Extracellular Hco₃ ⁻ Dependence of Electrogenic Na/Hco₃ Cotransporters Cloned from Salamander and Rat Kidney

Abstract

We studied the extracellular [HCOabstract ₃ ⁻] dependence of two renal clones of the electrogenic Na/HCO₃ cotransporter (NBC) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential (ΔV_m) elicited by the NBC cloned from the kidney of the salamander Ambystoma tigrinum (akNBC) and by the NBC cloned from the kidney of rat (rkNBC). We used a two-electrode voltage clamp to measure the change in current (ΔI) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCOabstract ₃ ⁻/CO₂ (0.33–99 mM HCOabstract ₃ ⁻, pH_o 7.5) elicited an immediate, DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid)-sensitive and Na⁺-dependent hyperpolarization (or outward current). In ΔV_m experiments, the apparent K _m for HCOabstract ₃ ⁻ of akNBC (10.6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent K _m for HCOabstract ₃ ⁻ of rkNBC was less (6.5 mM). Because it has been reported that SOabstract ₃ ⁼/HSO abstract ₃ ⁻ stimulates Na/HCO₃ cotransport in renal membrane vesicles (a result that supports the existence of a COabstract ₃ ⁼ binding site with which SOabstract ₃ ⁼ interacts), we examined the effect of SOabstract ₃ ⁼/HSO abstract ₃ ⁻ on rkNBC. In voltage-clamp studies, we found that neither 33 mM SOabstract ₄ ⁼ nor 33 mM SOabstract ₃ ⁼/HSOabstract ₃ ⁻ substantially affects the apparent K _m for HCO abstract ₃ ⁻. We also used microelectrodes to monitor intracellular pH (pH_i) while exposing rkNBC-expressing oocytes to 3.3 mM HCOabstract ₃ ⁻/0.5% CO₂. We found that SO abstract ₃ ⁼/HSOabstract ₃ ⁻ did not significantly affect the DIDS-sensitive component of the pH_i recovery from the initial CO₂ -induced acidification. We also monitored the rkNBC current while simultaneously varying [CO₂]_o, pH_o, and [COabstract ₃ ⁼]_o at a fixed [HCOabstract ₃ ⁻]_o of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [COabstract ₃ ⁼]_o . However, a pH titration curve nicely fitted the data expressed as current versus pH_o. Thus, rkNBC expressed in Xenopus oocytes does not appear to interact with SOabstract ₃ ⁼, HSOabstract ₃ ⁻, or COabstract ₃ ⁼.

INTRODUCTION

Since its first description in the renal proximal tubule of the salamander Ambystoma tigrinum ( Boron and Boulpaep 1983), the electrogenic Na/HCO₃ cotransporter has been functionally identified in a wide variety of cell types (for reviews, see Boron and Boulpaep 1989; Boron et al. 1997). After the expression cloning of the electrogenic Na/HCO₃ cotransporter (NBC) from Ambystoma kidney (Romero et al. 1997a), closely related cDNAs have been cloned from human kidney ( Burnham et al. 1997), rat kidney ( Romero et al. 1998), and human pancreas and heart ( Abuladze et al. 1998a; Choi et al. 1999). An in situ hybridization study showed the presence of NBC mRNA in the renal proximal tubule of the rabbit (Abuladze et al. 1998b). Immunocytochemical studies with polyclonal NBC antibodies have localized the NBC protein to the basolateral membrane of the Ambystoma, rat, and rabbit renal proximal tubule (Schmitt et al. 1999), rat epididymis (Jensen et al. 1999), and human pancreatic duct (Marino et al. 1999).

The electrogenic Na/HCO₃ cotransporter plays the major role in HCO₃ ⁻ reabsorption by the renal proximal tubule (Alpern 1985; Yoshitomi et al. 1985). Several groups have determined the apparent K _m for HCO ₃ ⁻ [K _m(HCO₃ ⁻)] of the Na/HCO₃ cotransporter, as naturally expressed in cells. Working on monkey kidney epithelial (BSC-1) cells, Jentsch et al. 1985 measured DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid)-sensitive ²² Na⁺ uptake and estimated an apparent K _m (HCO₃ ⁻) of 7–14 mM for extracellular HCO₃ ⁻ at a [Na]_o of 151 mM. Later, they demonstrated an inverse relationship between K _m(HCO₃ ⁻) and [Na⁺] _o (Jentsch et al. 1986). Akiba et al. 1986, in a study of ²²Na⁺ fluxes in basolateral membrane vesicles from rabbit kidney cortex, obtained an apparent K _m(HCO₃ ⁻ ) of 10 mM at a [Na]_o of 8 mM. Using a fluorescent probe thought to react with an amino acid near the substrate-binding site of NBC in solubilized membrane proteins from rabbit renal basolateral vesicles, Stim et al. 1994 obtained an apparent K _m (HCO₃ ⁻) of 15 mM. The only study of the intracellular HCO₃ ⁻ dependence of NBC is that of Gross and Hopfer 1998. These authors measured short-circuit current in an apically permeabilized monolayer of rat proximal-tubule (SKPT-0193) cells grown on filters. They obtained an apparent K _m(HCO₃ ⁻) of 19 mM ([Na⁺] _i = 10 mM, V_hold = −60 mV).

The cloning of NBC has made it possible to address the physiology of Na/HCO₃ cotransport heterologously expressed in Xenopus oocytes. Under the conditions of our experiments, no other acid–base transporters are active in the oocyte. In the present study, in which we expressed Ambystoma kidney NBC (akNBC) or rat kidney NBC (rkNBC) in Xenopus oocytes, we had two major goals. The first was to determine the apparent K _m of the two NBCs for extracellular HCO₃ ⁻ under conditions of net HCO₃ ⁻ influx. This uptake of HCO₃ ⁻ is in the direction opposite the net HCO₃ ⁻ efflux that normally occurs in the renal proximal tubule. Our approach was to measure the change in membrane potential (ΔV_m) or change in current (ΔI) (under voltage-clamp conditions) as we added varying levels of HCO₃ ⁻ /CO₂ to the extracellular solution at a constant pH.

Our second goal was to examine the possibility that NBC can transport sulfite (SO₃ ⁼), bisulfite (HSO₃ ⁻), or carbonate (CO₃ ⁼); SO ₃ ⁼ and HSO₃ ⁻ would presumably substitute for CO₃ ⁼ and HCO₃ ⁻, respectively. An earlier study with microelectrodes and ²²Na⁺ fluxes provided no evidence that SO₃ ⁼ substitutes for CO₃ ⁼ or HCO₃ ⁻ on the electrogenic Na/HCO₃ cotransporter of cultured bovine corneal endothelial cells ( Jentsch et al. 1986). However, a later ²²Na⁺ -uptake study on basolateral membrane vesicles isolated from rabbit kidney cortex led to the hypothesis that the electrogenic Na/HCO₃ cotransporter has a binding site for CO₃ ⁼, and that SO₃ ⁼ can substitute for CO₃ ⁼ (Soleimani and Aronson 1989). In our experiments, we examined the effect of SO₃ ⁼/HSO₃ ⁻ on both the apparent K _m(HCO₃ ⁻) and on the pH_i changes caused by rkNBC heterologously expressed in Xenopus oocytes. We found that SO₃ ⁼/HSO₃ ⁻ had no significant effect on either, making it unlikely that rkNBC, as expressed by itself in oocytes, transports either SO₃ ⁼ or HSO ₃ ⁻. To investigate a potential role for CO ₃ ⁼, we monitored rkNBC current while simultaneously varying [CO₂]_o, pH_o, and [CO₃ ⁼]_o at a constant [HCO₃ ⁻ ]_o. Based on the data from these experiments, we suggest that increased pH_o stimulates NBC with a pK of 7.5, but that NBC does not interact with CO₃ ⁼.

METHODS

Preparation of Xenopus Oocytes

We prepared oocytes from Xenopus laevis (NASCO) by incubating small pieces of ovary for 45 min in a Ca²⁺-free ND96 solution (pH 7.5, room temperature) containing 2 mg/ml collagenase (Type IA, #C-2674; Sigma Chemical Co.). We washed the oocytes three times for 10 min each in Ca²⁺-free ND96, and then washed them again for an additional 20 min in Ca²⁺-containing ND96. Until we used the oocytes, we incubated them at 18°C in OR3 media. This medium is a 1:2 dilution in water of Leibovitz's L15 Medium (41300-039; GIBCO BRL), supplemented with 50 U/ml of penicillin-streptomycin (15140-122; GIBCO BRL), 10 mM of HEPES, and titrated to pH 7.5 with NaOH. 1 d after this isolation procedure, we injected stage V or VI oocytes with either water (50 nl/cell) or 0.2 μg/μl cRNA (50 nl/cell) encoding rkNBC or akNBC. We used oocytes expressing NBC in electrophysiological experiments 3–10 d after injection.

Solutions

Table summarizes the composition of standard solutions used in the present study. For experiments conducted in the absence of SO₄ ⁼ or SO₃ ⁼/HSO₃ ⁻, our HEPES-buffered HCO₃ ⁻ /CO₂-free solution was Solution 1. This solution was noteworthy in that it contained only 7.6 mM Cl⁻, but 99 mM gluconate. Our standard HCO₃ ⁻/CO₂ Solution 2 contained 66 mM gluconate and 33 mM HCO₃ ⁻ (i.e., compared with Solution 1, 33 mM HCO₃ ⁻ replaced 33 mM gluconate) and was equilibrated with 5% CO₂, pH 7.5. We varied [HCO₃ ⁻] _o from 0.66 to 99 mM at constant pH_o by always maintaining the same ratio of [HCO₃ ⁻]/[CO ₂]. For example, the solution containing 16.5 mM HCO₃ ⁻ also contained 2.5% CO₂. We maintained a constant [Cl⁻]_o by exchanging HCO₃ ⁻ for gluconate in the solutions. The CO₂ /O₂ mixtures with which we equilibrated our solutions were primary standard grade and analyzed; the mixing tolerance for the CO₂ was 1% (TechAir). In all solutions, pH was 7.5.

Table 1.

Composition of Standard Solutions^‡

Component	1 standardHEPES	2 standardHCO3 −	3 standardSO4 + HEPES	4 standardSO4 + HCO3 −	5 standardSO3 + HEPES	6 standardSO3 + HCO3 −
	mM	mM	mM	mM	mM	mM
Na+	104	104	104	104	97.4	97.4
K+	2	2	2	2	2	2
Mg2+	1	1	1	1	1	1
Ca2+	1.8	1.8	1.8	1.8	1.8	1.8
Total cations, meq	111.6	111.6	111.6	111.6	105	105
Cl−	7.6	7.6	7.6	7.6	7.6	7.6
Gluconate−	99	66	33	0	33	0
HCO3 −	0	33	0	33	0	33
SO4 =	0	0	33	33	0	0
SO3 =	0	0	0	0	26.4	26.4
HSO3 −	0	0	0	0	6.6	6.6
HEPES−	5	5	5	5	5	5
Total anions, meq	111.6	111.6	111.6	111.6	105	105
Mannitol	0	0	33	33	33	33
HEPES (neutral)	5	5	5	5	5	5
pH	7.5	7.5	7.5	7.5	7.5	7.5

^‡Solutions containing 33 mM HCO₃ ⁻ were equilibrated with 5% CO₂.

The solutions containing SO₄ ⁼ were similar to those described above, except that we replaced 66 of the 99 mM gluconate in the HEPES-buffered solution (Solution 3 in Table ) with 33 mM SO₄ ⁼ and 33 mM mannitol. In the HCO₃ ⁻ /CO₂-containing solutions, we kept [SO₄ ⁼ ] fixed at 33 mM, and substituted HCO₃ ⁻ for gluconate. Because the solutions contained a maximal [gluconate]_o of only 33 mM, we were limited to HCO₃ ⁻ concentrations no higher than 33 mM.

The so-called “sulfite” solutions actually contained both SO ₃ ⁼ and HSO₃ ⁻ (pK 6.9). Thus, a pH 7.5 solution containing 33 mM “total SO₃ ⁼ ” actually contains 26.4 mM SO₃ ⁼ and 6.6 mM HSO₃ ⁻. Similar to the situation for the SO ₄ ⁼ solutions, we replaced 66 of the 99 mM gluconate in the HEPES-buffered solution with 33 mM total SO₃ ⁼ /HSO₃ ⁻ and 33 mM mannitol (Solution 5 in Table ). In the HCO₃ ⁻/CO₂ -containing solutions, we kept [total SO₃ ⁼/HSO ₃ ⁻]_o fixed at 33 mM, but substituted HCO₃ ⁻ for gluconate. In all solutions, pH was 7.5.

To determine the pH_o or [CO₃ ⁼]_o dependence of the NBC current, we used solutions containing a constant 33 mM HCO₃ ⁻. We varied pH_o from 9.2 to 6.2 by equilibrating with gas mixtures having [CO₂] values of 0.1–100%; as a result, [CO₃ ⁼] _o varied from ∼3.5 μM to ∼3.5 mM. Our standard 33 mM HCO₃ ⁻/5% CO₂ (Solution 2 in Table ) contained ∼70 μM CO₃ ⁼ at pH 7.5.

In all solutions, [Cl⁻] was 7.6 mM, osmolarity was 225 mOsm, and temperature was 22°C. We delivered solutions continuously at a rate of 7 ml/min through Tygon (Tygon Norton Co.) tubing, which has a low permeability to CO₂.

Voltage and pH-sensitive Microelectrodes

V_m measurements.

In some experiments, we used the change in membrane potential (ΔV _m) elicited by switching from a HEPES-buffered solution to a HCO₃ ⁻/CO₂-buffered solution as an index of the electrogenic flux mediated by NBC. We made the voltage microelectrodes by pulling borosilicate glass capillary tubing, 1.16 mm i.d. × 2.0 mm o.d. (GC200F-10; Warner Instruments Corp.) on a microelectrode puller (P-97; Sutter Instrument Co.), and then filling with 3 M KCl. The electrodes had resistances of 1–10 MΩ.

pH_i measurements.

In some experiments, we used the rate of pH_i increase (d pH_i/dt) as an index of the flux of HCO₃ ⁻ into oocytes expressing rkNBC. We made the pH microelectrodes using the same glass as described above, using an approach described previously (Siebens and Boron 1987; Nakhoul et al. 1998). We silanized the glass by exposing it to vapors of bis-(dimethylamino)-dimethylsilane (14755; Fluka Chemical Corp.). We filled the tips of these electrodes with hydrogen-ionophore-I (Cocktail B, #918882/1; Fluka Chemical Corp.) and back-filled the electrodes with a solution containing 15 mM NaCl, 230 mM NaOH, and 40 mM KH₂PO₄, pH 7.0. The pH microelectrodes had slopes of −54 to −59 mV per pH unit between pH values of 6.0 and 8.0. They had resistances of up to 100 MΩ. The voltage- and pH-sensitive microelectrodes were connected to high-impedance electrometers (FD223; World Precision Instruments, Inc.). The bath reference electrode was a calomel reference electrode (1362079; Fisher Scientific). We corrected for bath junction potentials.

Two-Electrode Oocyte Voltage Clamp

We voltage clamped oocytes using a two-electrode voltage clamp (OC-725B Oocyte Clamp; Warner Instrument Corp.). We impaled cells with microelectrodes filled with 3 M KCl (resistance = 0.3–1.0 MΩ). The holding potential (V_hold) was −60 mV. The currents were filtered at 20 Hz (four-pole Bessel filter).

Data Acquisition

The pH_i, V_m, and I_out data were recorded digitally on 80486-based personal computer. The analogue-to-digital converter (ADC-30; Contec Microelectronics U.S.A., Inc.) sampled the V_m and pH_i data at a rate of 0.4 Hz, and sampled the current data at a rate of 1 Hz. Software for data acquisition and analysis, as well as for fitting of the data, was developed in our laboratory.

Statistics and Data Analysis

We determined rates of pH_i change (dpH_i/ dt) by fitting a line to pH_i versus time data using a linear least-squares method. All average dpH_i/dt , ΔV_m, and ΔI data are reported as mean ± SEM. For ratios, we present the averages as log-normal means. The statistical significance of log-normal data was determined using an unpaired Student's t test.

In analyzing ΔV_m (or ΔI) data obtained in the absence of SO₄ ⁼ or SO₃ ⁼/HSO₃ ⁻ (i.e., when gluconate and HCO₃ ⁻ were the major anions), we normalized absolute values of ΔV_m (or ΔI) obtained under “test” conditions to bracketing values of ΔV_m (or ΔI) obtained under “standard” conditions of 33 mM HCO₃ ⁻. As noted in the discussion, the simplest equation that adequately fitted our data was a model having a Michaelis-Menten dependence on [HCO₃ ⁻]_o , plus a linear component:

1

where v is an absolute value of the velocity of the reaction (i.e., ΔV_m or ΔI) at each value of [HCO₃ ⁻], v _max is the maximum velocity, and α is a constant. We can rearrange to obtain α as follows ():

2

Under our standard conditions of [HCO₃ ⁻] = [HCO₃ ⁻]_std = 33 mM, α becomes:

3

Substituting into yields:

4

We define the normalized velocity (v*) to be the ratio of the observed velocity to the velocity under standard conditions (i.e., v * = v/v _std). Substituting this definition of v* into , we have:

5

where the normalized v _max is defined as v *_max = v _max/v _std. We used a nonlinear least-squares curve fitting approach to obtain v* _max and K _m, and then computed the normalized α* = α/v _std, using the following equation:

6

In analyzing normalized ΔI data obtained in the presence of sulfate or sulfite, ([HCO₃ ⁻]_o between 0 and 33 mM), we fitted the data with a normalized version of a function similar to , except that we assumed that α* was fixed to the same value obtained from the curve fit of the data obtained in the absence of sulfate or sulfite (see Table ). In this case, the equation for normalized data is:

7

where α* = 0.00577 mM⁻¹. After obtaining K _m by curve fitting, we obtained the value of v* _max using :

8

Table 3.

Effect of SO₄ ⁼ and SO₃ ⁼/HSO₃ ⁻ on Kinetic Parameters of rkNBC ^‡

Major extracellular anion	K m(HCO3 −)	Relative v max	α*	n
	mM		mM−1
66 mM gluconate−	6.5 ± 0.7	0.97 ± 0.03	0.00577	58
33 mM SO4 =	7.1 ± 0.5	0.98	0.00577 (fixed)	24
26.4 mM SO3 =/6.6 mM HSO 3 −	7.6 ± 0.6	0.99	0.00577 (fixed)	48

^‡The parameter values for 66 mM gluconate were obtained using and , whereas those for SO₄ ⁼ and SO₃ ⁼/HSO₃ ⁻ were obtained using and .

RESULTS

[HCO₃ ⁻]_o Dependence of akNBC, Based on Changes in V_m

Effect of adding 33 mM HCO₃ ⁻/5% CO₂ on pH_i and V_m.

Fig. 1 A illustrates the results of an experiment on a water-injected (i.e., control) oocyte. As described previously (Romero et al. 1997a), switching the extracellular solution from one buffered with HEPES to one buffered with HCO₃ ⁻/CO₂, at a constant pH_o, causes a slow and sustained fall in pH_i, as well as a slowly developing depolarization. These changes in pH_i and V_m are fully reversible. Fig. 1 B illustrates the results of a similar experiment, but performed on an oocyte injected 3 d earlier with cRNA encoding rkNBC. Although there is a modest recovery of pH_i from the initial CO₂-induced acidification, the major difference between this experiment and the one in Fig. 1 A is that applying HCO₃ ⁻ /CO₂ elicited an immediate hyperpolarization of 85 mV. This hyperpolarization partially decayed over the course of 12 min. The slow pH_i recovery and the large hyperpolarization are both consistent with the electrogenic influx of Na⁺ and HCO₃ ⁻. Previous work has shown that pretreating akNBC-expressing oocytes with DIDS blocks both the pH_i recovery and the V_m changes (Romero et al. 1997a).

Figure 1.

Membrane potential and pH_i of Xenopus laevis oocytes during a superfusion of 33 HCO₃ ⁻/5% CO₂ solution. (A) Water-injected oocyte. The CO₂/HCO₃ ⁻ solution is Solution 2 in Table . Typical of six experiments. (B) Oocyte expressing rkNBC. Typical of nine experiments. pH_o 7.5, 22°C.

Effect on V_m of adding graded levels of HCO₃ ⁻/CO₂ at a constant pH_o of 7.5.

The expression level of the akNBC clone, as judged in voltage-clamp experiments (not shown), was not sufficiently high to allow us to measure NBC currents accurately at low values of [HCO₃ ⁻ ]_o. To obtain a first approximation of the [HCO₃ ⁻]_o dependence of akNBC, we monitored changes in V_m while briefly applying extracellular solutions containing various levels of HCO₃ ⁻/CO₂ . Fig. 2 shows a typical experiment. We began with the oocyte in our standard gluconate-HEPES solution ( Table , Solution 1). We then switched to our standard gluconate-HCO ₃ ⁻/CO₂ solution, which was buffered to pH 7.50 with 33 mM HCO₃ ⁻ and 5% CO₂ (Solution 2), and determined the maximal change in V_m (ΔV_m). After returning the oocyte to the HEPES-buffered solution and waiting for V_m to stabilize, we exposed the cell to the first of five test HCO₃ ⁻/CO₂ solutions, each having a pH of 7.50. During the rest of the experiment, we bracketed each test HCO₃ ⁻/CO₂ pulse with a standard HCO₃ ⁻/CO₂ pulse. To compensate for differences in the expression level of akNBC in individual oocytes, we then obtained a normalized ΔV_m by computing the ratio of the ΔV_m of the test pulse to the mean ΔV_m of the two bracketing standard pulses.

Figure 2.

Membrane potential of akNBC-expressing Xenopus laevis oocytes during superfusion of solutions with different levels of HCO₃ ⁻/CO₂. In our assay, we bracketed each test pulse with a pulse of the standard (std) CO₂/HCO₃ ⁻ solution (33 mM HCO₃ ⁻/5% CO ₂, Solution 2 in Table ). We normalized the ΔV_m under test conditions to the mean ΔV_m for the bracketing std pulses. The HEPES-buffered solution was Solution 1 in Table . Typical of nine experiments.

Curve fitting.

As noted in the discussion, we attempted to fit the normalized akNBC data (Fig. 3, ▪) with a variety of “single-enzyme” rapid-equilibrium kinetic models. Visually, none of these fits was fully satisfactory. We also fitted our normalized ΔV_m data with a model for two enzymes catalyzing the same reaction. Although this fit was visually satisfactory (not shown), the higher of the two K _m(HCO₃ ⁻ ) values (i.e., ∼231 mM) was far higher than the highest [HCO ₃ ⁻]_o tested (i.e., 99 mM), and the standard deviation of this K _m(HCO₃ ⁻) was more than twofold higher than the K _m value. Therefore, we fitted the normalized ΔV_m akNBC data with a kinetic model for a single Michaelis-Menten process plus a linear component (). The result of this fit is shown as the solid curve in Fig. 3. As summarized in Table , the apparent K _m(HCO₃ ⁻) was 10.6 mM.

Figure 3.

[HCO₃ ⁻]_o dependence of akNBC and rkNBC, based on ΔV_m data. The solid curve represents the result of a nonlinear least-squares curve fit of to the akNBC data (▪) similar to those shown in Fig. 2 . The broken curve represents the result of a similar fit to the rkNBC data (□). Each symbol represents the mean of six to nine data points, obtained in separate experiments. The vertical bars represent SEMs; the bars are omitted when they are smaller than the size of the symbol. The kinetic parameters are summarized in the first two lines of Table .

Table 2.

[HCO₃ ⁻]_o Dependence of akNBC and rkNBC^‡

NBC	K m (HCO3 −)	Relative v*max	α*	n	Method
	mM		mM−1
akNBC	10.6 ± 1.2	1.22 ± 0.06	0.00226	33	Membrane voltage
rkNBC	10.8 ± 4.4	1.25 ± 0.21	0.00177	45	Membrane voltage
rkNBC	6.5 ± 0.7	0.97 ± 0.03	0.00577	58	Membrane current

^‡The parameter values were obtained using and .

[HCO₃ ⁻]_o Dependence of rkNBC, Based on Changes in V_m

To compare the HCO₃ ⁻ dependencies of rkNBC and akNBC, we used a protocol identical to that used in Fig. 2, except that we used oocytes expressing rkNBC rather than akNBC. The results of this series of rkNBC experiments are summarized (Fig. 3, □). The broken curve represents the result of the nonlinear least-squares fit of . The result is an apparent K _m(HCO₃ ⁻) of 10.8 mM, which is not different from the value obtained for akNBC (Table ).

[HCO₃ ⁻]_o Dependence of rkNBC, Studied in Voltage-clamped Oocytes

Because NBC is voltage dependent (Heyer et al. 1999), negative shifts in V_m produced by NBC would slow the very transporter responsible for the V_m change. Because the expression of rkNBC (as judged by NBC-dependent currents obtained under voltage-clamp conditions) was much higher than for akNBC, we elected to use the voltage-clamp approach to study the [HCO₃ ⁻ ]_o dependence of rkNBC.

Effect of 99 mM HCO₃ ⁻ on membrane current.

Fig. 4 A shows that briefly exposing a control (i.e., H₂O-injected) oocyte to a solution containing 99 mM HCO₃ ⁻/15% CO₂ caused very little change in the membrane current (V_hold = −60 mV). However, as shown in Fig. 4 B, the same maneuver elicited an outward current of ∼500 nA in an oocyte expressing rkNBC. Fig. 4 C shows the results from a second oocyte expressing rkNBC. Here, the HCO₃ ⁻/CO ₂ exposure caused almost no change in membrane current in the absence of Na⁺. Restoring the extracellular Na⁺ substantially increased the current elicited by adding 99 mM HCO₃ ⁻/15% CO₂. Thus, the current elicited by HCO₃ ⁻ depends on the expression of rkNBC and requires Na⁺. Previous work has shown that pretreating oocytes with 200 μM DIDS blocks the activity of rkNBC ( Romero et al. 1996, Romero et al. 1997b).

Figure 4.

Dependence of HCO₃ ⁻-evoked currents on the expression rkNBC and the presence of Na⁺. (A) H₂ O-injected, control oocyte. (B) Oocyte expressing rkNBC. (C) Effect of removing Na⁺ in an oocyte expressing rkNBC. In each case, we pulsed the oocyte with a pH 7.5 solution containing 99 mM HCO₃ ⁻/15% CO₂. V_hold = −60 mV, 22°C.

Effect on membrane current of adding graded levels of CO₂ /HCO₃ ⁻ at a constant pH_o of 7.5.

We used the peak amplitude of the current induced by exposing oocytes to HCO₃ ⁻/CO₂ as a measure of the inward, electrogenic transport of Na⁺ and HCO₃ ⁻ via rkNBC. Otherwise, the protocol we used was the same as in Fig. 2. A typical experiment is shown in Fig. 5. We computed a normalized ΔI by dividing the ΔI of the test pulse to the mean ΔI of the two bracketing standard pulses ([HCO₃ ⁻]_o = 33 mM). The normalized ΔI data are summarized in Fig. 6 (•). The curve represents the result of a nonlinear least-squares fit of . The apparent K _m (HCO₃ ⁻) was 6.5 mM (Table ). This K _m(HCO₃ ⁻) value for rkNBC in ΔI experiments is substantially less than for the same clone in ΔV_m experiments.

Figure 5.

Membrane current of rkNBC-expressing Xenopus laevis oocyte during superfusion of solutions with different levels of HCO₃ ⁻/CO₂. The protocol for changing the extracellular solutions was the same as in Fig. 2 . The standard (std) solution contained 33 mM HCO₃ ⁻/5% CO₂ (Table , Solution 2). Typical of eight experiments. V_hold = −60 mV, 22°C.

Figure 6.

[HCO₃ ⁻]_o dependence of rkNBC current. The solid curve represents the result of a nonlinear least-squares curve fit of to the data (•) similar to those shown in Fig. 5. Each symbol represents the mean of five to eight data points obtained in separate experiments. The vertical bar represents the SEM; the bars are omitted when they are smaller than the size of the symbol. The kinetic parameters are summarized in the last line of Table .

Effect of Sulfate and Sulfite/Bisulfite on the [HCO₃ ⁻]_o Dependence of the rkNBC Current

To test the hypothesis (see introduction) that NBC can transport SO₃ ⁼ or HSO₃ ⁻, we first examined the effect of SO₃ ⁼/HSO₃ ⁻ on membrane currents carried by rkNBC expressed in Xenopus oocytes. As a control, we examined the effects of another divalent anion, sulfate (SO₄ ⁼).

Effect of SO₄ ⁼ and SO₃ ⁼ on rkNBC current evoked by 33 mM HCO₃ ⁻.

Fig. 7 shows a voltage-clamp experiment in which we examined the effect of SO₄ ⁼ and SO₃ ⁼/HSO₃ ⁻ on the peak current produced by 33 mM HCO₃ ⁻ in an oocyte expressing rkNBC. The changes in current evoked by 33 mM HCO₃ ⁻ were virtually identical regardless of whether the dominant background anion was 66 mM gluconate, 33 mM SO₄ ⁼ or 33 mM total SO₃ ⁼/HSO₃ ⁻ (i.e., 26.4 mM SO₃ ⁼ + 6.6 mM HSO ₃ ⁻). In a total of six similar experiments, the ratio of the current in SO₄ ⁼ to the bracketing-paired currents in gluconate was 0.982. Similarly, in seven experiments, the ratio of the current in SO₃ ⁼ /HSO₃ ⁻ to the bracketing-paired currents in gluconate was 0.999. The difference between these mean ratios is not statistically significant (P = 0.13, one tail t test). Thus, under the conditions of our experiments, the current carried by rkNBC in 33 mM HCO₃ ⁻ is virtually identical in the presence of SO₄ ⁼ or SO₃ ⁼ /HSO₃ ⁻. It is therefore likely that SO ₃ ⁼/HSO₃ ⁻ per se has no effect on the NBC current.

Figure 7.

Effect of SO₄ ⁼ and SO₃ ⁼/HSO ₃ ⁻ on the current carried by rkNBC. The oocyte was exposed five times to a solution containing 33 mM HCO₃ ⁻/5% CO₂. For the first, third, and fifth pulses, we switched from a HEPES solution (Table , Solution 1) to a solution containing 33 mM HCO₃ ⁻/5% CO₂ solution (Table , Solution 2). For the second HCO₃ ⁻/CO₂ pulse, we switched from a HEPES solution containing 33 mM SO₄ ⁼ (Table , Solution 3) to a 33 mM HCO₃ ⁻/5% CO₂ that also contained 33 mM SO₄ ⁼ (Table , Solution 4). For the fourth HCO₃ ⁻ /CO₂ pulse, we switched from a HEPES-containing 33 mM SO₃ ⁼/HSO₃ ⁻ (Table , Solution 5) to a 33-mM HCO₃ ⁻/5% CO₂ solution that also contained 33 mM SO₃ ⁼/HSO₃ ⁻ (Table , Solution 6). Typical of six experiments. V_hold = −60 mV, 22°C.

Effect of SO₄ ⁼ and SO₃ ⁼ on the extracellular HCO₃ ⁻ dependence of the rkNBC current.

Fig. 8 summarizes the results of typical voltage-clamp experiments in which we examined the [HCO₃ ⁻]_o dependence of the rkNBC current using the same protocol as in Fig. 5, but with either SO₄ ⁼ (Fig. 8 A) or SO ₃ ⁼/HSO₃ ⁻ (B) as the dominant anion. Because, in these experiments, [gluconate]_o was only 33 mM when [HCO₃ ⁻]_o was 0 mM, we could only investigate the HCO₃ ⁻ dependence of NBC in the [HCO₃ ⁻]_o range of 0–33 mM.

Figure 8.

Effect of SO₄ ⁼ and SO₃ ⁼/HSO ₃ ⁻ on the [HCO₃ ⁻] _o dependence of the current carried by rkNBC. (A) Experiments conducted in 33 mM SO₄ ⁼. The experimental protocol was the same as in Fig. 5, except that all solutions contained 33 mM SO₄ ⁼. Typical of eight experiments. V_hold = −60 mV, 22°C, pH 7.5. (B) Experiments conducted in 26.4 mM SO₃ ⁼/6.6 mM HSO ₃ ⁻. The protocol was the same as in A. Typical of 10 experiments. (C) Effect of SO₄ ⁼ and SO₃ ⁼/HSO₃ ⁻ on [HCO₃ ⁻]_o dependency of rkNBC. One of the solid curves is the same as that in Fig. 6, and represents the fit of to the data obtained in the absence of SO₄ ⁼ and SO₃ ⁼/HSO₃ ⁻ (○). The other two solid curves represent the fits of to the data obtained in SO₄ ⁼ (▵), as in A, and the data obtained in SO₃ ⁼/HSO ₃ ⁻ (▴), as in B. Each symbol represents the mean of 6–17 data points, obtained in separate experiments. The bars representing SEM are omitted because they are smaller than the size of the symbol.

Fig. 8 C summarizes the data as well as the curve fits. Because the SO₄ ⁼ and SO₃ ⁼/HSO₃ ⁻ data in the range of 0–33 mM HCO₃ ⁻ did not permit an accurate determination of a slope of the linear component (i.e., α*), we assumed that α* was the same as that obtained in the fit of the gluconate data in Fig. 6 ([HCO₃ ⁻]: 0–99 mM). The results of these curve fits ( ) are summarized in Table , and show that the apparent K _m and v_max values are virtually identical, regardless of whether HCO₃ ⁻ was varied in the presence of gluconate, SO₄ ⁼ or SO₃ ⁼ /HSO₃ ⁻.

Effect of Sulfite/Bisulfite on DIDS-sensitive pH_i Change in Oocytes Expressing rkNBC

Is it possible that SO₃ ⁼/HSO₃ ⁻ could ride rkNBC and yet not produce a change in current? If NBC could neither distinguish SO₃ ⁼ from CO₃ ⁼, nor HSO₃ ⁻ from HCO ₃ ⁻, then introducing SO₃ ⁼ /HSO₃ ⁻ would have no effect on the current carried by NBC if the transporter were already near v _max. However, because the pK values governing the reactions SO ₃ ⁼ + H⁺ ↔ HSO₃ ⁻ and HSO₃ ⁻ + H⁺ ↔ H₂SO₃ are so much lower than for the corresponding reactions involving CO₃ ⁼, HCO₃ ⁻, and H₂CO₃, the pH_i changes for NBC carrying SO₃ ⁼/HSO₃ ⁻ would be much slower than for NBC carrying CO₃ ⁼/HCO₃ ⁻ (see discussion ). We therefore examined the possibility that SO₃ ⁼ /HSO₃ ⁻ would slow the pH_i produced by NBC in the presence of CO₂/HCO₃ ⁻.

Our assay was to expose an rkNBC-expressing oocytes to an extracellular solution buffered with 3.3 mM HCO₃ ⁻/0.5% CO ₂. As shown in Fig. 9 A, an experiment conducted in the absence of SO₃ ⁼/HSO ₃ ⁻, applying HCO₃ ⁻ /CO₂ causes a rapid but small pH_i decrease (a–b), followed by a pH_i increase (b–c). After ∼30 min, when pH_i was recovering at a constant rate in the HCO₃ ⁻/CO₂ solution, we applied 1 mM DIDS for ∼15 min. This DIDS blocked the NBC-mediated alkalinization, unmasking a slow acidification (c–d). We took the difference between the alkalinization rate in the absence of DIDS (b–c) and the presence of DIDS (c–d) as an index of the net base influx mediated by rkNBC. In a total of five similar experiments, the DIDS-dependent alkalinization rate was 0.98 ± 0.27 × 10⁻⁴ pH U/s, with a mean initial pH_i value of 7.34 ± 0.04.

Figure 9.

Effect of SO₃ ⁼/HSO₃ ⁻ on the DIDS-sensitive recovery of pH_i from a CO₂ -induced acid load. (A) Absence of SO₃ ⁼/HSO ₃ ⁻. During the indicated time, the solution bathing an oocyte expressing rkNBC was switched from standard HEPES ( Table , Solution 1) to a solution containing 3.3 mM HCO₃ ⁻/0.5% CO₂. During the pH_i recovery from the CO₂-induced acid load, we blocked rkNBC by applying 1 mM DIDS. (B) Presence of 26.4 mM SO₃ ⁼/6.6 mM HSO ₃ ⁻. The protocol was the same as in A, except that all solutions contained 26.4 mM SO₃ ⁼/6.6 mM HSO ₃ ⁻.

The experiment in Fig. 9 B is the same as in A, except that the oocyte was exposed to 26.4 mM SO₃ ⁼ /6.6 mM HSO₃ ⁻ during the application of the 3.3 mM HCO₃ ⁻/0.5% CO₂ solution. In a total of six such experiments, the mean net base influx was 0.88 ± 0.38 × 10⁻⁴ pH U/s, which is not significantly different from the value in the absence of SO₃ ⁼/HSO₃ ⁻ (P = 0.28, an unpaired one tail t test). The mean initial pH_i in the SO₃ ⁼/HSO₃ ⁻ experiments was 7.39 ± 0.06, which also is not significantly different from the value in the absence of SO₃ ⁼/HSO₃ ⁻ (P = 0.20, unpaired two tail t test).

Effect of Altering [CO₃ ⁼]_o and pH_o on the Current Carried by rkNBC

Because the data introduced above make it unlikely that rkNBC, as expressed in Xenopus oocyte, interacts with HSO₃ ⁻ or SO₄ ⁼, we asked whether rkNBC transports CO₃ ⁼. Our approach was to hold [HCO₃ ⁻]_o constant at 33 mM while raising [CO ₂] from 0.1% (pH_o 9.2, [CO₃ ⁼] _o = ∼3,500 μM) to 100% (pH_o 6.2, [CO₃ ⁼]_o = ∼3.5 μM). Our protocol was similar to that in Fig. 5, with two pulses of our standard solution (Table , Solution 2, [CO₃ ⁼ ]_o = ∼70 μM) bracketing each test pulse. Because CaCO₃ precipitated from the pH 9.2 solution, which nominally contains ∼3,500 μM CO₃ ⁼, we replaced all Ca ²⁺ with Mg²⁺. Control experiments showed that this switch has no effect on the NBC current.

Fig. 10 A summarizes our results, expressed as normalized ΔI data as a function of [CO₃ ⁼] _o. The dashed curve, which represents the best fit of a normalized Michaelis-Menten equation (total residual variance = 0.0341), systematically passes above or below points, depending where they lie along the curve. The solid curve, which represents the best fit of the normalized Michaelis-Menten equation plus a linear component [total residual variance (trv) = 0.0088], also systematically misfits the data. On the other hand, when we plot the same data as a function of pH _o (Fig. 10 B), whether the best-fit pH titration curve (pK = 7.50 ± 0.05) passes above or below a point does not depend systematically on the position of the point. In addition, the total residual variance of this fit (trv = 0.0055) is comparable with that in Fig. 6 (trv = 0.0043).

Figure 10.

Effect of varying [CO₃ ⁼]_o and pH_o on the rkNBC current. (A) Relative rkNBC current as a function of [CO₃ ⁼]_o. • represent data obtained in the presence of Ca²⁺, and ○, with Mg²⁺ replacing Ca²⁺. The dashed curve is the result of a nonlinear least-squares fit of the data by a normalized Michaelis-Menten equation. The best-fit value for K _m(CO₃ ⁼) was 6.1 ± 1.5 μΜ, and for I_max, 1.09. The solid curve represents the best fit of the data by a normalized Michaelis-Menten equation plus a linear component. The best-fit value for K _m(CO₃ ⁼) was 4.5 ± 0.6 μΜ, for I_max was 1.05, and for α was 0.000122 μΜ⁻¹. (B) Relative rkNBC current as a function of pH_o. The solid curve is the result of a nonlinear least-squares fit of the data by a normalized pH titration curve ( Boron and Knakal 1992). The best-fit value for pK was 7.50 ± 0.05. The number of determinations is given in parentheses. The vertical bars indicate SEM values; they are omitted where the length of the bar is smaller than the size of the symbol.

DISCUSSION

Why Is the NBC Current–[HCO₃ ⁻] _o Relationship Not Sigmoidal?

As expressed in the Xenopus oocyte, rkNBC is electrogenic. This observation is consistent with a Na⁺:HCO₃ ⁻ stoichiometry of 1:2, or perhaps 1:3, as has been observed in membrane vesicles prepared from rabbit kidney ( Soleimani et al. 1987). Recent voltage-clamp experiments suggest that rkNBC, at least as expressed in oocytes, has a stoichiometry of 1:2 (Heyer et al. 1999; Sciortino and Romero 1999). Thus, one would not be surprised if the relationship between NBC current and [HCO₃ ⁻ ]_o were more complex than a simple right-rectangular hyperbola, for example. In fact, we found that the current–[HCO ₃ ⁻]_o relationship is well described by the sum of a hyperbola and a line. Why did we not observe a sigmoidal current–[HCO₃ ⁻]_o relationship?

First, it is possible that, as expressed in Xenopus oocytes, rkNBC has but a single HCO₃ ⁻-related substrate. If rkNBC carried a single CO₃ ⁼ (equivalent to two HCO₃ ⁻), then the Na⁺ :HCO₃ ⁻ stoichiometry would be 1:2, and thus one would expect the current–[HCO₃ ⁻ ]_o relationship to be a right-rectangular hyperbola. Thus, our data are consistent with the hypothesis that rkNBC binds a single HCO₃ ⁻ related species, CO₃ ⁼.

Second, if rkNBC carried two HCO₃ ⁻ ions (for a stoichiometry of 1:2) or one HCO₃ ⁻ and one CO₃ ⁼ (for a stoichiometry of 1:3), then the current–[HCO₃ ⁻]_o relationship might show a foot at low [HCO₃ ⁻]_o, but only if the K _m values for the two binding sites were sufficiently similar and high. For example, if rkNBC carried two HCO ₃ ⁻ ions, and the K _m values for one binding site was 6.5 mM (as observed), but the K _m for the other was only 0.1 mM, then we would not have been able to detect a foot, given the precision of our data. Thus, our results are consistent with the hypothesis that rkNBC binds two HCO₃ ⁻-related species, but that we cannot detect a foot due to a low K _m value.

Third, if rkNBC carried three HCO₃ ⁻ ions (for a stoichiometry of 1:3), then the current–[HCO₃ ⁻]_o relationship might show a foot, but, again, only if the K _m values for all three were sufficiently similar and high. For example, if the K _m values were 6.5, 6.5, and 0.1 mM, or 6.5, 0.1, and 0.1 mM, we would not have been able to detect a foot. Thus, our data are consistent with the hypothesis that rkNBC binds three HCO₃ ⁻ , but that we cannot detect a foot due to a low K _m value.

Fourth, it is possible that a systematic error in the way we monitored rkNBC activity may have masked a foot. For example, when we expose a cell to HCO₃ ⁻/CO₂, rkNBC transports Na ⁺ and HCO₃ ⁻ into the cell, and the passive entry of CO₂ leads to the production of HCO₃ ⁻ and H⁺. We attempted to minimize such effects by making our measurements very soon after exposing the cell to HCO₃ ⁻/CO₂. Nevertheless, any buildup of intracellular Na⁺, HCO₃ ⁻ , and/or H⁺ that might have occurred in the vicinity of rkNBC would have slowed the cotransporter; the effect would have been greater at higher HCO₃ ⁻/CO₂ levels.

The “Linear Component”

The present study represents the first kinetic experiments on a member of the newly cloned NBC family. As suggested above, we would not have been surprised had the current–[HCO₃ ⁻]_o relationship been sigmoidal. Instead, the shape of the relationship appears to be the sum of a hyperbola and a line. We could not adequately fit the current versus [HCO₃ ⁻]_o data using any of several rapid-equilibrium models for random or ordered binding of HCO₃ ⁻/CO₃ ⁼ to the cotransporter. We therefore suggest that some additional process, which is a first-order function of [HCO₃ ⁻]_o, contributes to the current, especially at [HCO₃ ⁻]_o values above 33 mM. This linear component is not present in water-injected oocytes. As shown in Fig. 4 A, the transition from HEPES to 99 mM HCO₃ ⁻ caused a slow and small (∼6 nA) outward current in control oocytes. In contrast, as shown in Fig. 4 B, the same maneuver caused a rapid and large (∼490 nA) outward current in rkNBC-expressing oocytes.

The linear component also requires Na⁺. As shown in Fig. 4 C, a transition from Na⁺ -free HEPES to Na⁺-free 99 mM HCO₃ ⁻ caused only slow and small (∼9 nA) outward current in rkNBC-expressing Xenopus oocytes. However, in the presence of Na ⁺, the transition from HEPES to 99 mM HCO₃ ⁻ produced a much larger current (∼140 nA). Because virtually the entire HCO₃ ⁻-induced current at 99 mM HCO₃ ⁻ requires both rkNBC and Na⁺ , it is very likely that the linear component is carried by rkNBC or a closely related protein. What are the possible sources of the linear component of the current?

First, expression of rkNBC might induce the expression of a previously silent, endogenous NBC-like protein with a low affinity for HCO₃ ⁻. As described in several reports, the expression of exogenous membrane proteins induces various endogenous channels in Xenopus oocytes (Attali et al. 1993, Attali et al. 1995; Shimbo et al. 1995; Tzounopoulos et al. 1995; Buyse et al. 1997).

Second, the linear component could represent a parallel HCO₃ ⁻-conductance pathway that is part of rkNBC. The glutamate transporters (Fairman et al. 1995) have an intrinsic Cl⁻ conductance, and the electroneutral Na/HCO₃ cotransporter has an intrinsic conductance to Na⁺ (Choi, I., C. Aalkjaer, E.L. Boulpaep, and W.F. Boron, personal communication).

Third, it is possible that increases in [CO₂] and/or [HCO ₃ ⁻] cause the Na⁺:HCO₃ ⁻ stoichiometry of rkNBC to shift from, say, 1:2 to 1:3. If the turnover of rkNBC were governed by a classical kinetic model, then the shift in stoichiometry would lead to greater currents at greater values of [HCO₃ ⁻]_o.

Fourth, it is possible that changes in the concentrations of gluconate and CO₃ ⁼, both of which chelate Ca²⁺, led to changes in free [Ca²⁺]_o that affected NBC. However, in the series of experiments summarized in Fig. 10, we showed that replacing all Ca²⁺ with Mg²⁺ has no effect on the current carried by NBC.

Effect of Extracellular HSO₃ ⁻/SO₃ ⁼ on rkNBC

We found that SO₃ ⁼/HSO₃ ⁻ affected neither the currents (Fig. 7) nor the pH_i changes (Fig. 9) produced by rkNBC as it functions as a heterologously expressed protein in Xenopus oocytes. To assess these results, we examined a series of models (Fig. 11, A–G and A′–G′) for how SO₃ ⁼/HSO₃ ⁻ might interact with NBC, and predicted the effects of these interactions on the currents and pH_i changes produced by NBC. In A and A′, we assume that neither SO₃ ⁼ nor HSO₃ ⁻ is capable of interacting with NBC, so that SO₃ ⁼/HSO₃ ⁻ should have no effect on either NBC-mediated currents or pH_i changes, as we in fact observed.

Figure 11.

Predicted effects of SO₃ ⁼ and/or HSO₃ ⁻ on the pH_i changes mediated by NBC. A–G refer to a general scheme in which NBC transports one Na⁺ , one CO₃ ⁼, and one HCO₃ ⁻. (A) Neither SO₃ ⁼ nor HSO₃ ⁻ interact with cotransporter. The entering CO ₃ ⁼ can neutralize two H⁺, and the entering HCO₃ ⁻ can neutralize an additional H ⁺, for a total of three H⁺ neutralized. (B) SO₃ ⁼ replaces CO₃ ⁼. To the extent that SO₃ ⁼ replaces CO₃ ⁼, only 1.24 H⁺ are neutralized, and thus the expected rate of pH_i increase will be 41% of that in A. (C) HSO₃ ⁻ replaces HCO₃ ⁻. Only two H⁺ are neutralized, and thus the expected rate of pH_i increase will be only 67% of that in A. (D) SO₃ ⁼ and HSO ₃ ⁻ replace, respectively, CO₃ ⁼ and HCO₃ ⁻. Only total 0.24 H⁺ ions are neutralized, and thus the expected rate of pH_i increase will be only 8% of that in A. (E) SO₃ ⁼ acts as a competitive inhibitor of CO₃ ⁼. (F) HSO₃ ⁻ acts as a competitive inhibitor of HCO₃ ⁻. (G) SO₃ ⁼ and HSO₃ ⁻ both are competitive inhibitors. A′–G′ refer to a general scheme in which NBC transports one Na⁺ and three HCO₃ ⁻. (A′) Neither SO₃ ⁼ nor HSO₃ ⁻ interact with cotransporter. A total of three H⁺ are neutralized. (B′) HSO₃ ⁻ replaces one HCO ₃ ⁻ _. Only two H⁺ are neutralized, and thus the expected rate of pH_i increase will be 67% of that in A′. (C′) Two HSO₃ ⁻ replace two HCO₃ ⁻ _. Only one H⁺ is neutralized, and thus the expected rate of pH_i increase will be only 33% of that in A′. (D′) Three HSO₃ ⁻ replace three HCO₃ ⁻. No H⁺ ions are neutralized, and thus the expected rate of pH _i increase will be 0% of that in A′. In E′–G′, HSO₃ ⁻ acts as a competitive inhibitor at one, two, and three HCO₃ ⁻ -binding sites, respectively.

Figure 11a.

Figure 11b.

In Fig. 11b–D and Fig. b′– D′, NBC transports SO₃ ⁼ and/or HSO₃ ⁻. Although it is conceivable that SO₃ ⁼/HSO₃ ⁻ might not affect the currents that NBC carries (depending on the concentrations and K _m values for SO₃ ⁼ , HSO₃ ⁻, CO₃ ⁼, and HCO₃ ⁻), the pH_i changes would be appreciably slower for three reasons. (a) The pK of the reaction SO₃ ⁼ + H⁺ ↔ HSO₃ ⁻ is ∼6.9, compared with ∼10 for the equilibrium CO₃ ⁼ + H⁺ ↔ HCO₃ ⁻ . (b) The pK of the reaction HSO₃ ⁻ + H ⁺ ↔ H₂SO₃ is ∼1.6, compared with ∼3.4 for the equilibrium HCO₃ ⁻ + H⁺ ↔ H₂CO₃. (c) [H₂SO₃ ]_i is so low that the net efflux of H₂SO₃ is expected to be negligible. In contrast, H₂CO₃ forms CO₂, which is present at relatively high concentrations and can rapidly exit the cell. The net effect is that incoming SO₃ ⁼ and/or HSO₃ ⁻ will neutralize fewer H⁺ than incoming CO₃ ⁼ and/or HCO₃ ⁻. Because we found that SO₃ ⁼/HSO₃ ⁻ had no effect on NBC-mediated pH_i changes, Fig. 11b–D and Fig. b ′–D′, must be incorrect (i.e., NBC cannot transport SO₃ ⁼ and/or HSO₃ ⁻ under the conditions of our experiments).

In Fig. 11E–G and Fig. E′–G′, SO₃ ⁼ and/or HSO₃ ⁻ are competitive inhibitors for the transport of CO₃ ⁼ and/or HCO₃ ⁻, respectively. In these cases, adding SO₃ ⁼/HSO₃ ⁻ should decrease both the NBC current and pH_i changes. Inasmuch as we found that SO₃ ⁼/HSO₃ ⁻ had no effect on either, E–G and E′–G′ must be incorrect (i.e., neither SO₃ ⁼ nor HSO₃ ⁻ can competitively inhibit NBC under the conditions of our experiments).

Thus, we conclude that neither SO₃ ⁼ nor HSO₃ ⁻ interacts with rkNBC under the conditions of our experiments. This conclusion is in agreement with an observation of Jentsch et al. 1986 on bovine corneal endothelial cells. However, Soleimani and Aronson 1989, in studies on renal basolateral membrane vesicles, reported that SO₃ ⁼ /HSO₃ ⁻, when applied in the presence of HCO₃ ⁻, stimulates ²²Na uptake mediated by the Na/HCO₃ cotransporter. Based on this and other data, those authors concluded that NBC transports Na⁺, CO₃ ⁼, and HCO₃ ⁻ in a stoichiometry of 1:1:1, and that SO₃ ⁼ can substitute for CO₃ ⁼ at the CO₃ ⁼ binding site. This line of reasoning was the first, and probably the strongest, evidence that NBC can transport CO₃ ⁼.

We could reconcile our data and those of Jentsch et al. 1986 with the data of Soleimani and Aronson 1989 by proposing that (a) oocytes and corneal endothelial cells have a “factor” that prevents the SO₃ ⁼/HSO₃ ⁻ –rkNBC interaction, or (b) oocytes and corneal endothelial cells lack a factor required for the SO₃ ⁼/HSO₃ ⁻–rkNBC interaction. We think that the latter is more likely. The missing factor could be an enzyme(s) that catalyzes a posttranslational modification of NBC (e.g., phosphorylation) that is essential for the NBC–SO₃ ⁼/HSO₃ ⁻ interaction, or the missing factor could be an additional NBC subunit that confers sensitivity to SO₃ ⁼ /HSO₃ ⁻. Alternatively, the missing factor triggered by SO₃ ⁼/HSO₃ ⁻ could be part of a purely regulatory pathway that modulates NBC (i.e., not an intrinsic part of NBC). Thus, although the rkNBC protein expressed in Xenopus oocytes can carry out all other known functions of the renal NBC, rkNBC by itself cannot interact with SO₃ ⁼ or HSO₃ ⁻.

Effect of Altering [CO₃ ⁼]_o and pH_o on rkNBC

Fig. 10 shows the effect on the current carried by rkNBC of simultaneously varying [CO₃ ⁼] _o and pH_o. The best-fit Michaelis-Menten curve, with or without a linear component, fails to adequately fit the data, expressed in terms of [CO₃ ⁼]_o (Fig. 10 A). On the other hand, the best-fit pH titration curve nicely fits the data, expressed in terms of pH_o, over the entire range of pH_o values. One possible explanation for these results is that rkNBC transports CO₃ ⁼ with a K _m of ∼6 μM, but that an idiosyncratic pH_o sensitivity is responsible for the poor fits at the extreme [CO ₃ ⁼]_o values. However, the most straightforward explanation for these data is that rkNBC is not sensitive to [CO₃ ⁼]_o in the range 3.5–3,500 μM, but has a single titratable site that inhibits NBC when protonated. For example, this site could be an HCO₃ ⁻ -binding site that has a lower affinity for its substrate when protonated. Note that we cannot rule out the possibility that rkNBC transports CO₃ ⁼ with an extremely high affinity (i.e., a K _m << 3.5 μM).