INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

INTRACELLULAR CHLORIDE CONCENTRATION ([Cl]_i) is maintained at a higher level than its electroneutral value, as calculated by the Nernst equation, indicating that it is maintained by mechanisms other than simple diffusion (17). [Cl]_i must therefore be regulated by the relative activities of plasma membrane Cl-influx and Cl-efflux pathways. In previous studies, we showed that rat thymic lymphocytes possess both Na⁺-dependent and Na⁺-independent Cl/HCO₃ exchangers and examined the roles they play in the regulation of intracellular pH (pH_i) (3, 41). With the availability of a chloride-sensitive dye, we now explore the role of these and other potential exchangers on the pathways for Cl entry and exit.

Two classes of Cl/HCO₃ exchangers have been identified in mammalian cells. The band 3 family of exchangers [erythroid (AE1, AE2, and AE3) isoforms] is sodium independent. Band 3 protein-mediated anion exchange has been previously demonstrated in lymphocytes (17, 27, 28), which, as in most other cell types, express the AE2 isoform (1, 2, 27). AE2 is expressed in all regions of the kidney (6). In contrast, -intercalated cells in the kidney, like erythrocytes, express the AE1 isoform (13). The activity of this family of transporters is governed by the relative concentration gradients of Cl and HCO₃ across the cell membrane. Under normal physiological conditions this transporter is said to be inactive (17). It is activated by cellular alkalinization and normally acts as a Cl-influx mechanism (the exchange of intracellular HCO₃ for extracellular Cl). This protein is capable of transporting Cl or mediating pH_i changes that reflect Cl/HCO₃ exchange (27, 28). Thus in lymphocytes, AE2 is the likely candidate for regulation of pH_i and [Cl]_i (23). A second class of Cl/HCO₃ exchanger that has been identified is a Na⁺-dependent Cl/HCO₃ (41). This antiporter acts as a Cl-efflux mechanism (intracellular Cl exits the cell in exchange for HCO₃) (33).

Another family of transport proteins capable of regulating [Cl]_i is the Na⁺-K⁺-2Cl transporters (38). Two isoforms of the Na⁺-K⁺-2Cl transporter have been identified to date. NKCC1 (also dubbed BSC-2) is a widely distributed isoform. In renal cells, it is located in basolateral membranes and has been named a "secretory" isoform. This transporter is involved in maintenance of cell volume, and its activity is greatly increased on cell shrinkage (29, 39). A distinct isoform, NKCC2, (also dubbed BSC-1) exists only in the apical membrane of the thick ascending limb in the kidney where it is involved in the reabsorption of sodium chloride. It is sometimes referred to as the "reabsorptive" isoform. The Na⁺-K⁺-2Cl transporters are sensitive to inhibition by the loop diuretics furosemide and bumetanide. Studies that examined the role of Na⁺-K⁺-2Cl transport in the regulation of [Cl]_i in lymphocytes have resulted in conflicting findings (14, 17). One study suggested that a bumetanide sensitive and Na⁺- and Cl-dependent influx of ⁸⁶Rb, a measure of K⁺ transport, accounted for roughly 75% of the ⁸⁶Rb uptake (14). In contrast, others reported that Cl uptake in lymphocytes was largely unaffected by omission of extracellular Na⁺ and K⁺ or by the addition of bumetanide.

Functional characterization of the Cl/HCO₃ exchangers for the most part has been limited to experiments that evaluated fluorimetric measurements of pH_i changes during the removal and restoration of extracellular chloride or to experimentally driven alterations in unidirectional Cl fluxes, as measured by using radiolabeled Cl. Similarly, studies examining the activity of Na⁺-K⁺-2Cl transporters have centered largely on cell volume. There is little information on how the interplay of these Cl-transport mechanisms impact on [Cl]_i. In the present study, we examined the mechanisms involved in Cl influx and efflux, by using the fluorimetric indicators N(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) and 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), to determine net changes in [Cl]_i and pH_i in lymphocytes during the acute exposure to Cl-free media and during reexposure to external Cl after Cl_i depletion. We show that Cl efflux is totally inhibited by DIDS and is mediated by a Na⁺-dependent Cl/HCO₃ exchanger. Cl influx, which is partially DIDS sensitive and partially furosemide sensitive, is mediated by both a Na⁺-independent Cl/HCO₃ exchanger and by a Na⁺-K⁺-2Cl cotransporter.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Preparation of cells. Thymic lymphocytes were isolated from Sprague-Dawley rats, 6-9 wk of age, as previously described (4, 41). Briefly, the rats were anesthetized by an intraperitoneal injection of pentobarbital (50 mg/kg body wt), and the chest cavity was opened by cutting the ribs along the sternum. The thymus was removed with forceps and cleared of blood vessels. Contaminating blood was removed by rinsing with RPMI-1640. The thymus was then minced and pipetted vigorously with a syringe several times. Large cell aggregates and connective tissue were removed by passage through four layers of surgical gauze. The resulting suspension of lymphocytes was washed free of red blood cell contamination, by centrifugation three times in RPMI-1640 at 150 g for 10 min each.

Loading of lymphocytes with MQAE, a chloride-sensitive dye. After the final wash, the lymphocytes were resuspended in RPMI-1640 containing 5 mM MQAE (Molecular Probes, Eugene, OR) and incubated at 37°C for 120 min. After loading, the cell suspension was centrifuged for 10 min to remove the external dye solution. The cell pellet was then split and the cells resuspended in either a bicarbonate containing physiological salt solution (solution 1, Table1), in a Cl-free buffer solution (solution 2, Table 1), or their bicarbonate-free buffer equivalents. All solutions were kept at 37°C by using a water bath. Bicarbonate-containing buffers were preequilibrated with 5% CO₂-95% O₂ by directly bubbling the gas mixture through the buffer solution for at least 30 min before use. A constant stream of CO₂-O₂ over the cuvette was used to keep the solutions equilibrated with CO₂ during fluorometer readings. The composition of the standard buffer solutions used for this study is listed in Table 1. All solutions were filtered through a 0.2-µm filter before use to reduce autofluorescence of solutions.

Fluorescence measurements. Fluorescence measurements were obtained with an SLM DMX-1000 spectrofluorometer at an excitation wavelength of 352 nm and an emission wavelength of 450 nm (49). The dye-loaded cells were kept under constant magnetic stirring in a thermostatically controlled cuvette (37°C).

Calculation of [Cl]_i. [Cl]_i was calculated by using a modification of the procedure described by others (8, 11, 12, 30, 32). The F_t was obtained by subtracting the autofluorescence (after addition of KSCN) from the F_max, which was obtained as fluorescence signal in Cl-depleted cells assayed in a Cl-free solution. Cl depletion was accomplished by incubation of the cells in a Cl-free solution for 60 min before use.
The F_Cl was determined by the equation
where F was fluorescence signal of cells assayed in a normal physiological buffer solution.

Determination of Cl efflux and influx. Basal [Cl]_i was determined from the first reading of fluorescence of efflux curves. Chloride efflux rates were determined as the initial decline in [Cl]_i after suspending the cells in a chloride-free media. The initial rate of Cl efflux was defined as change in [Cl]_i during the first 90 s after exposure to the Cl-free solution. For these experiments isethionic acid (2-hydroxyethanesulfonic acid; sodium salt) was used to replace extracellular chloride (solution 2, Table 1).

Measurement of pH_i. The pH-sensitive fluorescent probe, BCECF was used to measure pH_i (4, 41). The cells were loaded with 1 µg/ml BCECF-AM (Molecular Probes) for 30 min at 37°C. After loading, the cells were washed three times by centrifugation with the assay buffer and then kept for at least 30 min before proceeding. Samples of cells were washed again immediately before use to remove any external dye.

Chemicals. MQAE, ethylisopropylamiloride (EIPA), and H₂DIDS were purchased from Molecular Probes, Eugene, OR. All other chemicals were purchased from Sigma or Aldrich, St. Louis, MO.

Statistical analysis. The n for each set of experiments was determined as average from duplicate readings from each animal used in the study. All data are expressed as means ± SE. Statistical differences between groups were determined by using a Student's t-test for paired or unpaired data when appropriate. Differences were considered significant when P  0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Intracellular Cl. There was no difference in the basal [Cl]_i between lymphocytes kept for 1 h in HCO₃-containing and those maintained in a HCO₃-free solution [HEPES buffered; 24.3 ± 1.3 vs. 26.2 ± 1.5 mM, respectively, n = 11, not significant (NS)]. On the basis of an external [Cl] of 101 mM (Table 1) and a resting membrane potential of 55 mV for lymphocytes (17), the electrochemical equilibrium for [Cl]_i is 13 mM as calculated by the Nernst equation. Thus [Cl]_i is maintained at a higher level than its electroneutral value indicating that it is regulated by mechanisms other than simple diffusion.

Cl efflux. After the removal of extracellular chloride (solution 2, Table 1), the initial decline in [Cl]_i was used to estimate the net chloride efflux rate. In cells maintained in a HCO₃-containing solution, this rate was markedly faster than that of paired cells kept in HCO₃-free solution ([Cl]_i 2.7 ± 0.36 vs. 1.1 ± 0.29 mM/90 s, respectively, n = 11, P < 0.01). (Fig. 1).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Chloride efflux from cells assayed in the presence or nominal absence of HCO3/CO2. Thymic lymphocytes were isolated and loaded with N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) for the measurement of intracellular Cl concentration ([Cl]i)as described in METHODS. Paired aliquots of cells were kept in either a HCO3/CO2 (, n = 11) solution or one that was nominally HCO3 free (HEPES) (, n = 11). Injection of cells into a Cl-free solution resulted in a faster decline in [Cl]i in cells assayed in the presence of HCO3/CO2 than in cells assayed in its nominal absence.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effect of DIDS on net Cl efflux and increase in intracellular pH (pHi). Cells were either pretreated for 25 min with DIDS (125 µM; ) or maintained in a control HCO3/CO2 solution () before injection into a Cl-free HCO3 containing solution. A: there was a rapid decline in [Cl]i (net Cl efflux) when cells were injected into a Cl-free solution. Preincubation with DIDS (n = 6) had no effect on the initial [Cl]i compared with controls (n = 6), but resulted in the complete inhibition of net Cl efflux that was observed in control cells that were exposed to a Cl-free solution. B: there was a rapid increase in pHi when the cells were injected into a Cl-free solution. Preincubation with DIDS (n = 5) had no effect on the initial pHi, but almost completely blocked the rise in pHi that was observed in control cells (n = 5) that were exposed to a Cl-free solution.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effect of acute Na+ removal on net Cl efflux and increase in pHi. Cells were maintained in a sodium-containing HCO3/CO2 solution and then either injected into a Na+-free, Cl-free, HCO3-containing solution () or injected into a control Cl-free HCO3 containing solution (). A: removal of external Na+ (n = 7) resulted in the complete inhibition of net Cl efflux that was observed in control cells (n = 7) that were exposed to a Cl-free solution. B: removal of external Na+ (n = 3) resulted in a transient cell acidification followed by only a slight rise in pHi that failed to alkalinize beyond the initial value.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effect of furosemide on net Cl efflux and increase in pHi. Cells were either pretreated for 60 min with furosemide (200 µM; ) or maintained in a control HCO3/CO2 solution () before injection into a Cl-free HCO3-containing solution. A: preincubation with furosemide (n = 5) resulted in a significantly lower initial [Cl]i than controls (n = 5), and a slower initial rate of net Cl efflux than controls. When normalized for [Cl]i, net Cl efflux was similar between the 2 groups of cells. B: preincubation with furosemide (n = 5) had no effect on either the initial pHi or the increase in pHi that occurred after injection into a Cl-free HCO3 containing solution.

Cl influx. The mechanisms involved in Cl entry into lymphocytes were examined by chloride depleting the cells for 1 h (solution 2, Table 1) and then reexposing them to a Cl-containing solution (solution 1, Table 1) . This protocol resulted in initial levels of Cl_i close to 0 (0.54 ± 0.11 mM, n = 39). Resuspension of Cl-depleted cells in a Cl-containing solution resulted in a rapid increase in [Cl]_i. The increase in [Cl]_i was significantly faster in cells resuspended in a bicarbonate buffer than in those exposed to a bicarbonate-free (HEPES) buffer ([Cl]_i 10.4 ± 1.29 vs. 5.8 ± 0.62 mM/90 s, respectively, n = 10, P < 0.01) (Fig. 5).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Cl influx in cells assayed in the presence or nominal absence of HCO3/CO2. Paired aliquots of cells were kept in either a HCO3/CO2 solution (, n = 10) or one that was nominally HCO3 free (HEPES) (, n = 10). The cells were Cl depleted for 60 min and then injected into a Cl-containing solution. Injection of cells into a Cl-containing solution resulted in a faster increase in [Cl]i in cells assayed in the presence of HCO3/CO2 than in cells assayed in its nominal absence.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Effect of DIDS on net Cl influx and decline in pHi. Cl-depleted cells were either pretreated for 25 min with DIDS (125 µM; ) or maintained in a control Cl-free HCO3/CO2 solution () before injection into a Cl-containing, HCO3-containing solution. A: there was a rapid rise in [Cl]i in control cells (n = 6) that began to plateau after ~2 min. In cells pretreated with DIDS (n = 6), the rise in [Cl]i was nearly linear and did not show any apparent saturation. The initial rate of Cl influx, at 90 s, was decreased in cells pretreated with DIDS by ~60% compared with controls. B: the initial pHi was increased compared with cells that had not been Cl depleted (see Fig. 4B). Reexposure to Cl resulted in a rapid decline in pHi in control cells (n = 5). Preincubation with DIDS (n = 5) had no effect on the initial pHi, but completely blocked the decline in pHi that was observed in control cells.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Effect of acute Na+ removal on net Cl influx and decline in pHi. Cells were Cl depleted in a Na+-containing HCO3/CO2 solution for a total of 60 min and then either injected into a Na+ free, Cl-containing solution () or injected into a control Na+-containing, Cl-containing solution (). A: removal of external Na+ (n = 5) resulted in ~50% inhibition of the net Cl influx that was observed in control cells (n = 5). Note that the shapes of the 2 curves were similar. B: removal of external Na+ (n = 3) has no effect on either the initial pHi or the decline in pHi that occurred in control cells (n = 3) that were reexposed to Cl.

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Effect of furosemide on net Cl influx and decline in pHi. Cells were Cl depleted for a total of 60 min. The cells were either pretreated for 15 min with furosemide (200 µM; ) or maintained in a control Cl-free HCO3/CO2 solution () before injection into a Cl-containing, HCO3-containing solution. A: pretreatment with furosemide (n = 5) resulted in ~50% inhibition of the net Cl influx that was observed in control cells (n = 5). Note that the shapes of the 2 curves were similar. B: pretreatment with furosemide (n = 5) had no effect on either the initial pHi or the decline in pHi that occurred in control cells (n = 5) that were reexposed to Cl.

View larger version (17K): [in this window] [in a new window]   Fig. 9.   Effect of furosemide and H2DIDS on net Cl influx. Cells were Cl depleted for a total of 60 min. The cells were either pretreated with furosemide (200 µM) and H2DIDS (125 µM) together (, n = 9) or maintained in a control Cl-free HCO3/CO2 solution (, n = 9) before injection into a Cl-containing, HCO3-containing solution. Preincubation with furosemide and H2DIDS together almost completely inhibited the net Cl influx that was observed in control cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In thymic lymphocytes, pH_i is regulated by both Na⁺-independent and Na⁺-dependent Cl/HCO₃ exchangers (41) as well as the NHE-1 isoform of the Na⁺/H⁺ exchange family (34, 43). Evidence for Cl/base exchange has accumulated largely on the basis of either flux studies or indirectly by measurements of pH_i during the removal and readdition of Cl from the media. [Cl]_i, however, was not measured in those studies. The present study was undertaken to examine the pathways involved in Cl entry and exit into thymic lymphocytes. Examination of Cl efflux pathways was performed by acutely exposing the cells to a Cl-free media and determining the change in [Cl]_i and pH_i under various experimental conditions. Similarly, examination of Cl influx pathways was performed by Cl depleting the cells for 1 h and then acutely exposing the cells to a Cl-containing media. Our data show that, in thymic lymphocytes, [Cl]_i is regulated by a balance of Cl efflux via a Na⁺-dependent Cl/HCO₃ exchanger and Cl influx via a Na⁺-independent Cl/HCO₃ exchanger and a furosemide-sensitive mechanism consistent with a Na⁺-K⁺-2Cl cotransporter.

The basal [Cl]_i reported in the present study (24.3 ± 1.3 mM) by using thymic lymphocytes was similar to values reported for other cell types (25-36 mM) including tonsillar B lymphocytes (20), astrocytes (5, 50), and vascular smooth muscle cells (10, 30), but lower than some other studies on lymphocytes (17, 25, 40). These differences are likely due to the methods used to measure [Cl]_i or the assay conditions under which they were determined. Methods that determine total Cl content in the cells, photometrically, with electrodes, or via radiolabeled Cl dilutions generally yield higher estimates of [Cl]_i than fluorimetric techniques that determine free [Cl]_i .

Interestingly, in the present study the basal [Cl]_i was not different between cells maintained in a HCO₃/CO₂-buffered solution and those maintained in the absence of HCO₃/CO₂, indicating that the presence of HCO₃/CO₂ in the external media is not an absolute requirement for the maintenance of normal [Cl]_i. pH_i, is also similar under both conditions (4) although it can be shown to be significantly lower in the nominal absence of HCO₃. A number of possibilities that could account for this observation include 1) HCO₃-independent mechanisms predominate the regulation of [Cl]_i in these cells; 2) Cl/HCO₃ exchangers do not have an absolute requirement for HCO₃ but rather can use other counteranions to exchange for Cl; and 3) both Cl influx and Cl efflux are equally affected by the removal of HCO₃ from the media, thus resulting in no net alteration in [Cl]_i . Evidence from our study and others (18) suggests that all of these possibilities may explain the observed phenomenon.

Pretreatment of the cells with furosemide for 1 h resulted in a 38% decline in [Cl]_i but had no effect on pH_i. In contrast, pretreatment with DIDS had no effect on either [Cl]_i or pH_i. These findings indicate that basal [Cl]_i, but not pH_i, is highly dependent on the activity of a Na⁺-K⁺-2Cl cotransporter, whereas inhibition of the Cl/HCO₃ exchangers had no net effect on the basal [Cl]_i. This is not to say that the sodium-dependent and -independent Cl/HCO₃ exchangers are not active under basal conditions. The finding that their coinhibition had no discernible affect on [Cl]_i is likely due to their counterbalancing each other. These findings are consistent with previous studies. In rabbit aorta, it was found that [Cl]_i is decreased in the presence of furosemide but not in the presence of DIDS or in HCO₃-free media (18). It had also been shown that the reduction in Cl transport observed in the rectal gland cells of Squalus acanthias after administration of furosemide is due to the fall in the [Cl]_i and not a direct effect on the chloride current (19).

Under control conditions, in the presence of a HCO₃/CO₂ solution, acute exposure of the cells to an isotonic Cl-free solution resulted in a rapid decline in [Cl]_i that was accompanied by a rise in pH_i (increase in [HCO₃]_i). When the cells were pretreated with furosemide and then subsequently exposed to a Cl-free solution, the rate of decline in [Cl]_i (net Cl efflux) was reduced compared with control cells, whereas furosemide pretreatment had no effect on the change in pH_i. As stated above, however, the control cells had a higher initial [Cl]_i than cells pretreated with furosemide. When the rate of decline in [Cl]_i for control cells and those pretreated with furosemide was compared at similar [Cl]_i, there was no difference, suggesting that the decrease in net Cl efflux was caused by the lower [Cl]_i because of reduced Cl influx, rather than a direct effect of furosemide on Cl efflux.

To determine whether preincubation with furosemide reduced Cl influx, the rate of Cl influx was measured in cells that had been Cl depleted and then reexposed to a Cl-containing solution. Preincubation of the cells with furosemide for 15 min resulted in a 45% decrease in net Cl influx, while having no effect on the pH_i. Similarly, blockade of Na⁺-K⁺-2Cl cotransport by the acute removal of external Na⁺ resulted in a 46% inhibition of net Cl influx, while having no effect on the pH_i. Our findings that inhibition of Na⁺-K⁺-2Cl cotransport reduces [Cl]_i and Cl influx into thymic lymphocytes, but does not directly affect Cl efflux, are in agreement with other studies that examined the effects of Na⁺-K⁺-2Cl-cotransport inhibition in lymphocytes (14) or other cell types (18, 19, 30). In contrast to these studies, others were unable to demonstrate any effect of bumetanide on [Cl]_i or Cl influx in rat lymphocytes (17).

Evidence for the existence of a Na⁺-dependent Cl/HCO₃ exchanger comes from our studies examining the effects of DIDS or Na⁺ removal on net Cl efflux and pH_i. Pretreatment of the cells with DIDS before exposure to a Cl-free solution completely inhibited both the decline in [Cl]_i and the rise in pH_i that were observed in control cells, suggesting that a Cl/HCO₃ exchange mechanism was responsible for Cl efflux in these cells. These findings are consistent with what others have shown, examining the effects of DIDS and SITS on pH_i and radiolabeled Cl fluxes in lymphocytes (37).

It is not likely that differences in the orientation of the inhibitor binding site accounts for the different inhibitor effects on Cl efflux and influx, but rather our data are best explained by the presence of two different transporters. Studies indicate that the stilbene binding site is located on the outer surface of the membrane rather than buried within the pocket formed by the tertiary complex of the protein (7, 44). It is also distinct from the anion binding site(s). The best model for explaining the transport of anions by the AE exchangers is the ping pong model, in which the anion binding site changes its orientation. To our knowledge, however, present models do not predict a change in the orientation of the inhibitor binding site.

Two classes of Cl/HCO₃ exchangers have been identified in mammalian cells. The band 3 family of exchangers (AE1, AE2, and AE3) is sodium independent. The activity of this family of transporters is governed by the relative concentration gradients of Cl and HCO₃ across the cell membrane. Under normal physiological conditions, this transporter is said to be inactive (17). It is activated by cellular alkalinization and normally acts as a Cl-influx mechanism (the exchange of intracellular HCO₃ for extracellular Cl). However, under experimental conditions of external Cl removal, this antiporter can be reversed (17, 33, 37). A second Cl/HCO₃ exchanger that has been identified is a Na⁺-dependent Cl/HCO₃ exchanger. This antiporter acts as a Cl-efflux mechanism (the exchange of intracellular Cl for extracellular Na⁺ and HCO₃) both physiologically and under experimental conditions of external Cl removal (33). Ion flux studies suggest that the Na⁺-dependent Cl/HCO₃ exchanger transports one Cl out of the cell in exchange for one Na⁺ and two base equivalents into the cell. The functional mode of the exchanger may be HCl extrusion in exchange for NaHCO₃, or Cl extrusion in exchange for 1 Na⁺ and 2 HCO₃ ions (42). In either case, the transporter is electroneutral. To distinguish which of these transporters could account for the decline in [Cl]_i when the lymphocytes were exposed to a Cl-free solution, the sodium dependence of net chloride efflux was examined.

Our data show that the net Cl efflux and rise in pH_i were both completely blocked by the acute removal of Na⁺ from the external media, indicating that Cl/HCO₃ exchange was due to a sodium-dependent mechanism. To rule out the possibility that these effects were caused by inhibition of the Na⁺/H⁺ antiporter, additional experiments were performed with EIPA, an inhibitior of the Na⁺/H⁺ antiporter. EIPA had no effect on net Cl efflux or the rise in pH_i. Taken together, these data support the contention that in thymic lymphocytes, Cl efflux is totally mediated by a Na⁺-dependent Cl base exchanger.

Our study shows that, in the nominal absence of HCO₃/CO₂ in the media, Cl efflux is still mediated by a Na⁺-dependent, DIDS-sensitive mechanism. The nature of this mechanism has not been elucidated, but the evidence suggests that it may be a different functional mode of the Na⁺-dependent Cl/HCO₃ exchanger. To date, this transporter has not been isolated and cloned. However, if one were to extrapolate the findings on other anion exchangers to that of the Na⁺-dependent Cl/HCO₃ exchanger then it would be likely that this transporter would be capable of using a number of different anions to exchange for Cl_i.

Studies have shown that the band 3 protein family of Cl/HCO₃ exchangers is capable of transporting a number of different anions and working in a number of different modes in addition to Cl/HCO₃ exchange (e.g., Cl/Cl