Cloning of a Na+-driven Cl/HCO3 exchanger from squid giant fiber lobe

Leila V. Virkki, Inyeong Choi, Bruce A. Davis, and Walter F. Boron

Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520

Submitted 24 September 2002 ; accepted in final form 7 May 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We extracted RNA from the giant fiber lobe (GFL) of the squid Loligo pealei and performed PCR with degenerate primers that were based on highly conserved regions of Na+-coupled HCO3- transporters. This approach yielded a novel, 290-bp sequence related to the bicarbonate transporter superfamily. Using an L. opalescens library, we extended the initial fragment in the 3' and 5' directions by a combination of library screening and PCR and obtained the full-length clone (1,198 amino acids) by PCR from L. pealei GFL. The amino acid sequence is 46% identical to mammalian electrogenic and electroneutral Na-HCO3 cotransporters and 33% identical to the anion exchanger AE1. Northern blot analysis showed strong signals in L. pealei GFL, optic lobe, and heart and weaker signals in gill and stellate ganglion. To assess function, we injected in vitro-transcribed cRNA into Xenopus oocytes and subsequently used microelectrodes to monitor intracellular pH (pHi) and membrane voltage (Vm). Superfusing these oocytes with 5% CO2-33 mM HCO3- caused a CO2-induced fall in pHi, followed by a slow recovery. The absence of a rapid HCO3--induced hyperpolarization indicates that the pHi recovery mechanism is electroneutral. Ion substitutions showed that Na+ and Cl- are required on opposite sides of the membrane. Transport was blocked by 50 µM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The characteristics of our novel clone fit those of a Na+-driven Cl/HCO3 exchanger (NDCBE).

intracellular pH; microelectrodes; giant axon; 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; bicarbonate


THE SQUID GIANT AXON (6) and the snail neuron (30) were the preparations in which investigators first described the dynamic regulation of intracellular pH (pHi): the recovery of pHi from an imposed intracellular acid load. Work on the giant axon of Loligo pealei—involving measurements with microelectrodes and manipulations of the ionic composition of the axoplasm by dialysis—showed that the activity of the transporter responsible for this pHi regulation requires HCO3- (5), intracellular Cl- (26), and extracellular Na+ (10). Comparable isotopic flux experiments showed that both Na+ and Cl- are transported (10) and not just required for acid-base transport activity. The ionic stoichiometry of the transporter is the influx of one Na+ ion and the equivalent of two HCO3- ions and the efflux of one Cl- ion, resulting in electroneutral transport. The entire process is blocked by disulfonic stilbenes (26). This work, along with parallel work on the snail neuron (31), led to the initial description of the Na+-driven Cl/HCO3 exchanger.

After the cloning of the first Na+-dependent HCO3- transporter, the electrogenic Na-HCO3 cotransporter NBC1 from salamander kidney (23), Romero et al. (24) cloned a Na+-driven Cl-/anion exchanger (NDAE1) from Drosophila. This Drosophila clone is more promiscuous than NBC1 in its selectivity for a base, inasmuch as it appears to transport both HCO3- and OH-. Moreover, expression of NDAE1 in oocytes is associated with a small leak conductance. More recently, Grichtchenko et al. (16) cloned a human Na+-driven Cl/HCO3 exchanger (NDCBE1) that has an absolute requirement for HCO3- or a HCO3--related species (e.g., CO32-).

Despite the advances in cloning and identification of new HCO3- transporters, the Na+-dependent HCO3- transporter whose function, arguably, has been most thoroughly studied is the NDCBE of the squid giant axon. Realizing the potential value of having the cDNA that encodes such a well-studied transporter for the understanding of the function of all the other members of the bicarbonate transporter (BT) superfamily—and exploiting the existing sequence information on other members of the NBC family—we set out to clone the squid axon NDCBE (sqNDCBE).


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study was conducted in accordance with the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society.

cDNA cloning. We harvested giant fiber lobes (GFL) from longfin inshore squid (L. pealei) obtained at the Marine Biological Laboratory (Woods Hole, MA). Total RNA was extracted with Trizol reagent (GIBCO-BRL, Life Technologies, Gaithersburg, MD) according to the manufacturer's directions. We synthesized cDNA by using Superscript reverse transcriptase (GIBCO-BRL) primed with (oligo)dT. This cDNA was used as a template for PCR with degenerate oligonucleotide primers that were designed to highly conserved regions of electrogenic and electroneutral NBCs. The first PCR was performed with the sense primer 3'-AARGGIKCIGGITWYCAYCTIGAY-5', which corresponds to a region between predicted transmembrane segments (TMs) 8 and 9, and the antisense primer 3'-ICCIGWIACRTAYTGYTCRTAIAC-5', which corresponds to a region between TMs 11 and 12. [The locations of the predicted TMs are based on the topological model of Taylor et al. (29) for anion exchanger (AE)1.] A second, nested reaction was run with the same sense primer and the antisense primer 3'-IGAIGIIAYICCCATRTADAGRAA-5' (corresponding to a region in TM 11). Resulting PCR products of the expected length were subcloned into pCR 2.1 TOPO vector (Invitrogen, Carlsbad, CA) and sequenced. All sequencing was performed by the Keck Biotechnology Resource Laboratory (Boyer Center for Molecular Medicine, Yale University) or Genewiz (New York, NY). Using this approach we identified a novel 290-bp sequence related to the BT superfamily.

To obtain the full-length coding region, we used a combination of library screening and PCR. We probed an L. opalescens library (a kind gift from Dr. William Gilly, Stanford University, Pacific Grove, CA) with the initial 290-bp sequence as a probe and identified a partial clone that extended the sequence in the 5' direction and into the 3' untranslated region (UTR) region. We extended the sequence in the 5' direction by performing PCR on the library with a gene-specific primer and a primer specific to the vector sequence adjacent to the 5' end of the insert. Resulting PCR products were cloned and sequenced as described above. This strategy extended the L. opalescens clone through the rest of the 5' coding region and into the 5' UTR. We designed primers complementary to the 5' and 3' UTRs just outside the coding regions. Using reverse-transcribed cDNA from L. pealei GFL as a template, we then obtained by PCR a product corresponding to the full-length (1,198 amino acids) sequence of sqNDCBE. To obtain a consensus sequence for sqNDCBE from L. pealei, we sequenced the full-length PCR product. The sequence was deposited in GenBank (accession no. AY151155 [GenBank] ). We then subcloned the full-length PCR product into the pCR 2.1 TOPO vector and sequenced five different clones. Using Chameleon and Quickchange mutagenesis kits (Stratagene, La Jolla, CA), we corrected all of the PCR errors in one of the full-length clones.

Northern blot analysis. Total RNA was extracted from L. pealei eye, gill, heart, testis, GFL, optic lobe. and stellate ganglion with Trizol (GIBCO-BRL). Total RNA (10 µg) was resolved by formaldehyde agarose (1%) denaturing gels and blotted to positively charged nylon membrane (Hybond XL; Amersham Biosciences, Piscataway, NJ) by capillary elution. Blots were prehybridized by incubation in ExpressHyb hybridization solution (Clontech Laboratories, Palo Alto, CA) at 68°C for 30 min and then hybridized with an {alpha}-32P-labeled cDNA probe (random primer labeling kit, GIBCO-BRL). The cDNA probe was the same 290-bp fragment that was obtained in the initial degenerate PCR and corresponded to base pairs 2503-2792 of the open reading frame.

Expression in oocytes. Capped cRNA was synthesized in vitro from the plasmid containing the sqNDCBE coding region in pCR 2.1 TOPO with a T7 Message Machine kit (Ambion, Austin, TX). Stage V-VI oocytes from Xenopus laevis were isolated as described previously (22). One day after isolation, the oocytes were injected with 50 nl of a solution containing 0.5 ng/nl of cRNA encoding sqNDCBE. Control oocytes were injected with 50 nl of sterile water. The oocytes were used in experiments 3-7 days after injection. All experiments were performed at room temperature (~22°C).

Solutions. Nominally HCO3--free ND96 solution contained (in mM) 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES, pH 7.50 (titrated with NaOH). HCO3--containing solutions were prepared by replacing 33 mM NaCl with 33 mM NaHCO3 in ND96 and equilibrating the solution with 5% CO2-balance oxygen. In some ND96 and HCO3- solutions, Na+ was replaced by N-methyl-D-glucamine (NMDG+), Li+, or K+. Cl--free solutions were prepared by replacing Cl- with gluconate. For butyrate-containing solutions, 30 mM Na butyrate replaced 30 mM NaCl in ND96. This butyrate concentration produced approximately the same degree of intracellular acidification as 5% CO2. Osmolality of all solutions was ~195 mosmol/kgH2O.

Measurements of pHi. An oocyte was placed in a chamber and constantly superfused with solution at a flow of 4 ml/min. Bath solutions were delivered with syringe pumps (Harvard Apparatus, South Natick, MA), and solutions were switched with pneumatically operated valves (Clippard Instrument Laboratory, Cincinnati, OH). In all experiments, the oocyte was initially superfused with the ND96 solution, which is nominally CO2/HCO3- free.

We assayed pH regulatory function of water-injected oocytes and oocytes expressing sqNDCBE by measuring pHi with pH-sensitive microelectrodes. pH-sensitive electrodes were fabricated and used as described previously (23, 28). Briefly, the oocyte was impaled with two microelectrodes, one for measuring the membrane potential (Vm) and the other for measuring pHi. The tip of the pH electrode contained a pH-sensitive liquid membrane (hydrogen ionophore I, cocktail B; Fluka Chemical, Ronkonkoma, NY), and the electrode was backfilled with a phosphate buffer (1). The two electrodes were connected to separate inputs of an FD223 dual electrometer (World Precision Instruments, Sarasota, FL). The voltage due exclusively to pHi was obtained by subtracting the analog signal of the Vm electrode from that of the pH electrode. The voltage due exclusively to Vm was obtained by subtracting the analog signal of a bath calomel electrode from that of the Vm electrode. The electrical signals, referenced to a platinum wire in the bath, were sampled by computer and data were acquired with software written in-house. The system was calibrated with buffered pH standards at pH 6.0 and 8.0 in the bath. An additional single-point calibration was performed with the standard ND96 solution of pH 7.50 in the bath before impaling the oocyte.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sequence data. The cDNA encoding sqNDCBE contains an open reading frame of 3,597 bp, encoding 1,198 amino acids. Figure 1 shows a sequence alignment of sqNDCBE with two other members of the BT superfamily, human NDCBE1 and human AE1. The transmembrane segments of AE1, based on the topology model developed by Taylor et al. (29), are underlined. The amino acid sequence of sqNDCBE is ~50% identical to that of human NDCBE1, ~32% identical to human AE1, and ~50% identical to Drosophila NDAE1 (sequence not shown). In the membrane-spanning regions (amino acids 503-1011) the homology is only slightly higher compared with human NDCBE (57%) and human AE1 (38%). The hydrophilic (presumably intracellular) COOH-terminal domain of sqNDCBE is 188 amino acids long, which is considerably longer than the COOH-terminal domain of AE1 (33 amino acids), NDCBE (48 amino acids), or Drosophila NDAE1 (92 amino acids).



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Fig. 1. Sequence alignment. The amino acid sequences of squid (sq) Na+-driven Cl/HCO3 exchanger (NDCBE), human (h)NDCBE, and human anion exchanger 1 (hAE1) were aligned with ClustalW and the BioEdit sequence alignment editor constructed by Hall (17). Black shading indicates an identical amino acid residue; gray shading indicates a conservative substitution. Predicted transmembrane regions (TMs) 1-14 of AE1 [based on the model developed by Taylor et al. (29)], are marked by black lines under the AE1 sequence. The 12th TM is predicted to be nonhelical and is marked by a dotted line. Three putative 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid DIDS motifs, either intact or disrupted, are located at the extracellular ends of TMs 3, 5, and 13 and are enclosed by boxes. The 4 predicted N-glycosylation sites in the 3rd exofacial loop are marked with an asterisk on top of the sqNDCBE sequence. The position of the 290-bp sequence, which was obtained in the initial PCR step of cloning and which was used as a probe in the Northern blot, is indicated by a dashed line on top of the sqNDCBE sequence.

 

The dendrogram of select members of the BT superfamily in Fig. 2A shows that sqNDCBE is more closely related to electroneutral NBCs and Na-dependent Cl/HCO3 exchangers, less closely related to electrogenic NBCs, and even less closely related to AE1 (AE2 and AE3, not shown, cluster with AE1). The hydropathy plot in Fig. 2B is consistent with a large NH2-terminal domain and 12-14 TMs. As is the case for mammalian Na+-coupled HCO3- transporters, but opposite the general pattern for the AEs, the third exofacial loop of SF1 is substantially longer than its fourth exofacial loop. The topology model in Fig. 2C is based on the hydropathy plot and on the alignment of sqNDCBE with human AE1 in Fig. 1. The NH2- and COOH-terminal domains are predicted to be intracellular and contain several consensus PKA and PKC phosphorylation sites. The third exofacial loop contains four consensus N-glycosylation sites. The topology of TMs 9-14 is not well defined and may include nonhelical membrane-spanning segments (29).



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Fig. 2. Sequence analysis. A: phylogenetic tree with the amino acid sequences of human NDCBE, human Na-HCO3 cotransporter (NBCn1), Drosophila Na+-driven Cl/anion exchanger (NDAE1), Loligo pealei sqNDCBE, human NBCe1, and NBCe2 (NBC4) and a functionally uncharacterized clone from Caenorhabditis elegans. B: hydropathy analysis with Kyte-Doolittle algorithm, window size 15. C: topology model of sqNDCBE. Membrane-spanning segments are predicted based on the AE1 topology model of Taylor et al. (29) and the hydropathy plot in B. The model also indicates the positions of the 4 consensus N-glycosylation sites, as well as the PKA and PKC consensus phosphorylation sites. Numbers indicate TMs 1-14.

 

Northern blot analysis. Figure 3 shows a Northern blot of total RNA from whole tissues of L. pealei; the blot was probed with a sqNDCBE-specific 290-bp fragment that was obtained by PCR in our initial cloning step. A strong signal, corresponding to an mRNA size of ~7.5 kb, is seen in GFL, heart and optic lobe, whereas a somewhat weaker signal is seen in gill and the portion of the stellate ganglion less the GFL. No signal is present in eye or testis.



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Fig. 3. Northern blot. Total RNA (10 µg/lane) from eye, giant fiber lobe (GFL), gill, heart, olfactory lobe (OL), stellate ganglion (SG), and testis was resolved on a denaturing agarose gel. The blot was hybridized to a 32P-labeled 290-bp cDNA fragment corresponding to part of the sqNDCBE sequence (indicated by the dashed line in Fig. 1).

 

Na+ and Cl- dependence of sqNDCBE. Figure 4A shows a recording of pHi and membrane potential in an oocyte expressing sqNDCBE. We initially superfused the oocyte with a nominally HCO3--free ND96 solution. As indicated in Fig. 4A, we first removed extracellular Na+ and then exposed the oocyte to a Na+-free solution (NMDG+ replacing Na+) containing 5% CO2-33 mM HCO3-. Exposing the oocyte to CO2 caused a decrease in pHi due to the influx of CO2 (25). However, in the absence of external Na+, we observed no pHi recovery. When we reintroduced external Na+, pHi started to recover rapidly, presumably because of the coupled entry of Na+ and HCO3- (or equivalent ions). When we again removed external Na+, this time in the presence of CO2/HCO3-, the transporter reversed and pHi decreased as the transporter presumably mediated the efflux of Na+ and HCO3-, the intracellular concentrations of which had risen earlier during the CO2/HCO3- exposure. Similar results were obtained whether the Na+ replacement ion was NMDG+ or K+, indicating that K+ does not support transport through sqNDCBE (not shown).



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Fig. 4. Na+ and Cl- dependence of sqNDCBE. A: representative recording of intracellular pH (pHi) and membrane potential (Vm) in an sqNDCBE-expressing oocyte. The oocyte was initially superfused with nominally HCO3--free ND96 solution. Solution changes were performed as indicated. B: values for rate of pHi change (dpHi/dt) were measured in 6 oocytes at times a-e, as indicated in A. C: representative recording of pHi and Vm in a water-injected oocyte. The oocyte was subjected to a solution protocol similar to that in A. D: values of dpHi/dt were measured in 5 water-injected oocytes at times a-e, as indicated in C. Error bars indicate SE. *Statistically significant (P < 0.05; paired 1-tailed t-test) difference between a dpHi/dt value and the previous value. Actual P values are shown in parentheses in B. Arrows highlight differential Vm between H2O-injected and sqNDCBE-expressing oocytes.

 

While the transporter was running in reverse, we removed external Cl- (in the continued absence of external Na+). This removal halted the pHi decrease, indicating that the transporter had stopped. When we then reintroduced external Cl-, pHi continued to decrease, indicating that the transporter was again running in reverse. Reintroducing the external Na+ caused pHi to increase once more, showing that the transporter was again running in the forward direction. These results show that the sqNDCBE—at least when running in the reverse direction—requires extracellular Cl- for activity. Because removing external Cl- when the transporter is running in the forward direction (i.e., in the presence of external Na+) has little effect on the rate of pHi recovery (not shown), it is unlikely that sqNDCBE requires Cl- as a nontransported activator that binds to an extracellular site on the transporter.

Figure 4B summarizes data on the rates of pHi change (dpHi/dt) in six different oocytes subjected to the protocol shown in Fig. 4A. We extracted the dpHi/dt values for the different conditions from the linear portions of the records indicated by points a-e in Fig. 4B. A negative dpHi/dt indicates that the transporter is running in reverse. There is a tendency for the dpHi/dt values obtained with a given extracellular solution (compare points a vs. e and b vs. d in Fig. 4B) to decrease during the time of the experiment. This apparent decrease in transport rate presumably does not reflect only a decay in transmembrane gradients for the transported ions, because if the experiment is prolonged by repeatedly removing external Na+ and Cl-, the transporter eventually stops completely (not shown).

Although not shown, we also found that removing extracellular Cl- in the presence of Na+ had no effect on the pHi recovery from a CO2-induced acid load (analogous to point a in Fig. 4A). The mean rate of pHi recovery was 22 ± 4 x 10-5 s-1 in the presence of Cl- and 24 ± 7 x 10-5 s-1 in the absence of Cl- (n = 9; P = 0.4 in a paired 1-tailed t-test).

Figure 4C shows a recording of pHi and Vm in a water-injected (control) oocyte subjected to a protocol similar to that in Fig. 4A. In contrast to the sqNDCBE-expressing oocyte, very little pHi recovery occurred in the presence of Na+ and subsequent removal of external Na+ and Cl- caused only small changes in pHi. There was a tendency for pHi to increase slowly during the exposure to CO2, indicating that oocytes have native, albeit very weak, pHi-regulating mechanisms. Figure 4D summarizes the mean dpHi/dt data for five water-injected oocytes.

The acid-base transport mediated by sqNDCBE appears to be electroneutral. When external Na+ was removed in the presence of HCO3, the changes in Vm were small (<10 mV) and similar in size in both sqNDCBE-expressing and water-injected oocytes, reflecting the presence of native ion channels in the oocytes. We did note a consistent difference in the Vm records between sqNDCBE-expressing and water-injected oocytes on removing CO2/HCO3-. In sqNDCBE-expressing oocytes, removing CO2/HCO3- always caused a slow depolarization (arrows in Fig. 4A) that was reversed on returning the CO2/HCO3- (not shown). In contrast, in water-injected oocytes removing CO2/HCO3- always caused a slow hyperpolarization. It is also curious that, in oocytes expressing sqNDCBE, the initial application of CO2/HCO3- did not elicit the inverse Vm change (i.e., a hyperpolarization), either when executed in the absence of Na+ (Fig. 4A; see Fig. 6A) or the presence of Na+ (see Fig. 7A). Thus the CO2/HCO3- dependence of Vm is history dependent. The effect could involve sqNDCBE alone, the interaction of sqNDCBE with oocyte protein(s), or oocyte protein(s) whose expression is increased in response to the expression of sqNDCBE.



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Fig. 6. Li+ transport by sqNDCBE. A: representative recording of pHi and Vm in an sqNDCBE-expressing oocyte. The oocyte was initially superfused with nominally HCO3--free ND96 solution. Solution changes were performed as indicated. In 2 of 6 experiments, NMDG+ was used instead of K+ between the Na+ and Li+ solutions. In some oocytes, the Li+ solution was used before the Na+ solution during the exposure to CO2/HCO3-. B: values of dpHi/dt were measured in 6 sqNDCBE-expressing oocytes at times a and b, as indicated in A. C: representative recording of pHi and Vm in an water-injected oocyte. The oocyte was subjected to a solution protocol similar to that in A. D: values of dpHi/dt were measured in 4 water-injected oocytes at times a and b, as indicated in C. The difference between the mean values in B and in D were not statistically significant at the 0.05 level in a paired, 1-tailed t-test.

 


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Fig. 7. DIDS sensitivity of sqNDCBE. A: representative recording of pHi and Vm in an sqNDCBE-expressing oocyte. The oocyte was initially superfused with nominally HCO3--free ND96 solution. Solution changes were performed as indicated. B: values of dpHi/dt were measured in 6 sqNDCBE-expressing oocytes before, during, and after the application of DIDS (50 µM). C: representative recording of pHi and Vm in an water-injected oocyte. The oocyte was subjected to a solution protocol similar to that in A. D: values of dpHi/dt were measured in 4 water-injected oocytes before, during, and after the application of DIDS. Error bars indicate SE. *Statistically significant (P < 0.05; paired 1-tailed t-test) difference between a dpHi/dt value and the previous value. Actual P values are shown in parentheses.

 

In summary, the data are consistent with the hypothesis that sqNDCBE carries out the electroneutral exchange of Na+ and HCO3- (or thermodynamically equivalent species) for Cl-.

HCO3- dependence. To determine whether sqNDCBE requires HCO3-for activity, we used a pH 7.5 solution containing 30 mM butyrate (calculated external butyric acid concentration = 60 µM assuming a pK of 4.8) to induce a pHi decrease that is similar to that produced by 5% CO2. If sqNDCBE requires HCO3-, then no pHi recovery should occur in butyrate. As shown in Fig. 5A, exposing an sqNDCBE-expressing oocyte to 30 mM butyrate caused an intracellular acidification without subsequent pHi recovery. When we then acidified the oocyte by using 5% CO2-33 mM HCO3-, pHi recovered rapidly after the initial acidification, indicating that H+/OH- are not sufficient to sustain transport through sqNDCBE. Figure 5B summarizes the mean rates of pHi change in butyrate and CO2/HCO3- for five different sqNDCBE-expressing oocytes.



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Fig. 5. HCO3- dependence of sqNDCBE. A: representative recording of pHi and Vm in an sqNDCBE-expressing oocyte. The oocyte was initially superfused with nominally HCO3--free ND96 solution. Solution changes were performed as indicated. B: values of dpHi/dt were measured in 5 sqNDCBE-expressing oocytes at times a and b, as indicated in A. C: representative recording of pHi and Vm in an water-injected oocyte. The oocyte was subjected to a solution protocol similar to that in A. D: values of dpHi/dt were measured in 5 water-injected oocytes at times a and b, as indicated in C. Error bars indicate SE. *Statistically significant (P < 0.05; paired 1-tailed t-test) difference between a dpHi/dt value and the previous value. Actual P values are shown in parentheses.

 

Figure 5C shows that, in water-injected oocytes, little pHi change occurred after the initial acidification induced by either butyrate or CO2. Figure 5D summarizes dpHi/dt data from five different water-injected oocytes.

Li+ transport. In Fig. 4A we saw that sqNDCBE does not transport the organic cation NMDG+. To determine whether sqNDCBE transports alkali metals other than Na+, we carried out experiments in which we replaced external Na+ with Li+, K+, or NMDG+. In the experiment shown in Fig. 6A, we first used CO2/HCO3- to acidify an sqNDCBE-expressing oocyte in an NMDG+ solution (as in Fig. 4A). When we replaced NMDG+ with Na+, pHi started to increase. Subsequently replacing Na+ with either NMDG+ or K+ caused pHi to decrease, showing that the transporter reversed. Thus K+ does not support significant sqNDCBE activity. Subsequently replacing the K+ with Li+ caused pHi to recover, indicating that Li+ does support transport through sqNDCBE.

Figure 6B summarizes dpHi/dt in Na+- and Li+-containing solutions obtained from six different sqNDCBE-expressing oocytes. To minimize possible effects of differences in pHi values and of concentration gradients for the transported ions, we always measured dpHi/dt at the same pHi in any one oocyte (Fig. 6A). To minimize bias resulting from a possible rundown of the transporter, we measured in two oocytes dpHi/dt first in the Li+ solution and then in the Na+ solutions. Also, the time period between the two measurements was kept as short as possible. The result shows that the pHi recovery rate in Li+ is ~75% of that seen in Na+, indicating that Li+ (but not K+) can support substantial transport of acid-base equivalents through sqNDCBE.

Figure 6C shows the results of an experiment in which we subjected a water-injected oocyte to the protocol shown in Fig. 6A. As we already saw in Fig. 4C, the pHi of the native oocyte recovered only slightly after the CO2-induced acidification. Figure 6D summarizes the dpHi/dt data from four different water-injected oocytes.

DIDS sensitivity. Because many other members of the BT superfamily are sensitive to disulfonic stilbenes, we examined the effect of 50 µM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) on the rate of pHi recovery in sqNDCBE-expressing oocytes. Figure 7A shows an sqNDCBE-expressing oocyte acidified by 5% CO2-33 mM HCO3-. In the continuous presence of external Na+, pHi started to recover after the initial acidification. Subsequently superfusing the oocyte with a solution containing DIDS caused a small initial decrease in pHi followed by a complete blockade of transport. The reason for the initial DIDS-induced acidification is unclear, but it might reflect the unmasking of an underlying acid-loading mechanism or perhaps the induction of a passive flux of HCO3- or H+. After we washed away the DIDS, pHi recovered once again, indicating that—at least over a time frame of ~5 min—DIDS interacts reversibly with sqNDCBE. Figure 7B summarizes dpHi/dt data from six different sqNDCBE-expressing oocytes.

Figure 7C shows the effect of DIDS on a water-injected oocyte. After the initial CO2-induced acidification, pHi recovered very little, although DIDS produced a slight inhibition. As summarized in Fig. 7D for four different water-injected oocytes, the dpHi/dt values in all categories for the control cells are very low compared with those in sqNDCBE-expressing oocytes. It should be noted that DIDS has a small but statistically significant effect, probably reflecting the presence of an endogenous DIDS-sensitive pHi-regulating mechanism.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Is sqNDCBE the transporter functionally described in squid giant axon? We report here the cloning and functional characterization of a Na+-driven Cl/HCO3 exchanger from the squid L. pealei; we call the clone sqNDCBE. From our Northern-blot analysis, the message for sqNDCBE is present in the GFL, the tissue that contains the neuron cell bodies that give rise to the giant axon. In the oocyte, sqNDCBE requires Na+, HCO3-, and Cl- for activity, and is thus similar in its ion requirements to the squid axon NDCBE. As is the transporter in the axon, sqNDCBE is very sensitive to blockade by disulfonic stilbenes, inasmuch as 50 µM DIDS completely blocked pHi recovery in sqNDCBE-expressing oocytes.

sqNDCBE differs, however, from the transporter described in the axon on two accounts. First, sqNDCBE readily reverses its direction of transport when the gradient for Na+ is inverted, whereas the transporter in the axon does not (unpublished observations cited in Ref. 4). Second, with sqNDCBE expressed in Xenopus oocytes, Li+ can support a pHi recovery from an acid load nearly as well as Na+, whereas in the axon, Li+ is a very poor substrate for the transporter (10).

Despite the above discrepancies, it is possible that sqNDCBE is, at least in part, responsible for Na+-driven Cl/HCO3 exchange in the squid axon. For example, differences in the molecules that interact with sqNDCBE in the oocyte vs. the axon may account for the aforementioned discrepancies. In addition, the osmolarity of the squid, a marine invertebrate, is near 1,000 mosM, whereas the osmolarity of an oocyte, from the freshwater frog Xenopus, is ~200 mosM. It is possible that these large differences in osmotic and/or ionic strength affect sqNDCBE itself or its physico-chemical interactions with ligands and/or other molecules that interact with the transporter.

On the other hand, it is possible that sqNDCBE makes no contribution to the Na+-driven Cl/HCO3 exchanger activity observed in intact squid axons. First, although the mRNA encoding sqNDCBE is present in the GFL, the protein may not be present in the axon. This issue will remain unresolved until an sqNDCBE antibody is available. Second, even if present in the axon, the sqNDCBE protein may not be active. Finally, we also have cloned from the GFL two partial cDNA clones that are related to sqNDCBE. It is possible that one (or both) of these is the molecular substrate of the Na+-driven Cl/HCO3 exchanger activity observed in axons or that one (or both) of these forms a heteromultimer with sqNDCBE and that only the heteromultimer exhibits the full range of properties previously observed in the intact axon. It should, however, be noted that heteromultimer formation has hitherto not been documented for any member of the BT family.

What does sqNDCBE transport? Transport by sqNDCBE is electroneutral, and one transport cycle involves the exchange of one external Na+ for one internal Cl- and the inward transport of two base equivalents. However, in our experiments it is impossible to separate the inward transport of OH-, HCO3-, CO32-, or the NaCO3- ion pair from the outward movement of H+. A series of kinetic studies (4, 7, 8, 10) are consistent with the model that the acid-base entity transported by the squid axon Na+-driven Cl/HCO3 exchanger is the NaCO3- ion pair. On the other hand, it appears that not all Na+-driven Cl/HCO3 exchangers operate by this mechanism: in barnacle muscle fibers, the NaCO3- ion pair model cannot account for the data (9). Instead, the data for this latter preparation are consistent with the model that Na+ and CO32- (or HCO3-) bind randomly to the transporter. Thus, although all "Na+-driven Cl/HCO3 exchangers"—including the squid and human versions named NDCBE—are thermodynamically equivalent, they may turn out to operate by a range of different ionic mechanisms.

Where does DIDS interact with sqNDCBE? Stilbene disulfonates are classic inhibitors of anion transport, and their interactions with the anion exchanger of erythrocytes (AE1) has been studied extensively (2, 3, 11, 12, 15, 19, 21, 27, 32). SITS (which has 1 rather than 2 isothiocyano groups) initially blocks AE1 transport as the result of a rapid ionic interaction with the transporter, which can be reversed by scavenging the SITS with an albumin-containing solution. Over the period of many minutes, SITS can covalently react with an amino group on AE1, after which block of function becomes permanent. Okubo et al. (21) demonstrated that H2DIDS covalently reacts with the lysine at position 539 (K539) of human AE1; after prolonged treatment at alkaline pH, DIDS cross-links with another lysine at K851. Furthermore, work by Passow and colleagues (32) showed that mutations of K558 in mouse AE1 (equivalent to K539 in human) and K869 (equivalent to K851) abolish most of the covalent (but not reversible) inhibition by H2DIDS.

Kopito et al. (18) first proposed a potential DIDS reaction motif KLXK (where the first K is K539 in human AE1 and X is I or Y) at the putative extracellular end of TM5. Subsequent to their cloning of the first Na+-dependent HCO3- transporter, Romero et al. (23) noted that the electrogenic NBCe1 from the salamander Ambystoma not only is functionally blocked by DIDS but also has an AE-like motif (KMIK) at the equivalent position, suggesting that the motif might be KXXK (where X might represent any of several hydrophobic residues). Preliminary work shows that in human NBCe1 this motif is involved in reversible block by DIDS (20). Mutating either the first or last lysine in this motif to asparagine increased the apparent binding constant and off-rate of DIDS, with the largest effects observed when both lysines were replaced. Finally, when Choi et al. (13) cloned the rat electroneutral Na-HCO3 cotransporter, they found that NBCn1 function is poorly sensitive to DIDS and that the DIDS reaction motif was a "disrupted" KLFH. Preliminary work shows that mutating KLFH to KLFK makes NBCn1 function very sensitively to DIDS (14).

Together, these data suggest that a site at the extracellular end of TM5 can play a role in both reversible and irreversible action of DIDS. Of the mammalian members of the BT superfamily, only AE1-3 and NBCe1 have a KXXK DIDS motif at the extracellular end of putative TM5. Interestingly, NDCBE1 and NCBE—both of which are DIDS sensitive—have the sequence KFCK at the extracellular end of putative TM3. sqNDCBE, despite being sensitive to DIDS, has disrupted DIDS motifs at both TM3 (NFCS) and TM5 (KLFH). However, sqNDCBE has a potential DIDS motif (KTIK) at the extracellular end of TM13 (see Figs. 1 and 2C). The first of these two lysines (K983) is at a position analogous to the K in human AE1 (K851) that reacts with DIDS at alkaline pH. It is intriguing to speculate that the four residues at the extracellular ends of TMs 3, 5, and 13 (boxed residues in Fig. 1), which tend to conserve charge (usually positive charge) at the first and fourth positions, may be spatially clustered in members of the BT superfamily, perhaps helping to form an electrostatic trap or binding site for HCO3--related species.


    DISCLOSURES
 
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-18400 and by Office for Naval Research Grant 1N00014-01-1-022. L. V. Virkki and I. Choi were supported by the American Heart Association.


    ACKNOWLEDGMENTS
 
Present address of I. Choi: Dept. of Physiology, Emory University School of Medicine, Atlanta, GA 30322


    FOOTNOTES
 

Address for reprint requests and other correspondence: L. V. Virkki, Institute of Physiology, Univ. of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.

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


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