Sodium/Bicarbonate Cotransporter NBCn1/Slc4a7 Inhibits NH₄Cl-mediated Inward Current in Xenopus Oocytes

Abstract

The electroneutral Na/HCO₃ cotransporter NBCn1 (SLC4A7) contributes to intracellular pH maintenance and transepithelial HCO₃⁻ movement. In this study, we expressed NBCn1 in Xenopus oocytes and examined the effect of NBCn1 on oocyte NH₄⁺ transport by analyzing changes in membrane potential, current, and intracellular pH mediated by NH₄Cl. In the presence of HCO₃⁻/CO₂, applying NH₄Cl (20 mM) produced intracellular acidification of oocytes. The acidification was faster in oocytes expressing NBCn1 than in control oocytes injected with water. However, NH₄Cl-mediated membrane depolarization was smaller in oocytes expressing NBCn1. In HCO₃⁻/CO₂-free solution, NH₄Cl produced a smaller inward current in NBCn1-expressing oocytes (56% inhibition by 20 mM NH₄Cl; measured at −60 mV), while minimally affecting intracellular acidification. The inhibition of the current by NBCn1 was unaffected when BaCl₂ replaced KCl. Current-voltage relationships showed a positive and nearly linear relationship between NH₄Cl-mediated current and voltage, which was markedly reduced by NBCn1. Large basal currents (before NH₄Cl exposure) were produced in NBCn1-expressing oocytes due to the previously characterized channel-like activity of NBCn1. Inhibiting this channel-like activity by Na⁺ removal abolished NBCn1’s inhibitory effect on NH₄Cl-mediated currents. The currents were progressively reduced over 72–120 h after NBCn1 cRNA injection, during which the channel-like activity was high. These results indicate that NBCn1 by its Na/HCO₃ cotransport activity stimulates NH₄⁺ transport, while reducing NH₄⁺ conductance by its channel-like activity.

INTRODUCTION

The kidney regulates blood pH by reclaiming filtered acid and base equivalents or by releasing them into urine. Two major acid-base components that are regulated by the kidney are HCO₃⁻ and NH₄⁺. HCO₃⁻ is filtered in the glomerulus, and filtered HCO₃⁻ is then reabsorbed in the proximal tubule (70–80%), thick ascending limb (10–15%), and cortical collecting duct (~5%) (Boron 1992). NH₄⁺, which is the main component of urinary net acid excretion, is synthesized and secreted in the proximal tubule, reabsorbed in the thick ascending limb, and is secreted again into the lumen in the collecting duct (Knepper et al. 1989). The kidney makes adaptive changes in HCO₃⁻ and NH₄⁺ absorption in response to acid-base disturbance, particularly to a low blood pH (Wagner 2007;Kraut & Kurtz 2005). Chronic metabolic acidosis stimulates HCO₃⁻ absorptive capacity and NH₄⁺ production/secretion in the proximal tubule and increases the net HCO₃⁻ and NH₄⁺ absorption in the thick ascending limb.

The absorption of HCO₃⁻ or NH₄⁺ is primarily governed by ion transporters/channels that move these ions across the plasma membrane (Weiner & Verlander 2011;Cordat & Casey 2009;Sindic et al. 2007;Pushkin & Kurtz 2006). In particular, the electroneutral Na/HCO₃ cotransporter NBCn1 (SLC4A7) plays an important role in transepithelial HCO₃⁻ movement in epithelia (Aalkjaer et al. 2004;Romero et al. 2004;Boron et al. 2009). NBCn1, which is a member of SLC4A bicarbonate transporters, normally moves HCO₃⁻ into the cytosol down the Na⁺ gradient in many different cells. Thus, the primary function of this transporter is to extrude acid (Pushkin et al. 1999;Lauritzen et al. 2010;Riihonen et al. 2010). In addition, the transporter has associated ionic conductance that is primarily governed by Na⁺ (Choi et al. 2000;Cooper et al. 2005). This channel-like activity can affect membrane potential and intracellular Na⁺ levels in heterologous expression systems (Choi et al. 2000;Cooper et al. 2005). The physiological role of this channel-like activity is unclear.

We recently reported that NBCn1 stimulates radiolabeled methylammonium uptake in Xenopus oocytes (Lee et al. 2010). Oocyte membranes have been considered to be less permeable to NH₃ than to NH₄⁺ (Burckhardt & Frömter 1992;Nakhoul et al. 2005). In response to NH₄Cl, oocytes exhibit membrane depolarization and intracellular acidification without an initial rapid alkalinization (Cougnon et al. 1996;Burckhardt & Burckhardt 1997;Nakhoul et al. 2010). On the other hand, NH₄⁺ currents are independent of NH₄Cl concentrations at constant NH₃ (Boldt et al. 2003), suggesting that oocytes possess two different pathways: an NH₃ entry and an NH₄⁺ conductance. It is thus unclear by what mechanism NBCn1 affects NH₄⁺ transport and/or conductance in oocytes.

In this study, we examined the effect of NBCn1 activity and expression on oocyte NH₄⁺ transport. We measured intracellular pH (pH_i), membrane potential (V_m), and current (I) during NH₄Cl application in both NBCn1-expressing oocytes and water-injected control oocytes and compared them. Our data show that NBCn1 inhibits oocyte NH₄⁺ conductance by the Na/HCO₃-independent mechanism, while stimulating NH₄⁺-mediated pH_i change by the intracellular buffering mechanism. A model for the effects of NBCn1 on oocyte NH₄⁺ transport and conductance is proposed in the Discussion.

METHODS

Ethical Approval

All experiments in this study were conducted under the NIH guidelines for research on animals, and experimental protocols were approved by the Institutional Animal Care and Use Committee at Emory University.

NBCn1 expression in oocytes

Female frogs (Xenopus laevis) were purchased from Xenopus Express (Brooksville, FL, USA). A frog was anesthetized with fresh 0.1% 3-aminobenzoic acid ethyl ester for 30 min or until the animal was fully anesthetized. An incision in the abdomen, lateral to the midline, was made and oocytes were collected. After suture, the frog was returned to a recovery tank containing 0.1 M NaCl. Oocytes were agitated in Ca²⁺-free ND96 solution (in mM; 96 NaCl, 2 KCl, 1 MgCl₂, and 10 Hepes, pH 7.5) five times for 20 min each. Follicles were removed by enzymatic digestion with collagenase type I (Sigma-Aldrich; St. Louis, MO, USA) twice for 20 min each at the concentration of 2 mg/ml. Oocytes were then washed with normal ND96 solution containing 1.8 mM CaCl₂ for 20 min. Oocytes at stages V-VI were sorted and stored in OR3 medium (Chang et al. 2008) at 18°C overnight. For cRNA injection, rat splice variant NBCn1-E (Cooper et al. 2005) was in vitro transcribed with T7 RNA polymerase using the mMessage/mMachine transcription kit (Ambion; Austin, TX, USA). The injection was done with 25 ng RNA (in 46 nl). The control was injection with sterile water. Injected oocytes were maintained at 18°C for 3–5 days before use.

Two-electrode voltage clamp

Two glass capillaries with 1 mm outer diameter (World Precision Instruments; Sarasota, FL, USA) were filled with 3 M KCl and had a tip resistance of ~1 MΩ. One electrode was connected to the voltage headstage and the other to the current cable. The two probes were connected to the voltage-clamp amplifier OC-725C (Warner; Hamden, CT, USA). Input offset voltage was adjusted by placing electrodes in the chamber containing the nominally HCO₃⁻/CO₂-free recording solution (mM; 76 NaCl, 20 mM NMDG-Cl, 2 KCl, 1 MgCl₂, 1.8 CaCl₂, 5 Hepes, pH 7.4; 195–200 mOsm/kg). An oocyte was impaled with current and voltage electrodes and superfused with the recording solution. Signals from voltage and current electrodes were sampled by the digitizer Digidata 1322A (Molecular Devices; Sunnyvale, CA, USA) interfaced to a computer, and data were acquired using pClamp 8 (Molecular Devices). The membrane voltage of the oocyte was monitored before and after applying 10 and 20 mM NH₄Cl, which replaced NMDG-Cl in the recording solution. For recording NH₄Cl-mediated current, the oocyte was clamped at −60 mV using the OC-725C. Current-voltage relations were determined by commanding voltages from −120 mV to +60 mV with 20 mV increments for 100 msec before and after NH₄Cl application. The voltage commands were imposed once the current value reached steady-state. For ionic conductance associated with NBCn1, current values were calculated by subtracting current values in control oocytes from values in NBCn1-expressing oocytes. Experiments were performed at room temperature.

Measurement of pH_i

An oocyte was impaled with three microelectrodes: a pH electrode, a voltage electrode, and a current electrode. The pH electrode was baked at 200°C, silanized with bis-(methylamino)dimethylsilane (Sigma-Aldrich), and filled with the proton ionophore 1 cocktail B (Sigma-Aldrich). The electrode was then back-filled with the phosphate buffer at pH 7.0. The pH electrode was connected to the high-impedance electrometer FD-223 (World Precision Instruments; Sarasota, FL, USA) and the signal was then routed to a custom-made subtraction amplifier. The voltage and current electrodes filled with 3 M KCl were connected to the OC-725C. Signals from pH, voltage, and current electrodes were sampled by the Digidata 1322A, and data were acquired using pClamp 8. The signal in the voltage electrode was subtracted from the signal in the pH electrode to calculate the voltage for pH. The slope of voltage to pH was calibrated by placing all three electrodes in the chamber filled with pH 6.0 and 8.0 standards (Fisher Scientific; Pittsburgh, PA, USA). The slope was typically at the range of 53 ± 3 mV/pH. For recording in the presence of HCO₃⁻/CO₂, an oocyte clamped at −60 mV was superfused with a solution buffered with 25 mM HCO₃⁻, 5% CO₂ (pH 7.4) and then with 20 mM NH₄Cl in the continued presence of HCO₃⁻/CO₂. NaCl was replaced mole for mole with NH₄Cl. NH₄Cl was applied ~2 min after pH_i reached the lowest point. LiCl or N-methyl D-glucamine+(NMDG⁺) was used to replace NaCl in the Na⁺-free experiments, and BaCl₂ was used to replace KCl in an effort to inhibit K⁺ channels. Oocyte NH₄⁺ transport activity was measured from the rate of acidification after NH₄Cl application. The rate of pH_i change (i.e., dpH_i/dt) was calculated by linear regression from the initial pH_i value over the first 2 min after NH₄Cl exposure. All experiments were performed at room temperature.

Statistical analysis

Data were reported as means ± standard error. The level of significance was assessed using the unpaired, 2-tailed Student t-test for comparison of dpH_i/dt, V_m, and I between control oocytes and NBCn1-expressing oocytes. The one-way ANOVA with Bonferroni post test was used to analyze daily I values for 5 days. The p value of less than 0.05 was considered significant.

RESULTS

NBCn1 cotransport activity stimulates NH₄Cl-mediated intracellular acidification in oocytes

To assess the effect of NBCn1 cotransport activity on NH₄⁺ transport, we expressed the rat renal splice variant NBCn1-E (hereafter, NBCn1) in Xenopus oocytes and simultaneously measured pH_i and V_m during NH₄Cl application in the presence of 25 mM HCO₃⁻, 5% CO₂ using the proton-selective pH microelectrode and the voltage electrode. Fig. 1A shows representative pH_i and V_m traces in a water-injected control oocyte. The oocyte was bathed initially in the HEPES-buffered (HCO₃⁻/CO₂-free) solution to maintain stable pH_i. Applying HCO₃⁻/CO₂-buffered medium caused pH_i to decrease as CO₂ entered the oocyte, hydrated, and produced H⁺. The pH_i then reached steady-state as intracellular CO₂ rose to equal extracellular CO₂. A slight increase in pH_i was often observed (a in the figure), possibly due to endogenous Na/H exchange (Busch 1997). Under this condition, subsequent exposure to 20 mM NH₄Cl induced intracellular acidification (b in the figure). NH₄Cl also caused the oocyte membrane to be depolarized. These responses to NH₄Cl (i.e., intracellular acidification and membrane depolarization) are comparable to the previous observations by others (Cougnon et al. 1996;Burckhardt & Burckhardt 1997;Nakhoul et al. 2010).

Fig. 1.

pH_i and V_m during NH₄Cl application in the presence of HCO₃⁻/CO₂. A) Representative pH_i and V_m traces in a control oocyte. The oocyte was superfused with a HEPES-buffered HCO₃⁻/CO₂-free solution and then exposed to a HEPES-free solution containing 25 mM HCO₃⁻, 5% CO₂. After pH_i reached steady state, the oocyte was exposed to 20 mM NH₄Cl in the continued presence of HCO₃⁻/CO₂. Intracellular acidification and membrane depolarization are shown. An initial rapid peak in V_m upon exposure to NH₄Cl is a solution delivery artifact. One of 7 oocytes is shown. B) Representative pH_i and V_m trances in an NBCn1-expressing oocyte. One of 6 oocytes is shown. dpH_i/dt due to NH₄⁺ transport was calculated by subtracting dpH_i/dt in region b (or b’) from dpH_i/dt in region a (or a’). dpH_i/dt values and other values are summarized in Table 1.

In an NBCn1-expressing oocyte (Fig. 1B), the application of HCO₃⁻/CO₂ also caused pH_i to decrease. However, the pH_i recovered from an initial CO₂-induced acidification (a’) as HCO₃⁻ transported via NBCn1 associated with intracellular H⁺. Applying NH₄Cl in the continued presence of HCO₃⁻/CO₂ induced a rapid acidification (b’). NH₄Cl-dependent intracellular acidification was evaluated by subtracting dpH_i/dt values before NH₄Cl application from values after NH₄Cl application (i.e., b – a or b’ – a’). The acidification rate was −10.33 ± 0.47 × 10⁻⁴/sec (n = 6) for NBCn1-expressing oocytes, which was 2.8-fold higher than −3.73 ± 0.49 × 10⁻⁴/sec (n = 7) for controls (p < 0.05). Nonetheless, a smaller depolarization mediated by NH₄Cl was observed in NBCn1-expressing oocytes (ΔV_m = 13.4 ± 1.2 mV for controls and 10.3 ± 2.4 mV for NBCn1-expressing oocytes). pH_i change rates and other values are summarized in Table 1.

Table 1.

pH_i and V_m measurements of control oocytes and NBCn1-expressing oocytes. Recordings were performed in the presence of 20 mM NH₄Cl, 25 mM HCO₃⁻, 5% CO₂.

	Control	NBCn1
Resting pHi	7.36 ± 0.01	7.41 ± 0.03
pHi in CO2/HCO3−	6.94 ± 0.03	6.99 ± 0.02
dpHi/dt before NH4Cl	0.51 ± 0.12	1.11 ± 0.11*
dpHi/dt after NH4Cl	−3.22 ± 0.45	−9.22 ± 0.37*
dpHi/dt due to NH4+ transport†	−3.73 ± 0.49	−10.33 ± 0.47*
Resting Vm	−28.7 ± 1.6	−22.4 ± 2.1*
ΔVm	13.4 ± 1.2	10.3 ± 2.4
n	7	6

^†Calculated by subtracting dpH_i/dt before NH₄Cl from dpH_i/dt after NH₄Cl.

^*p < 0.05.

NBCn1 reduces NH₄Cl-mediated inward current in oocytes

To further assess the effect of NBCn1 on NH₄⁺ transport, we then applied NH₄Cl to oocytes in the absence of HCO₃⁻/CO₂. For this analysis, oocytes were clamped at −60 mV, close to the mean resting potential of −61 mV for uninjected oocytes (Yang et al. 2009b), and pH_i and I were simultaneously recorded. Fig. 2A shows a representative pH_i trace in a control oocyte. A gradual intracellular acidification was observed in response to NH₄Cl, reflecting that NH₄⁺ transport is more favorable than NH₃ entry in oocytes (Nakhoul et al. 2001). Consistent with this, there was no immediate pH_i rise upon NH₄Cl exposure. The pH_i recovery after NH₄Cl removal was relatively slow probably due to low levels of accumulated NH₃ in the cells. An inward current was generated, which was abolished after NH₄Cl removal.

Fig. 2.

pH_i and I during NH₄Cl application in the absence of HCO₃⁻/CO₂. A) Representative pH_i and I in a control oocyte. Oocytes were exposed to 20 mM NH₄Cl at the holding potential of −60 mV, and pH_i and I were simultaneously measured using microelectrodes. One of 5 controls is shown. B) Representative pH_i and I in an NBCn1-expressing oocyte. One of 6 NBCn1 oocytes is shown. C) Summary of pH_i change rate. The pH_i change rate (dpH_i/dt) was calculated by linear regression analysis from the initial pH_i over 2 min after NH₄Cl application. D) Summary of I. NH₄Cl-mediated current (I_NH4Cl) was obtained by subtracting I before NH₄Cl application from I after NH₄Cl application. E) Summary of dpH_i/dt in the presence of BaCl₂. Experiments were done in the presence of BaCl₂, which replaced KCl mole for mole (n = 5 for each). F) Summary of I_NH4Cl in the presence of BaCl₂. Measurements were done as described in D (n = 9 controls and 5 NBCn1).

Fig. 2B shows a representative pH_i trace in an NBCn1-expressing oocyte. A gradual acidification in response to NH₄Cl was observed without an immediate pH_i rise. The mean dpH_i/dt during NH₄Cl application was similar between controls and NBCn1-expressing oocytes (Fig. 2C). The intracellular buffering power of Xenopus oocytes is reported to be similar at pH_i ranging between 7.2 and 7.5 and estimated to be 24 mM/pH unit (Cougnon et al. 2002). Thus, the rate of NH₄⁺ influx, which is equal to dpH_i/dt × buffering power, was estimated to be similar between the two groups of oocytes. Nonetheless, the NBCn1-expressing oocyte produced a smaller current in response to NH₄Cl. The mean I in these oocytes was −125.2 ± 25.8 nA, significantly smaller than −278.2 ± 16.7 nA for controls (p < 0.05) (Fig. 2D). NBCn1-expressing oocytes produced large holding currents before NH₄Cl application due to channel-like activity of NBCn1.

NH₄⁺ generally moves across the cell membrane by binding to the K⁺ site in K⁺ channels/transporters (Weiner & Hamm 2007). To test whether reduced NH₄Cl-mediated current by NBCn1 involves K⁺, we repeated experiments in a solution containing BaCl₂ instead of KCl (Fig. 2E and F). NBCn1-expressing oocytes had a slightly higher mean dpH_i/dt (p < 0.05; n = 5 for each) probably due to higher initial pH_i in these oocytes. Nonetheless, NBCn1-expressing oocytes had substantially smaller current mediated by NH₄Cl (p < 0.05). The reduction was 57%, comparable to 55% in the presence of KCl (Fig. 2D). Thus, barium has a negligible effect on the transporter’s ability to inhibit NH₄Cl-mediated current.

The inhibition of NH₄Cl-mediated current by NBCn1 was further examined using two-electrode voltage clamp. Fig. 3A shows representative recordings of the current caused by 10 mM NH₄Cl in oocytes (at −60 mV). NH₄Cl-mediated current was induced in both a control oocyte and an NBCn1-expressing oocyte, but the current amplitude was smaller in the NBCn1-expressing oocyte. This difference was more evident at 20 mM NH₄Cl (Fig. 3B), indicating that the inhibition depends on the amount of NH₄Cl. As summarized in Fig. 3C, the inhibition was 33% at 10 mM NH₄Cl and 56% at 20 mM NH₄Cl (p < 0.05 for each). In other experiments, we measured V_m during NH₄Cl application under the non-voltage clamp condition (Fig. 3D). NH₄Cl-mediated membrane depolarization was smaller in NBCn1-expressing oocytes, measured on the same day (3–4 days after cRNA injection). The reduction was 72% (p < 0.05; Fig. 3E)

Fig. 3.

Inhibition of NH₄Cl-mediated current by NBCn1. A) Representative inward currents during 10 mM NH₄Cl application (clamped at −60 mV). Oocytes were superfused with HCO₃⁻/CO₂-free solution and then exposed to NH₄Cl. Measurements were done on the same days after water or NBCn1 cRNA injection. One of 6 controls and one of 5 NBCn1-expressing oocytes are shown. B) Representative inward currents during 20 mM NH₄Cl application. One of 6 controls and one of 8 NBCn1 oocytes are shown. C) Comparison of currents induced by 10 mM and 20 mM NH₄Cl. D) Representative V_m traces during 10 mM NH₄Cl application in a water-injected control oocyte and an NBCn1-expressing oocyte. One of 6 controls and one of 8 NBCn1-expressing oocytes are shown. E) Summary of NH₄Cl-induced membrane depolarization (ΔV_m).

NH₄Cl-mediated current response to voltage is reduced by NBCn1

To analyze the NH₄Cl-mediated current response to voltage, we determined current-voltage (I–V) relations by imposing voltage steps from −120 to +60 mV before and after NH₄Cl exposure. The voltage command after exposure was made when the current reached steady-state (~2 min). In control oocytes (Fig. 4A), NH₄Cl caused the I–V plot to shift down with a progressive increase in the inward direction at more negative voltages. The difference before and after NH₄Cl application is a good estimate of the conductance for NH₄Cl-mediated current (G_NH4Cl; red line in the figure). The G_NH4Cl was positive and almost linear with a reversal potential of 33.2 ± 2.2 mV (n = 5). In NBCn1-expressing oocytes (Fig. 4B), the I–V plot before NH₄Cl application was already steeper. Upon the exposure to NH₄Cl, the I–V plot shifted down. However, the change was small and almost negligible at more positive voltages. Thus, the reversal potential shifted positively. Fig. 4C shows that the G_NH4 was reduced by 52% in NBCn1-expressing oocytes (measured between −80 and −40 mV; n = 5 for each).

Fig. 4.

NH₄Cl-mediated currents at different voltages. A) Current–voltage (I–V) relationships before and after NH₄Cl application in control oocytes (n = 5). Oocytes were clamped at −60 mV and voltages were stepped from −120 mV to +60 mM with 20 mV increments before (open circles) and 2 min after NH₄Cl application (closed circles). The red line is the mean difference between the two I–V plots. B) I–V relationships in NBCn1-expressing oocytes (n = 5). Measurements were done using the protocol in A. Large basal currents before NH₄Cl application (open circles) are due to NBCn1 channel-like activity. C) Average conductances for NH₄Cl-mediated current under the two conditions. The slope conductance (G_NH4Cl) was measured between −80 and −40 mV using the relationships between NH4Cl-mediated current and voltage

Channel-like activity of NBCn1 reduces oocyte NH₄⁺ currents

In the above data, NBCn1 caused the oocyte resting V_m to shift positively, producing a high holding current. NBCn1 also caused a steeper slope in the I–V plot before NH₄Cl application. These are hallmarks for channel-like activity of NBCn1 that occurs in the Na/HCO₃-independent manner (Choi et al. 2000;Cooper et al. 2005). Fig. 5A shows another example of the channel-like activity. Inward currents at negative potentials and outward currents at positive potentials were elicited in NBCn1-expressing oocytes. The zero-current voltage shifted positively by ~25 mV and the slope measured at the zero-current voltage was 6.96 ± 2.3 µS, larger than 1.13 ± 0.06 µS for controls (n = 5 for each). This channel-like activity is primarily caused by a Na⁺ current and secondarily by outwardly rectifying anion currents (Choi et al. 2000;Cooper et al. 2005). Consistent with this, NBCn1-expressing oocytes produced Na⁺ currents (Fig. 5B).

Fig. 5.

Channel-like activity of NBCn1 and its effect on NH₄Cl-mediated current. A) I–V relationships in HCO₃⁻/CO₂-free solution containing 96 mM Na⁺. Oocytes held at −60 mV were subjected to a step-voltage command from −120 to +60 mV. A large slope and a positive shift in the zero-current voltage are hallmarks for the channel-like activity of NBCn1 (n = 5 for each). B) Na⁺ component of NBCn1 channel-like activity. The Na⁺ component was measured by subtracting I in nominally Na⁺-free solution from I in 96 mM Na⁺ media (n = 4 controls and 5 NBCn1). NMDG⁺ was substituted for Na⁺. C) NH₄Cl-mediated currents at different voltages in Na⁺-free condition. Currents were calculated using the protocol in Fig. 4 and plotted versus the voltage (n = 4 controls and 5 NBCn1). D) NH₄Cl-mediated pH_i changes mediated by NH₄Cl in Na⁺-free solution. The dpH_i/dt was determined in oocytes exposed to 76 mM Na⁺ (n = 4 for each) and in other oocytes exposed to 0 mM Na⁺ (n = 5 for each).

To determine whether this Na⁺ current component affects NH₄Cl-mediated current, we performed experiments, in which oocytes were preincubated in Na⁺-free media for 20 min and subjected to the above I–V protocol. Fig. 5C shows the results. The two I–V plots corresponded to NH₄Cl-mediated currents at different voltages in control oocytes and NBCn1-expressing oocytes. The two plots were nearly superimposed after preincubation in Na⁺-free media. The current was not reversed at more positive voltages in NBCn1-expressing oocytes, probably due to incomplete washout of intracellular Na⁺ after 20 min preincubation. Nevertheless, the values at the resting membrane potentials were similar between the two groups of oocytes (p > 0.05). Parallel experiments were then performed to examine pH_i changes in Na⁺-free solution (Fig. 5D). The dpH_i/dt values were similar before and after Na⁺ removal (p > 0.05).

In other experiments, the time course of NH₄Cl-mediated current at −60 mV was measured daily after water or NBCn1 cRNA injection (Fig. 6A). The current appeared to increase over the initial 48 h after injection, the reason for which is unclear. Nonetheless, no significant difference was observed between controls and NBCn1-expressing oocytes during this period (p > 0.05). At 72 h, the current began to decrease in NBCn1-expressing oocytes (p < 0.05). The decrease was more profound at 96–120 h. We then determined the channel-like activity of NBCn1 during these time periods by subtracting the slope in the I–V plot for control oocytes from the slope for NBCn1-expressing oocytes (Fig. 6B). The channel-like activity was negligible over the initial 48 h, but significantly increased at 72–120 h (p < 0.05; one-way ANOVA with Bonferroni post test; 4–6 oocytes for each group at different time points). Values were similar at these time points, comparable to the earlier report (Yang et al. 2009a) that NBCn1 expression in oocyte membranes is saturated after 72 h. These data demonstrate that there is an inverse relationship between channel-like activity and NH₄Cl-mediated current in oocytes expressing NBCn1.

Fig. 6.

Time course of NH₄Cl-mediated current and NBCn1 channel-like activity. A) Time course of NH₄Cl-mediated currents after water or NBCn1 cRNA injection. Currents were daily measured in 4–6 oocytes from each group of oocytes. Measurements were done at −60 mV. B) Time course of NBCn1 channel-like activity. For this analysis, mean currents in control oocytes were first subtracted from currents of NBCn1-expressing oocytes at each voltage (n = 4–6 oocytes for each group at each time point). The subtracted values were then used to plot I–V relationships, from which the slope was calculated between −80 and −40 mV. The one-way ANOVA with Bonferroni post test was used to analyze the level of significance. Asterisks (*) represent p < 0.05 for comparison to the value at 48 h.

DISCUSSION

In our recent report (Lee et al. 2010), we demonstrated that NBCn1 increases HCO₃⁻/CO₂-dependent ¹⁴C-methylammonium uptake in oocytes. The goal of the present study was to further investigate the effect of NBCn1 on NH₄⁺ transport by electrophysiological measurements of V_m, I, and pH_i of oocytes. The major findings from this study are the following: 1) NBCn1 Na/HCO₃ cotransport activity enhances intracellular acidification mediated by NH₄Cl in oocytes; 2) In the absence of HCO₃⁻/CO₂, NBCn1 inhibits inward current mediated by NH₄Cl, while producing a negligible effect on intracellular acidification; 3) NBCn1-dependent inhibition of NH₄Cl current is unaffected by barium; 4) The inhibition is abolished by Na⁺ removal; and 5) NBCn1 channel-like activity appears to be responsible for inhibiting NH₄Cl-mediated current. These findings are novel and provide new information on the effect of NBCn1 on oocyte NH₄⁺ transport. In addition, the findings provide the first evidence for the functional significance of NBCn1’s channel-like activity. We propose that NBCn1 may play a key role in regulating NH₄⁺ transport and conductance in cells, where HCO₃⁻ and NH₄⁺ transport processes are tightly coupled.

In this study, we demonstrate that NBCn1 differentially affects pH_i and I (or V_m) caused by NH₄Cl in oocytes. The transporter stimulates intracellular acidification caused by NH₄Cl in the presence of CO₂/HCO₃⁻ (Fig. 1). NBCn1 moves Na⁺ and HCO₃⁻ into oocytes and causes pH_i to recover from a CO₂-induced acidification. NH₄⁺ transport is stimulated in this condition because HCO₃⁻ moved via NBCn1 compensates intracellular H⁺ load. Thus, the intracellular buffering mechanism can account for increased rates of acidification during NH₄Cl application in the presence of HCO₃⁻/CO₂. On the other hand, we observe a decrease in membrane depolarization under the same condition. Furthermore, NBCn1 inhibits NH₄Cl-mediated current without significantly altering dpH_i/dt in the absence of HCO₃⁻/CO₂ (Fig. 2 and Fig. 3). Therefore, the inhibition occurs regardless of Na/HCO₃ cotransport activity of NBCn1.

The finding that NBCn1 differentially affects pH_i and I leads us to revisit the mechanism of NH₄⁺ transport in Xenopus oocytes. Our data show that NH₄Cl induces membrane depolarization and intracellular acidification, consistent with previous reports by others (Cougnon et al. 1996;Burckhardt & Burckhardt 1997;Nakhoul et al. 2010). Given the reports that oocyte membranes are less permeable to NH₃ than to NH₄⁺ (Burckhardt & Frömter 1992), the changes in membrane depolarization and intracellular acidification during NH₄Cl application have been considered to be due to carrier-mediated NH₄⁺ transport. Upon NH₄Cl application, NH₄⁺ is preferentially transported into the oocyte cytosol and dissociates into NH₃ and H⁺. The dissociation results in intracellular acidification. Thus, the movement of charged NH₄⁺ into oocytes induces a depolarizing inward current. NH₄⁺ has been proposed to enter the oocyte cytosol via a nonselective cation channel (Burckhardt & Burckhardt 1997) or a mechanism that exhibits a linear relationship between pH_i change rate and NH₄⁺ concentration (Nakhoul et al. 2010). Thus, according to this paradigm, the two events following NH₄Cl application (i.e., intracellular acidification and inward current) are tightly coupled to each other.

In contrast, our data show that the cotransport activity of NBCn1 preferentially stimulates NH₄⁺-mediated intracellular acidification, while reducing inward current. Thus, the two events can be separated from each other. Our data also show that NBCn1 can reduce the current during NH₄Cl application, without altering intracellular acidification. This implies that oocyte NH₄⁺ transport does not necessarily have to be electrogenic. The crystal structure of the bacterial ammonium transporter AmtB suggests translocation of NH₃, but not NH₄⁺, via the transporter (Zheng et al. 2004). Mammalian NH₄⁺ transporter Rh glycoproteins have been proposed to be electroneutral (Weiner & Hamm 2007). Nonetheless, we are also aware that the Rh glycoproteins exhibit electrogenic NH₄⁺ transport (Nakhoul et al. 2005;Nakhoul et al. 2010). In this sense, it is also interesting to mention that we often observe negligible electrogenicity of NH₄⁺ transport in some preparations of oocytes, as shown in our previous report (Lee et al. 2010). The exact reason for this lack of electrogenicity in some preparations is unclear although oocytes are known to exhibit seasonal variances.

Our data show that NBCn1 exhibits channel-like activity (Fig. 4 and Fig. 5), consistent with previous reports (Choi et al. 2000;Cooper et al. 2005). Other HCO₃⁻ transporters also display channel-like conductance. The Cl/HCO₃ exchanger AE1 in erythrocytes mediates a conductive anion flux. The DIDS-insensitive component of net ion efflux increases with increasing membrane hyperpolarization (Freedman & Novak 1997). The Cl/HCO₃ exchangers Slc26a3 (DRA, CLD) and Slc26a6 (CFEX, PAT-1) also show channel-like activity, which mediates large NO₃⁻ and SCN⁻ currents that are uncoupled to OH⁻ or HCO₃⁻ transport (Ko et al. 2002;Shcheynikov et al. 2008). The channel-like activity of NBCn1 is known to increase intracellular Na⁺ levels and depolarize the membrane by 20–30 mV in Xenopus oocytes and HEK 293 cells (Choi et al. 2000;Cooper et al. 2005). Thus, Na⁺ influx via NBCn1 may lower the electrochemical Na⁺ gradient across the cell membrane, subsequently reducing the driving force for non-selective cation channels. Our finding that Na⁺ removal abolishes the capability of NBCn1 to inhibit NH₄Cl current support this idea. Whether the channel-like activity affects nonselective cation channels needs further investigation.

Based on the data obtained from this study, we propose a model for the effect of NBCn1 on oocyte NH₄⁺ transport (Fig. 7). NH₄⁺ moves to the cytosol via endogenous NH₄⁺ transport and dissociates intracellularly into NH₃ and H⁺. This process is stimulated by NBCn1 as HCO₃⁻ compensates H⁺. Intracellular NH₄⁺ or NH₃ then causes the oocytes to produce an inward current that can be affected by Na⁺ in the cytosol. The inward current can thus be inhibited by NBCn1 channel-like activity, which raises intracellular Na⁺. Our model assumes that oocyte NH₄⁺ transport is mainly electroneutral because NBCn1 reduces NH₄Cl-mediated current without significantly altering dpH_i/dt in the absence of HCO₃⁻/CO₂.

Fig. 7.

A model for the effect of NBCn1 on oocyte NH₄⁺ transport. NH₄⁺ enters into the cytosol via the endogenous NH₄⁺ transporter and dissociates into NH₃ and H⁺. Intracellular NH₄⁺ or NH₃ secondarily causes the oocyte to induce an inward current that is affected by intracellular Na⁺ (dashed line). The Na/HCO₃ cotransport activity of NBCn1 stimulates NH₄⁺ transport by buffering H⁺ dissociated from NH₄⁺. On the other hand, the channel-like activity of NBCn1 inhibits NH₄⁺ (or NH₃)-induced inward current by raising intracellular Na⁺ in the Na/HCO₃-independent manner. The molecule responsible for the inward current is possibly a non-selective cation channel. The model assumes that oocyte NH₄⁺ transport is mainly electroneutral.

What would be the physiological implication of the effect of NBCn1 on NH₄⁺ transport? In the thick ascending limb, where NBCn1 is localized to the basolateral side of the cells, NH₄⁺ is transported from the lumen mainly via the apical NKCC2 (Russell 2000;Kinne et al. 1986;Weiner & Hamm 2007). NH₄⁺ translocates through the K⁺ binding site in NKCC2 and is inhibited by bumetanide (Kinne et al. 1986). In addition, the thick ascending limb has amiloride-sensitive NH₄⁺ conductance (Attmane-Elakeb et al. 2001;Amlal et al. 1994). Together with verapamil-sensitive K⁺/NH₄⁺ exchange, the NH₄⁺ conductance contributes to apical NH₄⁺ transport by 35–50% (Attmane-Elakeb et al. 2001). Leipziger’s groups (Jakobsen et al. 2004;Odgaard et al. 2004) reported that the basolateral NBCn1 affects the apical NH₄⁺ transport by moving HCO₃⁻ into the cells and compensating H⁺ load. Our finding of NBCn1-mediated stimulation of intracellular acidification during NH₄Cl application is in good agreement with this paradigm. Nonetheless, it is unclear what physiological significance there is in the inhibition of NH₄⁺ conductance by NBCn1. One explanation is that the inhibition may help protect the tubules from membrane depolarization, which is deleterious to cells. This inhibition may be critical for cell function particularly during chronic metabolic acidosis, when NH₄⁺ and HCO₃⁻ absorptive capacity significantly increases (Wagner 2007).

In summary, our study shows that NBCn1 inhibits oocyte NH₄Cl-mediated current, while enhancing NH₄⁺ uptake by the compensation of intracellular H⁺ load. We envision that NBCn1 not only serves as an acid extruder to maintain intracellular pH within the physiological range, but it also plays a critical role in regulating HCO₃⁻ and NH₄⁺ transport. It will be interesting and important to test whether NBCn1 also inhibits NH₄⁺ conductance in the thick ascending limb cells.