitt, and M. F. Romero. Molecular cloning, biochemical characterization and immunolocalization of a rat pancreatic Na+:HCO-3 cotransporter (Abstract). FASEB J. 13: 73, 1999.

42.   Vince, J. W., and R. A. F. Reithmeier. Carbonic anhydrase II binds to the carboxyl terminus of human band 3, the erythrocyte Cl-/HCO-3 exchanger. J. Biol. Chem. 273: 28430-28437, 1998[Abstract/Free Full Text].

43.   Vince, J. W., V. E. Sarabia, and R. A. Reithmeier. Self-association of Band 3, the human erythrocyte anion exchanger, in detergent solution. Biochim. Biophys. Acta 1326: 295-306, 1997[Medline].

44.   Wang, D. N., W. Kuhlbrandt, V. E. Sarabia, and R. A. Reithmeier. Two-dimensional structure of the membrane domain of human band 3, the anion transport protein of the erythrocyte membrane. EMBO J. 12: 2233-2239, 1993[Abstract].

45.   Wang, D. N., V. E. Sarabia, R. A. Reithmeier, and W. Kuhlbrandt. Three-dimensional map of the dimeric membrane domain of the human erythrocyte anion exchanger, Band 3. EMBO J. 13: 3230-3235, 1994[Abstract].

46.   Wang, S., R. W. Raab, P. J. Schatz, W. B. Guggino, and M. Li. Peptide binding consensus of the NHE-RF-PDZ1 domain matches the C-terminal sequence of cystic fibrosis transmembrane conductance regulator (CFTR). FEBS Lett. 427: 103-108, 1998[Medline].

47.   Weinman, E. J., D. Steplock, K. Tate, R. A. Hall, R. F. Spurney, and S. Shenolikar. Structure-function of recombinant Na/H exchanger regulatory factor (NHE- RF). J. Clin. Invest. 101: 2199-2206, 1998[Abstract/Free Full Text].

48.   Yoshitomi, K., B. C. Burckhardt, and E. Frömter. Rheogenic sodium-bicarbonate cotransport in the peritubular cell membrane of rat renal proximal tubule. Pflügers Arch. 405: 360-366, 1985[Medline].

49.   Yoshitomi, K., and E. Frömter. How big is the electrochemical potential difference of Na+ across rat renal proximal tubular cell membranes in vivo? Pflügers Arch. 405: S121-S126, 1985[Medline].


Am J Physiol Renal Physiol 277(4):F611-F623
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society