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J Gen Physiol. May 2006; 127(5): 511–524.
PMCID: PMC2151520
Article
Coupling Modes and Stoichiometry of Cl/HCO3 Exchange by slc26a3 and slc26a6
Nikolay Shcheynikov,1 Youxue Wang,1 Meeyoung Park,1 Shigeru B.H. Ko,2 Michael Dorwart,1 Satoru Naruse,2 Philip J. Thomas,1 and Shmuel Muallem1
1Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390
2Department of Internal Medicine, Nagoya University, Showa-ku, Nagoya 466-8550, Japan
Correspondence to Shmuel Muallem:shmuel.muallem/at/utsouthwestern.edu
Received August 23, 2005; Accepted March 17, 2006.
The SLC26 transporters are a family of mostly luminal Cl and HCO3 transporters. The transport mechanism and the Cl/HCO3 stoichiometry are not known for any member of the family. To address these questions, we simultaneously measured the HCO3 and Cl fluxes and the current or membrane potential of slc26a3 and slc26a6 expressed in Xenopus laevis oocytes and the current of the transporters expressed in human embryonic kidney 293 cells. slc26a3 mediates a coupled 2Cl/1HCO3 exchanger. The membrane potential modulated the apparent affinity for extracellular Cl of Cl/HCO3 exchange by slc26a3. Interestingly, the replacement of Cl with NO3 or SCN uncoupled the transport, with large NO3 and SCN currents and low HCO3 transport. An apparent uncoupled current was also developed during the incubation of slc26a3-expressing oocytes in HCO3-buffered Cl-free media. These findings were used to develop a turnover cycle for Cl and HCO3 transport by slc26a3. Cl and HCO3 flux measurements revealed that slc26a6 mediates a 1Cl/2HCO3 exchange. Accordingly, holding the membrane potential at 40 and −100 mV accelerated and inhibited, respectively, Cl-mediated HCO3 influx, and holding the membrane potential at −100 mV increased HCO3-mediated Cl influx. These findings indicate that slc26a6 functions as a coupled 1Cl/2HCO3 exchanger. The significance of isoform-specific Cl and HCO3 transport stoichiometry by slc26a3 and slc26a6 is discussed in the context of diseases of epithelial Cl absorption and HCO3 secretion.
Anion transport and homeostasis is linked to the regulation of pHi, both of which are crucial for the proper function of the cells. In epithelia, these mechanisms also mediate transcellular Cl and HCO3 transport. The SLC26 transporters are members of a relatively new family of anion transporters consisting of 10 known members (Mount and Romero, 2004). Members of the family play important roles in many physiological functions, in particular in epithelia. This is evident from the diseases associated with members of the family. Mutations in this family of proteins can result in dystrophic dysplasia (SLC26A2; Superti-Furga et al., 1996), congenital chloride diarrhea (SLC26A3; Makela et al., 2002), Pendred's syndrome (SLC26A4; Everett et a1., 1997), and hearing loss (SLC26A5; Liu et al., 2003). SLC26A4 also participates in renal HCO3 absorption (Royaux et al., 2001) and the control of blood pressure (Verlander et al., 2003), whereas slc26a6 is involved in intestinal HCO3 and renal oxalate transport (Wang et al., 2005).
Although the anion selectivity is specific for each member of the family, most can transport Cl and HCO3 (Mount and Romero, 2004). Initially, the family was identified by searching for SO4= transporters and identifying SLC26A1 (Bissig et al., 1994). SLC26A2 was found by positional cloning of the gene associated with dystrophic dysplasia (Hästbacka et al., 1994) and was later shown to function as a SO4= transporter. Subsequent studies suggested that many SLC26 transporters function as Cl/HCO3 exchangers, including slc26a3 (Melvin et al., 1999; Ko et al., 2002), SLC26A4 (Soleimani et al., 2001; Ko et al., 2002), SLC26A6 (Ko et al., 2002; Wang et al., 2002), SLC26A7 (Petrovic et al., 2004), and SLC26A9 (Xu et al., 2005). However, more recent studies showed that SLC26A7 functions as an intracellular pH (pHi)–regulated Cl channel (Kim et al., 2005) and that SLC26A1 and SLC26A2 are specific SO4= transporters (Regeer et al., 2003; Forlino et al., 2005).
So far, all of the SLC26 transporters examined except SLC26A7 (Petrovic et al., 2004; but see the expression of SLC26A7 in the luminal membrane of the proximal tubules in Dudas et al., 2006) and SLC26A1 (Nakada et al., 2005) are expressed in the luminal membrane of epithelial cells (Mount and Romero, 2004). Transport of Cl and HCO3 by the SLC26 transporters raised the possibility that these transporters are the long sought luminal Cl/HCO3 exchangers that mediate epithelial Cl absorption and HCO3 secretion, such as in the pancreatic and salivary gland ducts (Cook et al., 1994; Melvin et al., 2005; Steward et al., 2005). Epithelial Cl absorption and HCO3 secretion is intimately regulated by CFTR (Kunzelmann and Mall, 2002; Irokawa et al., 2004; Melvin et al., 2005; Steward et al., 2005), as evident from the lack of these activities in cystic fibrosis (Wilschanski and Durie, 1998; Sokol, 2001). The importance of the SLC26 transporters in epithelial Cl absorption and HCO3 secretion is further highlighted by the finding that CFTR potently activates the SLC26 transporters (Ko et al., 2002), and, in turn, the SLC26 transporters are potent activators of CFTR (Ko et al., 2004). This mutual regulation is mediated by interaction of the CFTR R domain and the SLC26 solute transporter anti-σ factor antagonist (STAS) domain and is assisted by the binding of CFTR and the SLC26 transporters to PDZ-containing scaffolding proteins (Ko et al., 2004).
To understand the role of the SLC26 transporters in epithelial Cl absorption and HCO3 secretion, it is absolutely essential to understand their transport mechanism and their Cl/HCO3 transport stoichiometry. In an initial study, we reported that slc26a3 and slc26a6 are electrogenic Cl/HCO3 transporters with isoform-specific stoichiometry (Ko et al., 2002). At the same time, Xie et al. (2002) independently reported that slc26a6 is an electrogenic transporter. However, a recent study that examined the properties of slc26a6 and SLC26A6 concluded that slc26a6 and SLC26A6 are electroneutral Cl/HCO3 exchangers (Chernova et al., 2005). Although the later study contains some apparent internal inconsistencies (see Discussion), the confusion generated requires clarification. More importantly, many fundamental characteristics of these transporters are unknown. For example, we do not know the exact stoichiometry and mode of transport of any of the SLC26 transporters. It is also unclear whether the transport of Cl and HCO3 by these transports is obligatorily coupled.
The cardinal importance of the SLC26 transporters in epithelial and other cells' physiology demands clarification, especially the mode of transport and their Cl/HCO3 transport stoichiometry. The Cl/HCO3 transport stoichiometry will dictate their precise role in epithelial Cl absorption and HCO3 secretion (Ko et al., 2004; Steward et al., 2005). In this study, we determined the transport mode and Cl/HCO3 transport stoichiometry of slc26a3 and slc26a6. To this end, we simultaneously measured intracellular Cl (Cli), pHi, and membrane potential or current. We report that slc26a3 functions as a coupled 2Cl/1HCO3 exchanger that can also mediate uncoupled NO3 and SCN transport, whereas slc26a6 functions as an electrogenic coupled 1Cl/2HCO3 exchanger.
The slc26a3 and slc26a6 clones and their cRNA were the same as those used in previous studies (Ko et al., 2002, 2004). The slc26a3 in the pClneo vector was used for expression in human embryonic kidney (HEK) 293 cells and was shuttled to pXBG-ev1 for the preparation of cRNA. The slc26a6 in the pcDNA3 vector was used for expression in HEK293 cells and for the preparation of cRNA. HEK293 cells were transfected with 1 μg cDNA coding for the transporters and 0.5 μM cDNA coding for green fluorescent protein (GFP). The oocytes were injected with 4 ng cRNA per oocyte in a 50-nl volume. 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) was obtained from Invitrogen. The bath solution for HEK293 cells (solution A) contained 145 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, pH 7.4 (with NaOH), and 10 mM glucose. Cl-free solutions were prepared by the replacement of Cl with gluconate. Solutions containing NO3 or SCN were prepared by the replacement of NaCl and KCl with the respective NO3 and SCN salts. HCO3-buffered solutions were prepared by replacing 25 mM Na+-anion with 25 mM Na+-HCO3 and reducing HEPES to 5 mM. HCO3-buffered solutions were gassed with 5% CO2 and 95% O2. The osmolarity of all solutions was adjusted to 310 mosmol with the major salt. For experiments with Xenopus laevis oocytes, the standard HEPES-buffered medium was ND96 composed of 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 2.5 mM pyruvate, and 5 mM HEPES-Na, pH 7.5 (Shcheynikov et al., 2004; Kim et al., 2005). The HCO3-buffered solution contained 71.0 mM NaCl, 25.0 mM NaHCO3, 2.0 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, and 5.0 mM HEPES-Na, pH 7.5. The Cl-free and HCO3-buffered solution contained 71.0 mM Na-gluconate, 25.0 mM NaHCO3, 2.0 mM K-gluconate, 1.8 mM Ca2+-cyclamate, 1.0 mM MgSO4, and 5.0 mM HEPES-Na, pH 7.5. Solutions were gassed with 5% CO2/95% O2.
Cells
HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FCS and 1% penicillin and streptomycin. For functional studies, HEK293 cells were cotransfected with the SLC26 transporters and a plasmid coding for GFP. GFP fluorescence was used to identify the transfected cells. LipofectAMINE (Invitrogen) was used for transfections. Oocytes were obtained by partial ovariectomy of anesthetized female Xenopus. Follicles were removed and defolliculated as described previously (Shcheynikov et al., 2004; Kim et al., 2005). Healthy oocytes in stages V–VI were injected with 1–10 ng cRNA in a final volume of 50 nl. Injected oocytes were incubated at 18°C in a ND96 solution, and oocytes were used 48–120 h after injection.
Current Measurement in HEK293 Cells
The whole cell configuration of the patch clamp technique was used to measure the Cl, NO3, and oxalate currents in HEK293 cells as described previously (Ko et al., 2004). The pipette solution contained 140 mM NMDG+-Cl or NMDG+-NO3, 1 mM MgCl2, 2 mM EGTA, 5 mM ATP, and 10 mM HEPES, pH 7.3 (with Tris). The bath solution was Na+-free solution A. The current was recorded using a patch clamp amplifier (Axopatch 200A; Axon Instruments, Inc.) and digitized at 2 kHz. The membrane conductance was probed by stepping the membrane potential from a holding potential of 0 mV to membrane potentials between −80 and 60 mV at 10-mV steps for 200 ms, with 500-ms intervals between steps. Pipettes had resistance between 5–7 MΩ when filled with pipette solution, and seal resistance was always >8 GΩ. Current recording and analysis were performed with pClamp 6.0.3 software (Axon Instruments, Inc.). Results were analyzed, and figures were plotted with Origin 7.5 software (OriginLab).
Measurement of Current and Membrane Potential in Oocytes
Electrophysiological recordings were performed at room temperature with two-electrode voltage clamp or current clamp methods using an Oocyte Clamp System (OC-725C; Warner Instrument Corp.) as described previously (Ko et al., 2002; Shcheynikov et al., 2004). The microelectrodes were filled with 3 M KCl and had a resistance of 0.5–2 MΩ. Current and voltage were digitized via an A/D converter (Digidata 1322A; Axon Instruments, Inc.) and analyzed using the Clampex 8.1 system (Axon Instruments, Inc.).
Measurement of pHi and Cli in Oocytes
For pHi and Cli measurements, electrodes were prepared from single-barreled borosilicate glass tubes (outer diameter = 1.2 mm; inner diameter = 0.69 mm; Warner Instrument Corp.) as described previously (Shcheynikov et al., 2004). In brief, the electrodes were vapor silanized with bis(dimethylamino)dimethyl silane, and the tips of the pH electrodes were filled with 0.5 μl of a H+ exchanger resin (hydrogen ionophore I, cocktail B; Fluka Chemical Corp.). The electrodes were backfilled with a ND-96 solution and calibrated in standard fresh solutions of pH 6, 7, and 8 before and after each experiment. The electrodes were fitted with a holder with an Ag–AgCl wire attached to a high-impedance probe of a two-channel electrometer (FD-223; World Precision Instruments). A second channel was used for the measurement of membrane potential by standard reference microelectrodes. The bath was grounded via a 3-M KCl agar bridge connected to an Ag–AgCl wire. The signal from the voltage electrode was subtracted from the voltage of the pH electrode using Origin 5.0 or 7.5 software (OriginLab). Initial rates of pHi change were determined from the slope of the line obtained by fitting pH as a function of time to a linear regression line. The slope of the pH electrodes was between 56 and 57 mV (pH unit)−1. To calculate HCO3 fluxes, the total buffer capacity (βT) of the oocytes was determined from the change of pHi on exposure to CO2/HCO3 (Roos and Boron, 1981) and averaged 39.6 ± 1.3 mM/pH unit (n = 52) at the pHi attained by incubation in HCO3-buffered media of 6.81 ± 0.02.
Cli was measured with a Cl-sensitive liquid ion exchanger (477913; Corning) as described previously (Ianowski et al., 2002), with minor modifications. The tips of vapor-silanized electrodes were filled with the Cl-selective liquid ion exchanger and backfilled with 3 M KCl. The electrodes were calibrated in solutions prepared to contain 1, 3, 10, 30, and 100 mM Cl by mixing solutions containing 100 mM KCl and 100 mM K-gluconate. A similar procedure was used to prepare the NO3 calibration solutions. Fresh calibration solutions were prepared each experimental day. The slope of the Cl microelectrode was ~56 mV per 10-fold change in Cl concentration. An example of the calibration curve for Cl and NO3 is shown in Fig. 1 A. Intracellular Cl activity was calculated according to the equation Cli = Clcal × 10(ΔV/S) (Ianowski et al., 2002), where Cli is intracellular Cl activity, Clcal is the Cl activity in of the calibration solutions (the Cl activity coefficient for the 10- and 100-mM KCl solutions used are 0.77 and 0.901, respectively; Hamer and Wu, 1972), ΔV is the difference in voltage between the Cl electrode and reference electrode, and S is the slope measured in response to a 10-fold change in Cl activity. Cl calibrations were performed in HEPES- and HCO3-buffered solutions.
Figure 1.
Figure 1.
Stoichiometry of Cl/HCO3 exchange by slc26a3. (A) An example of a calibration of the Cl electrode and that the resin is not sensitive to 90 mM HCO3 (green triangles) and is more selective for NO3 (black circles) (more ...)
For simultaneous measurement of pHi and Cli in oocytes, a three-electrode method was used. In this case, two ion-sensitive electrodes were connected with the FD-223 electrometer, and one reference microelectrode was used to record membrane potential with the OC-725C amplifier (Warner Instrument Corp.).
Statistical Analysis
Results in all experiments are given as the mean ± SEM of the indicated number of experiments.
To understand the function of the SLC26 transporters in epithelial Cl absorption and HCO3 secretion, it is essential to know their transport mechanism (channel or coupled transporter) and, in particular, their Cl/HCO3 transport stoichiometry. The following paragraphs describe our recent efforts toward achieving these goals.
slc26a3 Cl/HCO3 Transport Stoichiometry Is 2Cl/1HCO3
To determine the precise Cl/HCO3 transport stoichiometry of the SLC26 transporters, it is necessary to measure the Cl and HCO3 fluxes, preferably simultaneously and in the same cells. Xenopus oocytes are ideal for this task because they can be impaled with two-ion selective microelectrodes and a reference electrode. In a previous study, we described our procedure of measuring pHi in Xenopus oocytes (Shcheynikov et al., 2004). Fig. 1 A shows an example of a calibration curve with a Cl-selective electrode with the particular Cl-selective resin used. Plotting the logarithm of the anion concentrations in standard solutions as a function of the electrode potential yielded a linear slope for Cl between 3–100 mM that was not affected by the presence of up to 90 mM HCO3 and for NO3 between 1–100 mM of ~56 mV/decade change in anion concentration. The slope obtained with NO3 was shifted to more negative potentials, indicating that the resin prefers NO3 over Cl. This precludes measurement of Cl in the presence of NO3 but should be useful for the measurement of Cl/NO3 exchange.
The simultaneous measurement of Cli and pHi in the oocytes is shown in Fig. 1 B. For controls, water-injected oocytes were incubated in HCO3-buffered media that reduced pHi to ~6.85 and had no effect on Cli, which averaged 26 ± 1 mM (n = 6). Exposing these oocytes to Cl-free medium resulted in very slow rates of increase in pHi and decrease in Cli (Fig. 1 B, black traces), which is consistent with the minimal pHi regulatory capacity of the oocytes reported previously (Boron, 1986). To validate our Cl and pH calibration procedures and HCO3 and Cli flux measurements, we expressed the Cl/HCO3 anion exchanger 1 (AE1) in the oocytes. It is well established that the Cl/HCO3 transport stoichiometry of this exchanger is 1:1 (Passow, 1986). In this study, when oocytes expressing AE1 and bathed in HCO3-buffered media were exposed to Cl-free media, there was a parallel increase in pHi and a decrease in Cli. Calculation of the Cl/HCO3 flux ratio from the initial rates of the changes in pHi and Cli yielded a ratio of 0.96 ± 0.04 (n = 10; Table I). This finding validates our measurement technique, calibration procedures, and determination of the net Cl and HCO3 fluxes.
TABLE I
TABLE I
Cl and HCO3 Fluxes and Stoichiometry of Cl/HCO3 Exchange by AE1, slc26a3, and slc26a6 Transporters
The expression of slc26a3 in Xenopus oocytes slightly reduced the resting pHi and set steady-state Cl at 29.5 ± 0.9 mM (n = 17), which is statistically different from water-injected oocytes (P < 0.05). However, invariably, when the oocytes were exposed to HCO3-buffered media, Cli was reduced to 25.0 ± 0.8 mM (n = 17; P < 0.01). Exposing slc26a3-expressing oocytes to Cl-free medium also increased pHi and reduced Cli. However, in the case of slc26a3, the Cl/HCO3 transport stoichiometry occurred at a ratio of 1.96 ± 0.06 (n = 12; Table I). In all stoichiometry experiments, the slc26a3 and AE1 stoichiometries were measured on the same day and with the same batch of oocytes to further validate the measurements. Hence, the results in Fig. 1 indicate that the slc26a3 Cl/HCO3 transport stoichiometry is 2Cl/1HCO3.
slc26a3 Functions as a Coupled Exchanger
The 2Cl/1HCO3 stoichiometry of slc26a3 may be the result of tight coupling of the transported ions or may reflect partially uncoupled Cl fluxes. The results in Figs. 2 and and33 indicate an initial tight coupling of Cl and HCO3 transport by slc26a3. It was reported previously that slc26a3 can transport both OH and HCO3, although it transports HCO3 better than OH (Melvin et al., 1999; Ko et al., 2002). This is confirmed in Fig. 2 A, in which we compared Cl/OH and Cl/HCO3 exchange by slc26a3. Water-injected oocytes had a resting membrane potential of −39 ± 4 mV (n = 15), and the expression of slc26a3 decreased the membrane potential to −23 ± 4 and −21 ± 4 mV (n = 12) in the absence and presence of HCO3, respectively. Incubation in HEPES- and HCO3-buffered Cl-free media depolarized the membrane potential by 13 ± 3 and 9 ± 3 mV, respectively. Incubating the oocytes in HCO3-buffered media similarly increased the rate of pHi and Cli changes on exposure to Cl-free medium.
Figure 2.
Figure 2.
Coupling of Cl and HCO3 transport by slc26a3. In A, an oocyte expressing slc26a3 was exposed to Cl-free medium while incubated in HEPES-buffered and HCO3-buffered media. Red trace, pHi; green trace, Cli; black (more ...)
Figure 3.
Figure 3.
Effect of the membrane potential on HCO3 transport by slc26a3. The protocol shown in Fig. 2 C was used to measure changes in pHi except that the changes in pHi at each Clo concentration were measured while holding the membrane potential (more ...)
Attenuation of the change in membrane potential by HCO3 was likely caused by the robust Cl/HCO3 exchange and rapid depletion of Cl from the oocytes. This conclusion is supported by comparing the current generated by Cli/OHo and Cli/HCO3o exchange. Because of the 2Cl/1HCO3 stoichiometry, the current was measured at a holding potential of −90 mV. Clamping control oocytes to −90 mV generated an inward current of 0.13 ± 0.04 μA (n = 6). Fig. 2 B shows that in control oocytes, the removal of extracellular Cl (Clo) resulted in a small outward current (black trace). On the other hand, oocytes expressing slc26a3 showed substantial inward current at −90 mV, and the removal of Clo further increased the inward current (Fig. 2 B, blue trace) as expected from the slc26a3 stoichiometry. Higher current was generated by Cl/HCO3 compared with Cl/OH exchange, which is consistent with the higher rate of Cl/HCO3 exchange in Fig. 2 A. Interestingly, after the initial rapid increase in current on the removal of Clo, the current continued to increase slowly for several minutes. Development of the slow current parallels the change in pHi. This may reflect the relief of inhibition of a current-carrying step by the reduction in Cli or increase in HCO3i (see models in Fig. 9) or the development of uncoupled HCO3 current at continuous incubation in Cl-free medium that requires the accumulation of HCO3 in the oocytes. Uncoupled anion current and channel-like behavior of slc26a3 are illustrated in more detail in the next section. In five experiments, the removal of Clo in oocytes bathed in HCO3-buffered media resulted in a Cl/HCO3 exchange current of 1.9 ± 0.3 μA. With the Cl and HCO3 fluxes in Table I, this would suggest that the effective oocyte Cl volume is ~285 nl. This relatively low value reflects, in part, the size of the oocytes used and can be influenced by the large surface area of the oocytes. Another potential contributing factor is the development of an uncoupled current with polarity opposite to that generated by slc26a3.
Figure 9.
Figure 9.
Models of coupled and uncoupled anion transport by slc26a3. (A) A model of the turnover cycle of coupled 2Cl/1HCO3 exchange by slc26a3. (B) A model for the turnover cycles of uncoupled NO3 and SCN transport by slc26a3. (more ...)
In Fig. 2 (C and D), we measured the Clo dependency of HCO3 and Cl transport. To measure the transport at close to the resting pHi of ~7.4, oocytes bathed in HCO3-buffered media were incubated in Cl-free medium to increase pHi. After the stabilization of pHi, the oocytes were alternately exposed to media containing between 7.5 and 75 mM Clo and to Cl-free medium. The resulting changes in pHi and Cli (Fig. 2 C) were used to determine the rates of the fluxes, which are plotted in Fig. 2 D. It is clear that the slc26a3-mediated Cl and HCO3 fluxes have the same dependency on Clo. Interestingly, the Clo dependence of both anions was similar and had an averaged Hill coefficient of 1.9 ± 0.4 (n = 6 from three experiments), suggesting that 2Cl ions are transported during each cycle of Cl/HCO3 exchange, which is consistent with the 2Cl/1HCO3 transport stoichiometry of slc26a3.
Similar acceleration of HCO3 and Cl transport rates by HCO3 and similar dependence of the transport of the two anions on Clo suggest tight coupling of Cl and HCO3 exchange. The 2Cl/1HCO3 transport stoichiometry implies that transport by slc26a3 should be sensitive to the membrane potential. This is illustrated in Fig. 3. In these experiments, oocytes expressing slc26a3 were incubated in HCO3-buffered media and then in Cl-free medium to increase pHi to physiological levels. Cl-dependent HCO3 efflux and influx were measured by the addition and removal of different concentrations of Clo, respectively. As expected from the 2Cl/1HCO3 transport stoichiometry of slc26a3, hyperpolarization inhibited Clo-dependent HCO3 efflux and accelerated Cli-dependent HCO3 influx, whereas depolarization had the opposite effects (Fig. 3 A). Measurement of the effect of membrane potential on the Cl dependence of Cl/HCO3 exchange showed that hyperpolarization decreased and depolarization increased the apparent affinity for Clo (Fig. 3 B). The implication of the effect of the membrane potential for the turnover cycle of Cl and HCO3 transport by slc26a3 is the stabilization of Cl- and HCO3-preferring conformations (see Discussion).
Uncoupled Anion Transport by slc26a3
slc26a3 was found to mediate uncoupled anion transport to generate large current, possibly functioning as an anion channel. Evidence for uncoupled transport by slc26a3 was obtained when NO3 and SCN were used as substrates. Fig. 4 (A and B) shows that replacing Cl with NO3 in HEPES-buffered media resulted in rapid increase in the outward current measured at +60 mV (NO3 influx) but a small increase in the inward current measured at −100 mV. However, the inward current increased with time, reaching a maximum after ~15 min. Immediately after replacing Cl with NO3, the reversal potential shifted from −19.8 ± 0.8 to −28.8 ± 1.1 mV (n = 14; P < 0.01). However, after a 15-min incubation in NO3, the reversal potential returned to that measured in the presence of Clo, indicating that the increase in inward current is caused by replacing Cli with NO3I and the permeability of slc26a3 to NO3 is higher than that for Cl. After a 15- min incubation with NO3, the outward and inward currents increase by 2.68 ± 0.21- and 2.7 ± 0.3-fold (n = 14), respectively. Examining the currents with other anions revealed that large currents could be recorded with SCN (Fig. 4 C). The current observed with SCN was similar to that observed with NO3 except that SCN increased the current more than NO3 (5.8 ± 1.4-fold; n = 4).
Figure 4.
Figure 4.
NO3 and SCN current by slc26a3. Xenopus oocytes expressing slc26a3 and bathed in HEPES-buffered media (A–C) were incubated in media in which Clo was replaced with NO3 (A and B) or SCN (C) while measuring (more ...)
In a previous study, we were not able to measure appreciable Cl current in HEK293 cells expressing slc26a3 (Ko et al., 2004). The increased current measured with NO3 (Fig. 4, A and B) offered a new opportunity to determine whether slc26a3 can mediate a current when expressed in HEK293 cells. We used NO3 because the cell tolerated NO3 better than SCN. In Fig. 4 D, the whole cell current was measured in HEK293 cells bathed in HEPES-buffered media and dialyzed with NO3. Incubating control cells in NO3 media resulted in a current of 32 ± 7 pA at −100 mV. On the other hand, incubating HEK293 cells expressing slc26a3 in NO3-containing media resulted in a current of 238 ± 32 pA at −100 mV, with a reversal potential of 0.3 ± 0.5 mV (n = 10). Replacing extracellular NO3 (NO3o) with gluconate eliminated the outward current and shifted the reversal potential to 64 ± 2 mV, indicating high selectivity of slc26a3 for NO3. Unexpectedly, in HEK293 cells dialyzed with NO3i, replacing NO3o with Clo markedly reduced both the outward and inward currents. Similarly, when HEK293 cells expressing slc26a3 were dialyzed with Cli and incubated with NO3o, only a small current was measured (unpublished data). The findings in Fig. 4 have several important implications. First, slc26a3 can function as a conductive transporter independent of the expression system. Equally important, the slow development of the inward NO3 current in oocytes and the inhibition of the inward NO3 current by Clo in HEK293 cells suggest that Cl/NO3 exchange is a slow mode of transport that limits the current and that Clo slows the dissociation of NO3 to the external medium to reduce the overall current in HEK293 cells. This also implies that our failure to observe slc26a3-dependent current in HEK293 cells (Ko et al., 2004) was a result of the slow rates of Cl/OH and Cl/HCO3 exchange and highlights the usefulness of the oocyte system in this respect.
An increased NO3 current could be caused by higher rates of NO3/OH and NO3/HCO3 exchange than the parallel Cl exchange rates or could be caused by uncoupling of the exchange by NO3. To distinguish between these possibilities, we measured the effect of NO3 on the membrane potential and pHi in HEPES- and HCO3-buffered media. Fig. 5 (A and B) shows that replacing Clo with NO3 in oocytes incubated in HEPES- or HCO3-buffered media resulted in a rapid but transient hyperpolarization by 10.7 ± 0.7 mV (n = 10) and 11.0 ± 1.2 mV (n = 12), respectively. Subsequent replacement of NO3o with Clo transiently depolarized the cells. The transients are likely caused by the slow accumulation and efflux of NO3, respectively, mediated by Cl/NO3 exchange. Replacing NO3 with gluconate markedly depolarized the cells in both HEPES- and HCO3-buffered media (Fig. 5, A and B; last part of the traces). However, in HEPES-buffered media, the depolarization was stable, whereas in HCO3-buffered media, it was transient. The simultaneous measurement of pHi revealed that the transient change in membrane potential was the result of NO3/HCO3 exchange that depleted the oocytes of NO3 and returned the membrane potential to the resting level.
Figure 5.
Figure 5.
Uncoupled NO3 and SCN fluxes by slc26a3. Xenopus oocytes expressing slc26a3 were bathed in HEPES-buffered (A) or HCO3-buffered media (B and C). As indicated by the solid bars, they were exposed to Cl-free media, and, (more ...)
Fig. 5 B shows that replacing Clo with NO3o further acidified the oocytes by ~0.1 pH units, suggesting that at the acidic pH of 6.85, the NO3 gradient is slightly more efficient than the Cl gradient in mediating HCO3 efflux. However, the addition of NO3 to oocytes incubated in Cl-free medium resulted in marked hyperpolarization but with no reduction in pHi in HEPES-buffered media and a slow reduction in pHi in HCO3-buffered media (Fig. 5 B, shaded area). Hence, it is clear that slc26a3 can mediate NO3/HCO3 exchange. However, NO3o/HCO3i exchange occurred at a rate 2.20 ± 0.15-fold (n = 8) slower than Clo/HCO3i exchange, whereas the current in the presence of NO3o was 2.6-fold higher than in the presence of Clo (Fig. 4). An even more dramatic dissociation between current, membrane potential, and HCO3 transport was found with SCN. Fig. 5 C shows that the addition of SCN to oocytes incubated in Cl-free medium stably hyperpolarized the cells by 16.5 ± 0.4 mV (n = 3), but the SCN/HCO3 exchange occurred at a rate 13.7 ± 1.6-fold slower than that of the Cl/HCO3 exchange. Uncoupled NO3 and SCN transport by slc26a3 indicates that slc26a3 has a channel-like activity.
slc26a6 Is an Electrogenic Transporter with a 2HCO3/1Cl Stoichiometry
Two groups reported that slc26a6 functions as an electrogenic Cl/HCO3 exchanger (Ko et al., 2002; Xie et al., 2002). In contrast, Chernova et al. (2005) used the SLC26A6 and slc26a6 orthologues to conclude that slc26a6 mediates an electroneutral Cl/HCO3 exchange. To address this controversy, we measured Cli, pHi, and membrane potential in oocytes expressing slc26a6. All of the following experiments were performed with slc26a6 because we and others (Waldegger et al., 2001) found that the SLC26A6 clone is inactive. Fig. 6 A shows that incubating oocytes expressing slc26a6 in HCO3-buffered media invariably increased Cli from 27.1 ± 0.9 to 29.7 ± 1.0 mM (P < 0.05; n = 11). This is the opposite from what was found with oocytes expressing slc26a3 (Figs. 1 and and2).2). Incubating the oocytes in Cl-free medium in which Cl was replaced with gluconate resulted in hyperpolarization of the oocytes from a resting membrane potential of −28 ± 3 to −44 ± 6 mV and −39 ± 5 mV in HEPES- and HCO3-buffered media, respectively (n = 10; P < 0.01). The hyperpolarization was associated with a significant increase in pHi but a slow reduction in Cli. Similar hyperpolarization and changes in pHi were observed when Cl was replaced with SO4=, indicating that the permeability of slc26a6 to SO4= is much lower that that for Cl and comparable with that of gluconate. The Cl/HCO3 flux ratio calculated from the slopes of the pHi and Cli changes was found to be 0.56 ± 0.03 (n = 10; Table I), indicating a slc26a6 transport stoichiometry of 2HCO3/1Cl. With a 2HCO3/1Cl stoichiometry, slc26a6 is expected to generate a current. Because of the relatively slow Cl/HCO3 exchange by slc26a6 (Table I), the slc26a6-mediated current was resolved at a membrane potential of 40 mV (Fig. 6 B). Incubation of oocytes expressing slc26a6 in HCO3-buffered Cl-free medium and holding the membrane potential at 40 mV resulted in an outward current of 0.76 ± 0.13 μA (n = 4), which is smaller than that mediated by slc26a3, as expected from the slower Cl/HCO3 exchange by slc26a6.
Figure 6.
Figure 6.
Stoichiometry of Cl/HCO3 exchange by slc26a6. (A) Xenopus oocyte expressing slc26a6 and bathed in HCO3-buffered media was incubated in Cl-free and Cl-containing medium as indicated. The rates of HCO3 (more ...)
We have previously reported that slc26a6 expressed in HEK293 cells mediates Cl/HCO3 and Cl/OH exchange (Ko et al., 2002). To determine the conductive properties of the exchange, we measured the effect of Cl on the current and reversal potential in HEK293 cells expressing slc26a6. Fig. 6 C shows that in symmetrical 150 mM Cl and at 60 mV, the expression of slc26a6 resulted in a current of 196 ± 36 pA (n = 8). As was found in oocytes expressing slc26a6 (Ko et al., 2002), the removal of Clo shifted the reversal potential from 0.5 ± 0.3 to −22 ± 4 mV (n = 6). Slc26a6 can also transport formate and oxalate (Ox−2), and Ox−2 hyperpolarizes oocytes expressing slc26a6 (Knauf et al., 2001; Jiang et al., 2002). The addition of 10 mM Ox−2 to the incubation medium increased the current at 60 mV to 525 ± 48 pA (n = 8). In the presence of Clo, 10 mM Ox−2 shifted the reversal potential to −9.2 ± 0.9 mV, and the removal of Clo resulted in a reversal potential of −43 ± 4 mV (n = 5; Fig. 6 B). Hence, slc26a6 also behaves as an electrogenic transporter in HEK293 cells.
Another finding presented in Fig. 6 A is that readdition of Clo resulted in slow rates of Cl influx and HCO3 efflux. This can be explained by the rapid depolarization of the membrane potential that disfavors a 2HCO3i/1Clo exchange that moves a negative charge out of the oocytes. This interpretation can be tested by examining whether changes in the membrane potential will have the predicted effect on the fluxes. The results of such tests are shown in Fig. 7 A. The first part of the top traces in Fig. 7 A show that holding the membrane potential at 40 mV accelerated the rate of HCO3o/Cli exchange and completely halted the HCO3i/Clo exchange initiated by the readdition of Clo. On the other hand, holding the membrane potential at −100 mV accelerated Clo/HCO3i exchange and completely stopped HCO3o/Cli exchange, which was relieved by switching the membrane potential to 40 mV (Fig. 7 B). These findings are the exact behavior predicted for a 2HCO3/1Cl exchanger.
Figure 7.
Figure 7.
Effect of the membrane potential on HCO3 and Cl transport by slc26a6. A Xenopus oocyte expressing slc26a6 was bathed in HCO3-buffered media. (A) After the stabilization of pHi, the membrane potential was clamped at −30 (more ...)
slc26a6 Is a Coupled Cl/HCO3 Exchanger
To determine whether Cl and HCO3 transport by slc26a6 are coupled, we measured the effect of the membrane potential on Cl transport. Fig. 7 C shows that incubating oocytes expressing slc26a6 in a HCO3-buffered Cl-free medium resulted in the typical Cl efflux, and the readdition of Clo resulted in a very slow Cl influx. Clamping the membrane potential at −100 mV markedly accelerated the rate of Cl influx, which stopped on clamping the membrane potential at 40 mV. Accelerating the influx of the negatively charged Cl by holding the membrane potential at −100 mV can occur only by an electrogenic process that tightly couples the transport Cl to the transport of another anion with a stoichiometry of at least 2:1.
Additional evidence for the coupling of Cl and HCO3 transport by slc26a6 is provided in Fig. 8. Fig. 8 A shows that HCO3 similarly accelerates the pHi and Cli changes initiated by incubating the oocytes in Cl-free medium. In Fig. 8 (B and C), inhibition by DIDS of Cl and HCO3 fluxes and membrane hyperpolarization were compared in the same cells. All parameters were similarly inhibited by 1 and 5 μM DIDS. DIDS similarly inhibited the hyperpolarization measured in HEPES-buffered media. The combined results in Figs. 7 and and88 allow us to conclude that slc26a6 functions as a coupled Cl/HCO3 exchanger.
Figure 8.
Figure 8.
Coupling of Cl and HCO3 transport by slc26a6. (A) An oocyte expressing slc26a6 was incubated in Cl-free medium while bathed in HEPES-buffered and HCO3-buffered media. Red trace, pHi; green trace, Cli; black (more ...)
Mutations in several SLC26 transporters are linked to human diseases, most of which involve epithelia dysfunction in specific organs. This indicates that SLC26 transporters play a central role in transepithelial fluid and electrolyte transport, including Cl absorption and HCO3 secretion by the kidney, the GI tract, and secretory glands (Kunzelmann and Mall, 2002; Ko et al., 2004; Melvin et al., 2005; Steward et al., 2005). To understand the function of the SLC26 transporters in epithelial Cl absorption and HCO3 secretion, it is essential to know their transport mechanism and Cl/HCO3 transport stoichiometry. Two of the most studied SLC26 transporters are slc26a3 and slc26a6. Both were shown to function as Cl/HCO3 exchangers (Melvin et al., 1999; Ko et al., 2002; Wang et al., 2002) and as electrogenic transporters (Ko et al., 2002; Xie et al., 2002) with isoform-specific stoichiometry (Ko et al., 2002). However, the electrogenicity of the transporters was called into question by a recent study claiming that slc26a6 mediates electroneutral Cl/HCO3 exchange based on the inability to measure OH or HCO3-dependent Cl current in Xenopus oocytes expressing slc26a6 (Chernova et al., 2005). The critical importance of resolving this issue for understanding epithelial Cl absorption and HCO3 secretion in the normal and disease states requires precise knowledge of the function of these transporters and their stoichiometry.
In this study, we measured all critical parameters, HCO3 and Cl fluxes, and membrane current and potential in the same cells to determine the Cl/HCO3 transport stoichiometry and transport mechanism of slc26a3 and slc26a6. The procedure for measuring Cl/HCO3 exchange stoichiometry was validated by determining a 1Cl/1HCO3 exchange stoichiometry for AE1 (Passow, 1986). With this technique, we proceeded to determine a 2Cl/1HCO3 exchange stoichiometry for slc26a3 and a 1Cl/2HCO3 stoichiometry for slc26a6. Indeed, transport by slc26a3 and slc26a6 was affected by the membrane potential in a manner consistent with their respective stoichiometries. Both transporters appear to function as coupled exchangers. For slc26a3, this conclusion is based on the similar acceleration of HCO3 and Cl transport by HCO3, similar dependence of the Cl and HCO3 transport on Clo (Fig. 2), and stimulation of HCO3 influx by clamping the membrane potential at −100 mV (Fig. 3). Tight coupling of Cl/HCO3 exchange by slc26a6 is supported by the similar acceleration of Cl and HCO3 transport by HCO3, similar inhibition by DIDS (Fig. 8), and stimulation of Cl influx by holding the membrane potential at −100 mV (Fig. 7).
Although the 2Cl/1HCO3 stoichiometry (Table I), a 1.9 Hill coefficient for Cl (Fig. 2 D), the stimulation of the negatively charged HCO3 influx by holding the membrane potential at −100 mV (Fig. 3 A), the effect of the membrane potential of the Cl and HCO3 fluxes (Fig. 3 B), and the very large uncoupled NO3 and SCN currents (Fig. 4) all point to electrogenic transport by slc26a3, two observations need further considerations. The first is the small change in membrane potential observed on the removal of Clo in slc26a3-expressing oocytes bathed in HCO3-buffered media (Figs. 2 A and 5, B and C). We note that completion of the depolarization as a result of the removal of Clo in HEPES-buffered media required >3 min with the large oocytes (Figs. 2 A and 5 A). At this time, the oocytes lost ~18 mM of their Cl, which can account for the small residual change in membrane potential. The second problematic observation is that the current mediated by slc26a3 is smaller than that expected from the coupled Cl and HCO3 fluxes. One possible explanation for this observation is the development of an uncoupled anion current during the incubation in Cl-free medium that may carry Cl and/or HCO3, which will result in an apparent reduced slc26a3-mediated current. Further work is needed to resolve this uncertainty.
The effect of the membrane potential on the apparent affinity for Clo suggests the turnover cycle for coupled Cl and HCO3 transport by slc26a3 that is depicted in Fig. 9 A. The model is based on the stabilization of a Cl- or HCO3-preferring conformation of slc26a3 by the membrane potential. The extracellular-facing substrate-binding sites of the empty transporter (Eo) prefers Cl over HCO3 and can bind 2Cl ions to form Eo[center dot]2Cl. Eo[center dot]2Cl undergoes a conformational change to Ei[center dot]2Cl and transfers the Cl into the cytosol. The cytosolic form of slc26a3 (Ei) prefers HCO3 over Cl to dissociate the Cl and bind HCO3 to form Ei[center dot]HCO3. Ei[center dot]HCO3 undergoes a conformational transition to Eo[center dot]HCO3 to transfer and release HCO3 to the external medium and complete the cycle. Clamping the membrane potential at 40 mV will favor the Cl-binding conformation of slc26a3 and shifts the steady-state levels toward the Ei[center dot]2Cl conformation, resulting in increased apparent affinity for Cl. On the other hand, clamping the membrane potential at −100 mV will favor the HCO3-binding conformation of slc26a3 to shift the steady-state levels toward the Eo[center dot]HCO3 conformation, resulting in decreased apparent affinity for Cl. Eo and Ei can have the same or different charge. For example, the substrate-binding site of Ei may have two positive charges, binds 1HCO3 to have a net positive charge, and will be stabilized by a negative membrane potential to reduce the apparent affinity for Cl. The substrate-binding site of Eo may have one positive charge, binds 2Cl to have a net negative charge, and will be stabilized by a positive membrane potential to increase the apparent affinity for Cl. The change in the substrate site charge takes place after dissociation of the respective anions. Alternatively, Eo and Ei may have two positive charges, and only the HCO3-bound Ei has a net positive charge to be stabilized by negative membrane potential and reduce the apparent affinity for Cl. At present, we cannot distinguish between the potential mechanisms.
Interestingly, slc26a3 can mediate a channel-like transport by functioning as an uncoupled anion transporter to mediate large NO3 and SCN currents that are not coupled to OH or HCO3 transport (Figs. 4 and and5).5). That is, slc26a3 functions as a NO3 and SCN conductive transporter rather than as an exchanger. slc26a3 can generate NO3 and SCN currents either by functioning as a NO3 and SCN channel or as an electrogenic carrier, as depicted in Fig. 9 B for NO3. Functioning as a carrier requires that after the dissociation of NO3 or SCN, the empty carrier can undergo a conformational change to display the substrate-binding sites facing the cell interior or exterior (Fig. 9 B, dashed arrow) and that in the presence of NO3 or SCN, the conformational change of the empty carrier is preferential to that of the carrier occupied with HCO3. In contrast, Cl disfavors the conformational change of the empty carrier to recouple the transport. This can be because the affinity of the carrier for Cl is higher than that for NO3 and SCN so that only a minute fraction or none of the carrier is empty. The cardinal difference between a channel and uncoupled carrier mode is that a carrier mode requires conformational changes to alternately display the substrate-binding sites to the cell interior and exterior, whereas a channel only requires the transporter to be in an open or closed state. Both modes of transport by the same protein have been described previously. For example, the neurotransmitter transporters can function as coupled carriers or as channels (Kanner and Borre, 2002), whereas a recent study showed that the prokaryotic homologue of the WT ClC Cl channels ClC-ec1 functions as an electrogenic H+-Cl exchanger, but its E148A mutant functions exclusively as a Cl channel (Accardi and Miller, 2004). This is reminiscent of the Cl and NO3/SCN transport by slc26a3. A simple way to distinguish between a channel and a carrier mode is to measure single channel activity and observe whether the transporter can switch between discrete open and closed states.
Despite extensive efforts and examination of many experimental conditions, we were unable to measure single channel activity with slc26a3 expressed either in oocytes or in HEK293 cells. Although this would favor an uncoupled carrier mode, negative results must be interpreted with caution. For example, it was suggested that the Cl/HCO3 exchanger AE1 may mediate a current by occasionally letting the ions move along a channel-like pathway in an uncoupled slippage-like manner (Frohlich, 1984). This may generate a small current that is difficult to detect by single channel measurement. NO3 and SCN transport by slc26a3 may be mediated, in part, by such a mechanism. Therefore, at present, our results are not sufficient to state with confidence which model more accurately describes the NO3 and SCN currents by slc26a3. Nevertheless, it is clear that slc26a3 can function as a coupled 2Cl/1HCO3 exchanger or as an uncoupled transporter to mediate anion currents. However, in the presence of physiological Cl and HCO3 gradients, the preferential mode of transport by slc26a3 is a coupled 2Cl/1HCO3 exchange.
The current findings concerning the properties of slc26a6 are in agreement with two previous studies (Ko et al., 2002; Xie et al., 2002) but contradict another (Chernova et al., 2005) concluding that slc26a6 is an electroneutral Cl/HCO3 exchanger. However, Chernova et al. (2005) did not measure the stoichiometry of the transport, and their findings have internal inconsistencies. For example, they reported that hSLC26A6 and mslc26a6 mediate the same Cl/HCO3 exchange activity, yet mslc26a6 showed close to 100-fold higher Cl fluxes than hSLC26A6 (Chernova et al., 2005). This calls into question their measurement of pHi with BCECF in the large oocytes and whether these measurements reflect net HCO3 transport by slc26a6. In addition, Chernova et al. (2005) reported similar Ox−2 transport by hSLC26A6 and mslc26a6 but also that Ox−2 affected the membrane potential of oocytes expressing mslc26a6 but not hSLC26A6. The size of the current in the oocytes expressing hSLC26A6 and mslc26a6 in the presence and absence of Ox−2 was small with poor signal/noise (Chernova et al., 2005). As shown in Fig. 6, Ox−2 must cause a large increase in the current, and the increase should be independent of the slc26a6 isoform used for the current measurements to be valid.
The Cl/HCO3 transport stoichiometry of slc26a3 and slc26a6 has profound significance for the mechanism of epithelial Cl absorption and HCO3 secretion. Thus, as discussed in a previous study (Ko et al., 2004) and reviewed in Steward et al. (2005), the axial distribution of these transporters in secretory epithelia, their interaction with CFTR, and regulation of their function (Ko et al., 2002, 2004) determines the final Cl and HCO3 concentrations of the secreted fluid. The stoichiometry of slc26a3 and slc26a6 is suitable for absorbing the Cl and concentrating HCO3 in the secreted fluid. At luminal membrane potentials more depolarized than −50 mV, slc26a6 in the proximal duct and slc26a3 in the distal duct will determine the final Cl and HCO3 concentrations of HCO3-rich and Cl-poor fluids such as those secreted by the pancreas and salivary glands (Ko et al., 2004). However, at more hyperpolarized voltages and at Cli that is at or <4 mM, the opposite arrangement is more favorable (Steward et al., 2005). The Cl and HCO3 content of fluids generated by epithelia are of vital importance for the integrity and function of these organs, as evident from their destruction in cystic fibrosis, a disease typified by aberrant Cl absorption and HCO3 secretion (Wilschanski and Durie, 1998; Sokol, 2001).
Acknowledgments
This work was supported by National Institutes of Health grants DE12309 and DK38938 and the Cystic Fibrosis Foundation grant MUALLE01G0.
Olaf S. Andersen served as editor.
Notes
Abbreviations used in this paper: AE1, anion exchanger 1; DIDS, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid; GFP, green fluorescent protein; HEK, human embryonic kidney.
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