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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut; and 2Department of Physiology, Emory University School of Medicine, Atlanta, Georgia
Submitted 23 October 2006 ; accepted in final form 31 January 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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intracellular pH; acid-base; axon; SLC4 family
Despite the important contributions of invertebrate models to our understanding of pHi regulatory mechanisms, relatively little is known about the molecular identity and molecular physiology of the proteins that mediate the transport processes in these systems. To date, a HCO3– transporter has been cloned and characterized from only one of these model systems, namely, the squid giant fiber lobe (GFL). Virkki et al. (49) cloned the cDNA encoding the Na+-driven Cl/HCO3– exchanger (sqNDCBE), which is 50% identical at the amino-acid level to both the Na-coupled HCO3– transporters of the solute carrier 4 (SLC4) family in humans and the Na+-driven anion exchanger (AE) of Drosophila melanogaster (39), drNDAE.
When sqNDCBE is expressed in Xenopus oocytes, the transporter mediates electroneutral Na+-driven Cl/HCO3– exchange that is inhibited by the stilbene derivate DIDS (49). However, the physiology of sqNDCBE in oocytes differs in some details from the observed physiology in squid axons inasmuch as, in oocytes, sqNDCBE can operate 1) with Li+ substituting for Na+ and 2) in the direction of HCO3– efflux when the Na+ gradient is reversed. One possibility is that the oocyte expression system or ionic strength could account for the discrepancies. Alternatively, the in situ physiology may require a factor not present in oocytes, or may be mediated by a different protein entirely. Our group provided preliminary evidence for two partial cDNA clones (16), suggesting that the squid giant-fiber system expresses at least two SLC4-like transporters besides sqNDCBE. Sequence analysis suggested that one of these additional squid clones ("SF3") is most similar to Na+-coupled HCO3– transporters in the SLC4 family, whereas the other ("SF4") is most similar to the anion exchangers. To determine whether one of these transporters could explain other aspects of the observed physiology in the squid axon, we set out to 1) obtain the full-length cDNA for SF3 from GFL cDNA and 2) characterize the electrophysiology of the encoded transporter when expressed in Xenopus oocytes. We show that the full-length amino acid sequence of SF3 is very similar to sqNDCBE. A phylogenetic analysis of membrane domains indicates that SF3 clusters with sqNDCBE and other electroneutral Na+-coupled SLC4 transporters. However, when we measure pHi and membrane potential (Vm) with microelectrodes—or use two-electrode voltage clamping to measure currents—on Xenopus oocytes expressing SF3 cRNA, the oocytes exhibit the hallmarks of an electrogenic NBC. That is, an exposure to extracellular CO2/HCO3– not only causes an abrupt fall in pHi followed by a recovery, but also 1) a rapid hyperpolarization and 2) a robust membrane current. The pHi recovery and current require HCO3– and Na+, and are blocked by DIDS. Furthermore, neither K+ nor Li+ can fully replace Na+ in supporting the pHi recovery, and extracellular Cl– is not necessary for the transporter to operate in the reversed direction. Therefore, SF3—hereafter referred to as sqNBCe—is an electrogenic Na/HCO3– cotransporter, the first electrogenic NBC characterized from an invertebrate.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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From longfin squid L. pealei collected at the Marine Biological Laboratory (Woods Hole, MA), we 1) extracted eye, optic lobe, stellate ganglion, GFL, gill, heart, and testis; 2) immersed the tissues in ice-cold TriZOL reagent (Invitrogen, Carlsbad, CA), and 3) stored the tissues at –80°C. To isolate total RNA, we homogenized the tissues in TriZOL and followed the manufacturer's protocol (for details, see Ref. 49).
Cloning of sqNBCe cDNA
To clone the full-length sqNBCe cDNA, we synthesized two pools of single-stranded cDNA using GFL RNA as a template. To generate the first pool of cDNA, we used Superscript Reverse Transcriptase (Invitrogen) primed with oligo-dT (Invitrogen). For the second pool, we used a GeneRacer Kit (Invitrogen) to 1) enrich full-length RNA transcripts, and 2) ligate a generic GeneRacer RNA Oligo "cap" to the 5' end of the RNA. With the full-length, capped RNA, we generated full-length cDNA using SuperScript III Reverse Transcriptase primed with GeneRacer oligo dT primer or random hexamers (all from Invitrogen).
On the first pool of cDNA we conducted nested-PCR with degenerate primers (for details, see Ref. 49) that were complementary to well-conserved regions of Na+-coupled HCO3– transporters and encompass a 290-bp region (see region between gray diamonds in Fig. 1). We ligated the nested-PCR products of the expected size into a pCR 2.1-TOPO TA vector (Invitrogen) and transformed the ligation product into TOP10 chemically competent Escherichia coli (Invitrogen) according to the manufacturer's protocol. We isolated and sequenced plasmid DNA from the resulting colonies. All sequencing was performed in both 5' and 3' directions by the W. M. Keck DNA Sequencing Center (Yale University School of Medicine, New Haven, CT). Using this approach, we identified multiple copies of 3 novel, partial clones of 290 bp that we provisionally named squid fiber (SF) clone 1 (SF1), SF3, and SF4 (16). Virkki et al. (49) cloned and characterized the full-length cDNA for SF1, which is an NDCBE. Below we describe the cloning and characterization of SF3, which we renamed sqNBCe on the basis of the functional analysis described in this paper.
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Once we extended the sequence of sqNBCe beyond the open-reading frame (ORF) and into the 5' and 3' untranslated regions, we designed primers to amplify the entire ORF by unnested PCR. Using the second pool of GFL cDNA as a template, we obtained a full-length PCR product spanning the entire ORF of 3,486 bp. We generated a consensus sequence for sqNBCe by sequencing the full-length PCR product from five individual clones, and deposited the consensus sequence into GenBank (accession no. DQ469799). In one of the clones—used as a template for subsequent subcloning—we: 1) identified PCR errors by aligning the clone's full-length nucleotide sequence with that of the consensus sequence using ClustalW (11), and 2) corrected these PCR errors using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA).
Northern Blot Analysis
We resolved total RNA (10 µg) isolated from all tissues using denaturing formaldehyde-agarose (1%) gel electrophoresis, and used capillary elution to blot the RNA to a Hybond XL nylon membrane (GE Healthcare Life Sciences, Piscataway, NJ). We prehybridized the blot by incubating the membrane at 68°C for 30 min in ExpressHyb solution (Clontech Laboratories, Palo Alto, CA), and then hybridized with a radiolabeled [
-32P]probe (Random Primer labeling kit, Invitrogen), corresponding to the 290-bp product from the initial degenerate-PCR.
Expression in Xenopus Laevis Oocytes
We subcloned cDNA containing the sqNBCe ORF into the pGH19 Xenopus expression vector (48). With the use of a T7 mMessage mMachine kit (Ambion, Austin, TX) and the modified-pGH19 vector, we synthesized capped cRNA encoding sqNBCe. We purified the cRNA using an RNeasy MinElute Cleanup Kit (Qiagen, Valencia, CA), and stored the cRNA in nuclease-free H2O (Ambion) at –80°C.
We isolated stage V and VI oocytes from Xenopus laevis as described previously (37), and the following day we injected oocytes with 50 nl of sqNBCe-cRNA (0.5 ng/nl) or nuclease-free H2O. We cultured the oocytes at 18°C for 3–6 days before conducting any experiments, which we performed at room temperature (
22°C).
Solutions
We prepared a nominally HCO3–-free ND96 solution composed of (in mM) 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES, pH 7.50 (titrated with NaOH). To generate a modified-ND96 solution containing 5% CO2-33 mM HCO3–, we replaced 33 mM NaCl in the ND96 solution with 33 mM Na HCO3– and equilibrated the solution with 5% CO2-95% O2. When required, we dissolved DIDS (Sigma-Aldrich, St. Louis, MO) in the 5% CO2-33 mM HCO3– solution to a final concentration of 200 µM. To generate Na+-free solutions, we replaced Na+ with N-methyl-D-glucammonium (NMDG+), K+, or Li+ (and titrated the HEPES with KOH or LiOH), whereas to generate Cl–-free solutions we replaced Cl– with gluconate. To acidify oocytes in the absence of CO2/HCO3–, we replaced 30 mM NaCl in the ND96 solution with 30 mM Na-butyrate. If necessary, we adjusted the osmolality of all solutions to 195 mOsm by adding dH2O or mannitol.
In all experiments, we held the solutions in 140-ml plastic syringes (Sherwood Medical, St. Louis, MO) and delivered solutions to the oocyte chamber with syringe pumps (Harvard Apparatus, S. Natick, MA) at a flow rate of 4 ml/min. We initially placed an oocyte in a chamber that was constantly superfused with ND96, and then switched solutions using pneumatically operated valves (Clippard Instrument Laboratory, Cincinnati, OH).
Measurements of pHi and Vm
For intracellular measurements, we fabricated pH-sensitive and voltage-sensitive electrodes from thin-walled borosilicate glass (part no. 30-0077, Harvard Apparatus, Holliston, MA), as described previously (49). Briefly, we filled the tip of a pH microelectrode with a H+-sensitive liquid membrane (hydrogen ionophore I, mixture B; Fluka Chemical, Ronkonkoma, NY), and backfilled the electrode with a phosphate buffer (3). We filled the voltage-sensitive electrode with 3 M KCl.
With an oocyte bath clamp (model 725I; Warner Instruments, Hamden, CT) we set the bath potential to 0 mV as a reference for the Vm electrode. With the use of an FD-223 electrometer (World Precision Instruments, Sarasota, FL), we measured the voltage from the pHi electrode. To obtain the pHi-specific voltage, we subtracted the response of the Vm electrode from that of the pHi electrode using a subtraction amplifier (model V3.1; Yale University). To obtain the Vm-specific voltage we subtracted the response of a bath electrode from that of the Vm electrode. Before impaling an oocyte with both microelectrodes, we calibrated the pHi electrode in the bath using pH standards (Fisher Scientific, Hampton, NH) of 6.0 and 8.0, and ND96 (pH 7.50) as an additional single-point calibration. Data acquisition was performed by software that was written in-house.
Two-Electrode Voltage Clamping
We recorded whole cell currents of oocytes with an oocyte clamp (model OC-725C, Warner Instruments), which was controlled by the Clampex module of pCLAMP software (version 8; Axon Instruments, Foster City, CA). We filled the current (I) microelectrodes and Vm microelectrodes with 3 M KCl, and their resistances ranged from 0.4–1.0 M
. We also used a KCl electrode with nominal tip resistance as the bath reference (i.e., Isense of OC-725C).
For all I-V relationships, we held oocytes at a potential close to the spontaneous Vm before initiating the voltage-clamp protocol, which consisted of stepping the holding potential from –160 mV to +20 mV in 20 mV increments (100-ms duration each). The clamp was turned off immediately after completion of the voltage-clamp protocol. For a given oocyte, we acquired such I-V relationships in ND96 immediately before the first solution change into 5% CO2-33 mM HCO3–, and 5 min after switching to CO2/HCO3–. In 
Fig. 6, we acquired the I-V relationships 1–2 min after switching to a solution containing DIDS, and then 1–2 min after removing DIDS. In Fig. 8, we acquired I-V relationships 1–2 min after switching to a solution without Na+, and then 1–2 min after returning Na+. We used the Clampfit module of pCLAMP to analyze the data.
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Using software written in-house, we calculated 1) the rates of pHi recovery by fitting a linear regression to the pHi vs. time traces, and 2) the 
Vm values by subtracting the Vm immediately before a solution change from the Vm recorded 90 s after. For I-V relationships, we used Microsoft Excel 2002 to calculate the slope conductance at 0 mV by fitting a linear regression to the current values obtained at voltage clamps of –20, 0, and +20 mV. All values are presented as means ± SE.
We used Prism 4 software (GraphPad, San Diego, CA) for all statistical tests. For comparisons involving two groups (e.g., CO2/HCO3– vs. butyrate, sqNBCe injected vs. H2O injected) we used paired or unpaired t-tests as appropriate. For comparisons within sqNBCe-injected or H2O-injected groups that contained more than two experimental conditions (e.g., slope conductance in ND96 vs. CO2/HCO3– vs. DIDS vs. DIDS removal), we used a two-way repeated-measures ANOVA, with a Bonferroni posttest for multiple comparisons. For pHi recovery rates and Vm measurements, we used this post test only to compare means within sqNBCe-expressing oocytes for which a significant difference was already found from control oocytes, and for which the directions of the means was similar. For example, in Fig. 7, we used the posttest to compare mean pHi recovery rates between the first and second Na+ exposure, as both were significantly different from their control counterparts (i.e., in H2O-injected oocytes) and both were in the same direction (i.e., mean pHi increase). However, we did not compare the mean for the first Na+ exposure to the mean for Na+ removal because the mean pHi changes were in opposite directions.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Using primers designed to the 5'- and 3'-untranslated regions of sqNBCe, we amplified a cDNA that contains an open-reading frame (3,486 bp) encoding 1,162 amino acids (Fig. 1). A multiple-sequence alignment of sqNBCe with sqNDCBE, hNBCe1, and hNDCBE reveals several characteristic features of SLC4-like proteins (see Fig. 1 for details). Results from a hydropathy analysis (data not shown) are consistent with those from similar analyses of other SLC4 proteins. On the basis of our analysis and the topology model of AE1 (52), we predict (Fig. 1) that sqNBCe has 1) a large cytoplasmic NH2-terminal domain (467 residues), 2) a membrane domain (516 residues) that consists of 13 TM segments connected by endo- and exofacial loops, including a large exofacial loop between TM5 and TM6, and 3) a cytoplasmic COOH-terminal domain (179 residues). The COOH-terminal domain of sqNBCe is notably longer than that of human SLC4 transporters (30–100 residues) and drNDAE (99 residues), but is slightly shorter than that of sqNDCBE (195 residues).
Among the Na+-coupled HCO3– transporters, sqNBCe is most identical to sqNDCBE, followed by drNDAE and human electroneutral NBCs, and least identical to human electrogenic NBCs (Table 1). An analysis of the predicted membrane domains of sqNBCe-including the endo- and exo-facial loops-reveals that these regions are most similar to those of sqNDCBE, followed by drNDAE and human Na+-coupled HCO3– transporters (Table 1). The multiple-sequence alignment in Fig. 1 shows that large segments of sqNBCe are extremely similar to homologous regions of sqNDCBE, hNDCBE, and hNBCe1-A. For example, in the cytoplasmic NH2 terminus, one region (Fig. 1; second group of four rows) begins 3 residues before the classic "E[T/S]ARW[I/V]KFEE" motif and extends 26 residues after. This motif is highly conserved among members of the SLC4 family (36), and mutations of residues within this motif in SLC4A2 (hAE2) indicate that this region of the transporter is important for sensing pHi, pHo, cell volume, and intracellular NH4+ (12, 44, 45). Another conserved region (Fig. 1, fourth group of 4 rows) extends from residue 347 (EIGR) to reside 402 (IRIEPP). Not surprisingly, sqNBCe also has several highly conserved regions within the membrane domain of the molecule, particularly from the fourth exofacial loop (between TMs 7 and 8) through TM12.
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Tissue Expression of sqNBCe mRNA
Northern blot analysis of total mRNA extracted from squid tissues (Fig. 3), using a sqNBCe-specific radiolabeled probe, shows no detectable hybridization in the eye. However, we detect a signal at 8 kb in each of the other tissues. Qualitatively, the signal appears strongest in the gill and heart, moderate in the GFL, and weakest in the optic lobe, stellate ganglion, and testis. Using an identical technique, Virkki et al. (49) observed a very different tissue distribution of the mRNA for sqNDCBE (7.5 kb). That signal was strongest in the GFL, heart, and optic lobe, weak in the gill and stellate ganglion, and undetectable in the eye and testis.
Electrophysiological Characterization of sqNBCe
Electrogenicity and HCO3– dependence.
In Fig. 4A, we initially superfuse an oocyte expressing sqNBCe with our ND96 solution, which nominally contains no CO2/HCO3–. Switching to a solution containing 5% CO2-33 mM HCO3– causes pHi to fall, due to CO2 influx and subsequent generation of H+ (40), and then rapidly recover (Fig. 4A, a), due to the active uptake of HCO3–. Switching back to ND96 causes the oocyte to alkalinize due to CO2 efflux. Regarding oocyte membrane potential, exposure to 5% CO2/33 mM HCO3– results in a rapid hyperpolarization of 60 mV, followed by a gradual depolarization. The initial hyperpolarization is characteristic of an electrogenic NBC that mediates the influx of more HCO3– than Na+ (4, 15, 28, 38, 50), and not of an electroneutral NBC. The slower depolarization most likely reflects the accumulation of intracellular Na+ and HCO3–. As expected for an electrogenic NBC, the removal of CO2/HCO3– causes a rapid depolarization—likely reflecting the efflux of more HCO3– than Na+. The following small hyperpolarization probably reflects the depletion of HCO3– and active removal of Na+, terminating the contribution of the NBC to Vm.
Although the subsequent exposure of the sqNBCe-expressing oocyte to 30 mM butyrate reacidifies the cell, both the pHi recovery (Fig. 4A, b) and membrane hyperpolarization (10 mV) are much reduced.
Repeating the above CO2/HCO3– exposure on a H2O-injected oocyte results in 1) no substantial pHi recovery (Fig. 4B, a) following the acidification, and 2) a slow membrane depolarization (6 mV) as opposed to a hyperpolarization. The subsequent application of butyrate produces pHi and Vm effects that are very similar to those produced by CO2/HCO3–.
Figure 4C summarizes the mean pHi recovery rates (corresponding to periods a and b in Fig. 4, A and B) for sqNBCe-expressing (shaded bars) and H2O-injected oocytes (open bars). Similarly, Fig. 4D summarizes the mean Vm measured 90 s after the solution change to CO2/HCO3– or butyrate. The mean pHi recovery rates (Fig. 4C) for sqNBCe oocytes are 1) greater in CO2/HCO3– and butyrate than in their H2O-injected counterparts, and 2) greater in CO2/HCO3– than in butyrate. As far as Vm is concerned (Fig. 4D), the hyperpolarization of sqNBCe oocytes in CO2/HCO3– is greater than either sqNBCe oocytes in butyrate or control oocytes in either CO2/HCO3– or butyrate. These data indicate that sqNBCe is primarily HCO3– dependent and electrogenic, but may have a minor capacity for the transport of butyrate. On the other hand, the minor sqNBCe activity observed during butyrate exposure may simply be a consequence of residual HCO3– in the solution.
Inhibition by DIDS.
In Fig. 5A, we again initially superfuse an oocyte expressing sqNBCe with ND96 solution. As seen above, switching to 5% CO2-33 mM HCO3– causes an intracellular acidification that is followed by a rapid recovery (Fig. 5A, a), as well as a rapid hyperpolarization. The addition of 200 µM DIDS markedly slows the pHi recovery (Fig. 5A, b) and depolarizes the membrane. The subsequent removal of DIDS 1) does not restore recovery of pHi (Fig. 6A, c) and 2) does not affect Vm, suggesting that sqNBCe has been irreversibly blocked.
In a H2O-injected oocyte, exposure to DIDS results in 1) no substantial effects on pHi recovery (Fig. 5B, a and b), and 2) a rapid, small hyperpolarization (5 mV) rather than a depolarization. DIDS removal does not influence the pHi trajectory and causes a rapid, small depolarization (3 mV).
Figure 5, C and D, summarize the mean pHi recovery rates and Vm for sqNBCe-expressing (shaded bars) and H2O-injected oocytes (open bars). The mean pHi recovery rate (Fig. 5C) in CO2/HCO3– is higher in sqNBCe than in control oocytes. In DIDS and after DIDS removal, the mean pHi recovery rates of sqNBCe-expressing oocytes are indistinguishable from control oocytes. The magnitudes of Vm (Fig. 5D) are greater in sqNBCe than in control oocytes both for the addition of CO2/HCO3– and the addition of DIDS. The Vm values during removal of DIDS are indistinguishable both from each other, and from the two other Vm values of control oocytes. These data show that—as is the case for several other SLC4 transporters—DIDS blocks the activity of sqNBCe. Moreover, the effect of DIDS on sqNBCe is not easily reversed.
Figure 6A shows the effect of DIDS on the current-voltage (I-V) relationship of sqNBCe. Using two-electrode voltage clamping, we measure current through the oocyte membrane during different phases of the pHi recovery and DIDS exposure of an experiment like that shown in Fig. 5A. The I-V relationship is nearly linear when the oocyte is in ND96 (open circles in Fig. 6A). About 5 min after application of CO2/HCO3–, after the zero-current potential had already begun shifting to more positive values (i.e., relative to 1 min after exposure to CO2/HCO3–), we took the I-V curve represented by the dark gray squares in Fig. 6A. Application of DIDS (light gray triangles in Fig. 6A) causes the current at positive voltages to fall nearly to the values previously observed in ND96. At negative voltages, however, DIDS has little effect on the current. Thus, DIDS produces a voltage-dependent block of sqNBCe, as has recently been described for human NBCe1-A (34). Subsequent removal of DIDS (black squares in Fig. 6A) has no effect on the current at positive voltages, and causes a slight positive shift in current at the negative voltages to values that are similar to ND96. In H2O-injected oocytes (Fig. 6B), the currents in ND96 are very small, and affected very little by applying CO2/HCO3– or DIDS.
Figure 6C summarizes the mean slope conductances at 0 mV measured in sqNBCe-expressing (shaded bars) and control (open bars) oocytes. The mean slope conductance of sqNBCe oocytes (Fig. 6C) is significantly greater 1) in CO2/HCO3– than under the other three conditions, and 2) in ND96 relative to DIDS. The mean slope conductances of the control oocytes (Fig. 6C) are small and statistically indistinguishable under all conditions. The mean slope conductance of sqNBCe oocytes is greater than that of control oocytes in CO2/HCO3– and after removal of DIDS (Fig. 6C). These voltage-clamp data confirm that DIDS blocks the activity of sqNBCe, and that its effect is not easily reversed.
Because we noted that the I-V relationship of sqNBCe oocytes in the presence of CO2/HCO3– exhibits a very slight degree of outward rectification, we examined the voltage dependence of sqNBCe more closely. For each of 24 sqNBCe-expressing oocytes in the present paper, we subtracted the I-V relationship in ND96 (open circles in Fig. 6A) from that in CO2/HCO3– (dark gray squares). Figure 6D summarizes the mean data, and confirms that sqNBCe exhibits slight outward rectification. Another group has reported the rectification of electrogenic NBCs when operating backwards in Xenopus oocytes (21).
Another observation from Fig. 6D is that the mean reversal potential (Erev) is close to –140 mV for sqNBCe. From this Erev value, the stoichiometry of an electrogenic NBC can be estimated (50). Given the Erev of sqNBCe and assuming similar estimates of [Na]i and [HCO3–]i as that for hNBCe2 (50), we estimate that sqNBCe operates with 2:1 stoichiometry (HCO3–:Na+), which is similar to that reported for 1) rat NBCe1-A (43) and hNBCe2 (50) when expressed in Xenopus oocytes, and 2) hNBCe1-A and hNBCe1-B when expressed in mouse collecting duct cell lines (25).
Na+ dependence.
The protocol in Fig. 7A is similar to that in Fig. 5A, except that—rather than adding DIDS—we remove extracellular Na+. As before, switching to 5% CO2-33 mM HCO3– causes an intracellular acidification that is followed by a rapid recovery (Fig. 7A, a), as well as a rapid hyperpolarization. Substituting NMDG+ for bath Na+ 1) dramatically reverses the pHi recovery (Fig. 5A, b), presumably due to the Na+-coupled efflux of HCO3–, and 2) rapidly depolarizes the membrane, a response that is characteristic of electrogenic efflux of more HCO3– than Na+. Return of Na+ 1) partially restores the pHi recovery (Fig. 7A, c) and 2) partially repolarizes the membrane. The small size of this repolarization is curious because the reversal of the transporter in period "b" should have reduced intracellular concentrations of Na+ and HCO3– in the oocyte. Thus, all else being equal, the return of Na+ should have given rise to an exaggerated repolarization, driving Vm to a more negative value than immediately before the Na+ removal—as previously observed for mammalian electrogenic NBCs (15, 28, 37, 50). A second curiosity is that, during the pHi recovery (c), Vm drifts slowly in the negative direction, rather than in the positive direction, as during the initial phase of pHi recovery (a). These observations suggest that the removal of Na+ has somehow reduced the activity of sqNBCe, and that the return of Na+ slowly tends to restore that activity.
In a H2O-injected oocyte, Na+ removal results in 1) minor effects on pHi recovery (Fig. 7B, a and b), and 2) a small hyperpolarization (5 mV) rather than a depolarization. Return of Na+ (c) does not influence the pHi trajectory and causes a rapid, small depolarization (3 mV).
Figure 7, C and D, summarize the mean pHi recovery rates and Vm measured in sqNBCe-expressing (shaded bars) and H2O-injected (open bars) oocytes. The mean pHi recovery rate (Fig. 7C) is greater for the first than the second Na+ exposure in sqNBCe-expressing oocytes, and both are greater than in their H2O-injected counterparts. Similarly, the rate of pHi decline in 0 Na+ (i.e., NMDG+) is greater in sqNBCe than in control oocytes. As far as Vm is concerned (Fig. 7D), the hyperpolarization during the first Na+ exposure (i.e., addition of CO2/HCO3–) is greater than during the second in sqNBCe oocytes, and both are greater than the depolarizations of the control oocytes. The depolarization in 0 Na+ (i.e., NMDG+) is greater in sqNBCe than the hyperpolarization in control oocytes. Thus these data indicate that the activity of sqNBCe is Na+ driven. Moreover, because the acidification rate for sqNBCe expressing in 0 Na+ (middle shaded bar in Fig. 7C) is substantially greater than in DIDS (middle shaded bar in Fig. 5C), Na+ removal does not simply block the transporter but causes it to run in the reverse direction (i.e., HCO3– efflux).
Figure 8A shows the effect of removing Na+ on the I-V relationship of sqNBCe. We measure current through the oocyte membrane during different phases of an experiment like that shown in Fig. 7A. Similar to that seen in Fig. 6, the I-V relationship is nearly linear when the oocyte is in ND96 (open circles in Fig. 8A) with a low slope conductance. After 5 min of applying 5% CO2-33 mM HCO3– (dark-gray squares in Fig. 8A), the slope conductance has increased and the zero-current potential has shifted to more negative values. Removing Na+ by substitution with NMDG+ (light-gray diamonds in Fig. 8A) causes the current at +20 mV to decrease to the value previously observed in ND96. At more negative voltages, the current in the Na+-free solution becomes more inward compared with the current in ND96. Thus, in the absence of extracellular Na+, sqNBCe mediates a relatively large inward current (i.e., outward Na+ and HCO3– movements) at negative voltages (e.g., point b in Fig. 7A).
Similar observations have been made for human NBCe1 (47) and human NBCe2 expressed in oocytes (50). However, for NBCe1 and NBCe2 examined at very negative voltages, the I-V curves in 0 Na+ are not only shifted downward compared with the I-V curves in CO2/HCO3–, but also are parallel (see Fig. 3 in Ref. 47 and Fig. 3 in Ref. 50). Examination of Fig. 8A reveals that although the I-V curve for sqNBCe in 0 Na+ (gray diamonds) is shifted downward compared with the I-V curve in CO2/HCO3– (dark gray squares), the two curves approach each other at very negative voltages. In other words, the sqNBCe inward currents in 0 Na+ are not as large as they should be. Thus, the removal of extracellular Na+ has not only blocked electrogenic Na/HCO3– influx as expected, it has somehow inhibited electrogenic Na/HCO3– efflux.
The return of Na+ to sqNBCe oocytes does not cause the I-V curve (black squares in Fig. 8A) to recovery fully. At positive voltages, the current is substantially lower than in the original CO2/HCO3– record (dark gray squares in Fig. 8A), and at very negative voltages, the current is almost identical to that seen in ND96. These observations are consistent with our findings in Fig. 7A, which indicate that the activity of sqNBCe after the readdition of Na+ is not as great as before the removal of Na+. Thus sqNBCe may require a long reexposure to Na+ before returning to its full activity. In H2O-injected oocytes (Fig. 8B), the currents in ND96 are very small, and affected very little by applying CO2/HCO3– or removing Na+.
Figure 8C summarizes the mean slope conductances at 0 mV measured in sqNBCe-expressing (shaded bars) and H2O-injected (open bars) oocytes. As we saw for Fig. 6C, the mean slope conductance of sqNBCe oocytes (Fig. 8C) is significantly greater in CO2/HCO3– than under the other three conditions, which in this case are indistinguishable. The mean slope conductances of the control oocytes (Fig. 8C) are small and statistically indistinguishable under all conditions. The mean slope conductance of sqNBCe oocytes is greater than that of control oocytes under all conditions (Fig. 8C). These voltage-clamp data confirm that sqNBCe cotransports Na+ in an electrogenic fashion, and that Na+ removal causes a long-lasting inhibition of the cotransporter.
Lack of Li+ and K+ involvement.
We designed the following experiment to determine whether, in the absence of Na+, either Li+ or K+ could support sqNBCe activity. We test Li+, because Virkki et al. (49) demonstrated that this ion can replace Na+ in supporting sqNDCBE activity. This finding is intriguing because, in the squid giant axon, replacing Na+ with Li+ abolishes NDCBE activity (7). We test K+, because previous investigators described K/HCO3– cotransport activity in the squid giant axon (30, 31), and the molecular identity of the transporter responsible for that activity is still not known.
In Fig. 9A, the protocol is similar to that in Fig. 7A, except that—rather than replacing extracellular Na+ with NMDG+—we replace Na+ with K+, followed by Li+. As seen before, switching to 5% CO2-33 mM HCO3– causes an intracellular acidification that is followed by a rapid recovery (Fig. 9A, a), and a rapid hyperpolarization. Substituting K+ for bath Na+ 1) reverses the pHi recovery (Fig. 9A, b), and 2) rapidly depolarizes the membrane by 90 mV to near 0 mV. This depolarization represents the combined effects of the positive shifts in EK and the reversal potential of sqNBCe (which by itself was responsible for the depolarization in Fig. 7A). Switching to Li+ after K+ 1) does not restore the pHi recovery (Fig. 9A, c), but 2) repolarizes the membrane close to the original resting potential in ND96. Finally, the return of Na+ causes 1) pHi to recover (Fig. 9A, d) and 2) the membrane to hyperpolarize, albeit to a lesser extent than seen with the original exposure to Na+ and HCO3– (i.e., Fig. 9A, a). We made similar observations in Fig. 7A. However, unlike the protocol of Fig. 7A, in which Vm consistently drifts in the negative direction after the return of Na+, in experiments with the protocol of Fig. 9A, Vm consistently drifts in the positive direction. Thus, the protocol of Fig. 9A may not allow the recovery of sqNBCe activity that presumably occurred in Fig. 7A. Note that, in the protocol of Fig. 9A, Na+ was absent for a longer period than in Fig. 7A, and was replaced by K+ and then Li+ rather than by just NMDG+.
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35 mV) that is of lesser magnitude than that seen in the sqNBCe-expressing oocyte. The addition of Li+ does not influence the pHi trajectory (Fig. 9B, c), but causes a rapid, large repolarization (40 mV), similar in magnitude to that seen in the sqNBCe oocyte. The return of Na+ does not influence pHi trajectory (Fig. 9B, d) and causes a slight depolarization (5 mV), rather than the enhanced pHi recovery and hyperpolarization seen in the sqNBCe oocyte.
Figure 9, C and D, summarize the mean pHi recovery rates and Vm measured in sqNBCe (shaded bars) and H2O-injected (open bars) oocytes. As we saw in Fig. 7, the mean pHi recovery rate (Fig. 9C) is greater for the first Na+ exposure (i.e., application of CO2/HCO3–) than the second in sqNBCe oocytes, and both are greater than in their H2O-injected counterparts. The rates of pHi decline in K+ and Li+ do not differ from one another in sqNBCe oocytes, are greater than in H2O-injected oocytes, and on average are 50% slower than those in which Na+ is substituted by NMDG+ (Fig. 7C). Regarding Vm (Fig. 9D), the hyperpolarization during the first Na+ exposure (i.e., addition of CO2/HCO3–) is greater than during the second in sqNBCe oocytes, and both magnitudes are greater than those for the H2O-injected oocytes. The depolarization in K+ is greater in sqNBCe than control oocytes, whereas the hyperpolarization in Li+ is not different than controls. Thus although these data clearly demonstrate that neither K+ nor Li+ can fully replace Na+ in supporting sqNBCe-mediated HCO3– transport, the slower acidification rates in K+ and Li+ (Fig. 9C) compared with NMDG+ (Fig. 7C) indicate that there may be a minor K+ or Li+ dependent component to sqNBCe activity. However, in preliminary experiments in which sqNBCe oocytes were initially acidified by CO2/HCO3– in the absence of extracellular Na+ (i.e., replaced with NMDG+), we found that neither addition of K+ nor Li+ could support pHi recovery (data not shown). This finding is similar to those from other groups who reported that K+ does not support, and Li+ minimally supports, HCO3–-senstive currents in oocytes expressing NBCe1-A from rat kidney (43), and pHi recovery in HEK-293 cells expressing NBCe1-A from human kidney (2).
Lack of Cl– involvement.
Our last task was to test the possibility that sqNBCe is an electrogenic Na+-driven Cl-HCO3– exchanger. The most straightforward approach would be to deplete the cell of Cl– and determine whether this treatment blocks Na+-coupled HCO3– uptake. Unfortunately, it is impractical to deplete an oocyte of Cl–, as overnight incubations of oocytes in 0 Cl– media often results in oocytes with membrane potentials that are inadequate for further physiological characterization (unpublished observation). Therefore, we used an approach employed by others to examine hNDCBE (24), sqNDCBE (49), and hNBCn1 (13): we force sqNBCe to run backwards and then determine whether removing extracellular Cl– blocks Na+-coupled HCO3– efflux.
In Fig. 10A, we subject a sqNBCe-expressing oocyte to a protocol that at first is identical to the one in Fig. 7A. Thus, switching to 5% CO2-33 mM HCO3– leads to a fall in pHi, followed by a rapid recovery ("a" in Fig. 10A) and a rapid hyperpolarization. Similar to Fig. 7A, replacing bath Na+ with NMDG+ 1) reverses the pHi recovery ("b" in Fig. 10A), and 2) rapidly depolarizes the membrane. With the transporter now reversed, we switch to a modified NMDG+ solution, in which we replace Cl– with gluconate. In the case of hNDCBE (24) and sqNDCBE (49), this maneuver blocks the pHi recovery. However, Fig. 10A shows that sqNBCe continues to acidify the cell in the combined absence of extracellular Na+ and Cl–. Note that the removal of Cl– causes a small paradoxical hyperpolarization that is also seen in H2O-injected oocytes (see below), and that has been reported previously (49).
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In a H2O-injected oocyte (Fig. 10B), the solution changes result in minor effects on pHi recovery. Replacement of Na+ with NMDG+ and of Cl– with gluconate both result in small, reversible membrane hyperpolarizations.
Figure 10, C and D, show the mean pHi recovery rates and Vm measured in sqNBCe (shaded bars) and H2O-injected (open bars) oocytes. As we saw in Figs. 7 and 9, the mean pHi recovery rate (Fig. 10C) is greater for the first Na+ exposure (i.e., application of CO2/HCO3–) than the second in sqNBCe oocytes, and both are greater than in their H2O-injected counterparts. For sqNBCe oocytes, the rate of pHi decline for the first 0 Na+ is greater than for 0 Na+/0 Cl–, but not for the second 0 Na+. The 50% slower acidification rates in 0 Na+/0 Cl– compared with 0 Na+ (Fig. 10C) indicate that there may be a minor Cl– dependent component to sqNBCe activity. However, in sqNBCe oocytes, the rate of pHi decline in 0 Na+/0 Cl– (Fig. 10C) was not significantly different than the second exposure to 0 Na+ (i.e., Cl– return) and was significantly greater than in DIDS (Fig. 5C, unpaired t-test, P < 0.01). Furthermore, the changes we noticed upon Cl– removal and return were relatively slow and subtle compared with the relatively rapid and robust effects that similar treatments had on sqNDCBE activity (49). These findings lead us to conclude that the pHi decline in 0 Na+/0 Cl– likely represents a continued reversal of the transporter, albeit at a slower rate, possibly due to partial inhibition of the transporter by 0 Na+ (see Na+ dependence). All magnitudes of pHi recovery rates in sqNBCe oocytes are greater than the corresponding values in H2O-injected oocytes. Regarding Vm (Fig. 10D), the hyperpolarization during the first Na+ exposure (i.e., addition of CO2/HCO3–) is greater than during the second in sqNBCe oocytes, and both magnitudes are greater than those for the H2O-injected oocytes. The depolarization in 0 Na+ is greater in sqNBCe than the hyperpolarization in control oocytes. However, the Vm changes associated with Cl– removal or return are not different between sqNBCe and control oocytes. These data indicate that external Cl– is not necessary for sqNBCe to operate, which rules out the presence of substantial NDCBE activity.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In this study we demonstrate that—despite having a sequence reminiscent of an electroneutral Na+-coupled HCO3– transporter—sqNBCe behaves like a classic, mammalian electrogenic Na/HCO3– cotransporter when expressed in Xenopus oocytes. That is, sqNBCe mediates a pHi recovery from an acid load that requires HCO3– and Na+, is independent of Cl–, and is blocked by DIDS. Moreover, sqNBCe carries current. Its I-V relationship is slightly outward rectifying, and the Na+-dependent and DIDS-inhibited currents are voltage dependent. The identification of an electrogenic Na+-coupled HCO3– transporter in the squid is exciting because it represents the first description of an electrogenic SLC4-like transporter from an invertebrate. Thus, sqNBCe provides valuable insights into the evolutionary and structure/function relations of SLC4 transporters.
Another reason that we did not expect sqNBCe to be electrogenic is that—although we cloned sqNBCe from the GFL and sqNBCe mRNA is present in the GFL (Fig. 3)—physiological studies on the squid giant axon indicate that the pHi recovery from an acid load is electroneutral (5–7, 41). The responsible transporter is a Na+-driven Cl-HCO3– exchanger with many of the properties of sqNDCBE, cloned by Virkki et al. (49). Possible explanations for the lack of measurable sqNBCe activity in the axon are that the sqNBCe protein is expressed: 1) in other parts of the neuron but not abundantly in the giant axon, 2) in the giant-axon cell membrane only under conditions not prevailing during the physiological experiments, or 3) only in GFL cells that do not give rise to the giant axon. Development of an antibody against sqNBCe will be necessary to determine whether the protein is translated by the GFL and where it is localized.
A factor that complicates comparisons between previous data from the intact axon and our data on squid transporters heterologously expressed in Xenopus oocytes is the differing experimental conditions used in the axon vs. the oocytes. For example, the experiments on squid axons were conducted under conditions of high ionic strength (i.e., [NaCl] of 500 mM) and an osmolality of nearly 1,000 mOsm, whereas the oocyte experiments were conducted under conditions of relatively low ionic strength (i.e., [NaCl] of 96 mM) and an osmolality of 195 mOsm. Differences in posttranslational processing or membrane lipids could also have an effect. Therefore, it is possible that sqNBCe functions differently under the conditions of the oocyte vs the axon experiments. However, until the development of an expression system that is more like the squid axon—and also allows the measurement of pHi, Vm, and I-V relationships—we are currently restricted to the above limitations of the Xenopus-oocyte system.
Role of sqNBCe in Other Tissues
Although sqNBCe-mRNA expression is found in the GFL (Fig. 3), the mRNA is even more abundant in the gills and heart. Detection of sqNBCe mRNA in the gills is intriguing because the gills of aquatic animals are known to play an important role in systemic pH and ion regulation (22, 35). Furthermore, in mollusks, the gill epithelium is considered an important site of HCO3– uptake for use in biomineralization (see Ref. 19). Thus it is possible that sqNBCe plays a role in HCO3– uptake by the gill. For example, if the transporter is localized to the basolateral membrane of gill epithelial cells, sqNBCe may contribute to HCO3– uptake in a manner similar to that of NBCe1-A in the vertebrate renal proximal tubule (4, 38, 42).
In the heart, sqNBCe may play a role in pHi regulation of cardiac muscle in a fashion similar to that of NBCe1-B, which is expressed in the mammalian heart (15, 32) and contributes to HCO3– uptake in mammalian cardiac myocytes (1, 9, 10). Given that the squid is an active predator and makes long, seasonal migrations, the expression of a HCO3– uptake mechanism in the heart may allow optimal cardiac muscle performance by counteracting the build-up of acid during extensive muscle activity. The development of a sqNBCe antibody would be useful to localize cellular expression of sqNBCe in both the heart and the gills to assess the putative function of sqNBCe in these tissues.
Interaction with DIDS
Previous researchers (13, 14, 38) suggested that the putative DIDS-binding motifs of Na-coupled HCO3– transporters consist of two lysines separated by two other residues (e.g., KXXK) and that the ends of TM segments 3, 5, and 12 (see Fig. 1) may spatially form a DIDS-binding pocket (49). Human NBCe1-A has the sequence KKMIK at the end of TM5. Even though the other 2 motifs in hNBCe1-A are "disrupted" (e.g., "NFSK" near TM segment 3, and "KSTV" near TM segment 12), the transporter is DIDS sensitive. Recent work on hNBCe1-A shows that mutating any one of the three TM5 Lys residues to Asn modestly reduces the affinity for reversible DIDS binding, and that mutating all three reduces the affinity by 10-fold (34). Moreover, mutating any one of the three TM5 Lys residues has little effect on irreversible DIDS inhibition. sqNBCe has NVCK at TM3, KKTFG at TM5, and KTIK at TM12 (completely conserved with sqNDCBE, ref. 49). Thus, it is possible that DIDS binds reversibly to sqNBCe at TM5 and/or TM12, and then quickly reacts with a Lys residue to produce a permanent blockade.
Effect of Removing and Returning External Na+ on sqNBCe
Whereas exposure to DIDS blocks sqNBCe-mediated transport, removal of Na+ reverses the direction of sqNBCe-mediated transport (Figs. 7 and 8). This phenomenon is indicated by the reversal of pHi recovery, depolarization of the cell, and enhanced inward currents at the most negative voltages. Upon returning Na+, one would expect a pHi recovery, hyperpolarization and enhanced outward currents, comparable to the initial CO2/HCO3– exposure. However, upon returning Na+ in sqNBCe-expressing oocytes, we find that overall transport appears to be inhibited. The basis for this phenomenon is unclear, and requires further study. It is possible that removal of Na+ triggers a rapid endocytosis of individual transporters, similar to the protein kinase induced endocytosis observed for Na+/glucose and Na+/phosphate cotransporters expressed heterologously in Xenopus oocytes (23, 29). Another possibility is that as sqNBCe operates backwards in the absence of Na+, it assumes a conformation from which—at least initially—it cannot maximally operate in the forward direction once the Na+ is restored. Perhaps once sqNBCe reverses, the affinity of the transporter for Na+ is reduced.
Evolution of Electrogenicity
The description of the first known electrogenic SLC4-like transporter in an invertebrate lineage might provide new insight into the evolution of this protein family. Na-coupled HCO3– transporters, including sqNBCe, from protostomes (e.g., molluscs, arthropods) closely cluster (Fig. 2) with the electroneutral SLC4 transporters of deuterostomes (e.g., chordates, echinoderms) and are less closely related to electrogenic SLC4 transporters of deuterostomes. This observation suggests that electroneutral Na+-coupled transporters may represent a basal condition from which the electrogenic transporters twice evolved independently, subsequent to the divergence of the protostome and deuterostome lineages. To test this hypothesis, it will be useful to identify and characterize SLC4-like cDNAs from common ancestors of both protostomes and deuterostomes to determine whether an electrogenic NBC was among the primordial mix of proteins. Furthermore, the functional characterization of NBCs from more "primitive" deuterostomes, such as the NBC recently identified in an echinoderm (26), should be useful in understanding the evolution of electrogenicity in the deuterostome lineage.
In addition to providing insights about SLC4 evolution, the sqNBCe clone is a potentially valuable template for deciphering the structural differences between electroneutral and electrogenic transporters. By comparing the amino-acid sequence of sqNBCe with other electrogenic SLC4 transporters from vertebrates, we find several residues for which sqNBCe is identical to vertebrate electrogenic transporters rather than to sqNDCBE and vertebrate electroneutral SLC4s (Table 2). These residues are primarily in predicted TM segments, which is consistent with a recent study showing that the electrogenic property of hNBCe1-A requires TM1-5 and TM6-13 (17). It will be interesting to know whether mutating a small number of residues conserved among electrogenic transporters to their counterparts in electroneutral transporters can convert an electrogenic NBC into an electroneutral NBC.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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), CO2/HCO3– solution (dark gray squares), CO2/HCO3– solution with 200 µM DIDS (light gray triangles), and CO2/HCO3– solution after removal of DIDS (
), using the voltage-clamp protocol described in MATERIALS AND METHODS. pHo was 7.50 throughout. B: steady-state I-V relationships from a representative H2O-injected (control) oocyte. C: slope conductance summary for sqNBCe (shaded bars) and control (open bars) oocytes. The bars represent the mean slope conductances (±SE) measured at 0 mV, with numbers of oocytes in parentheses. Solid horizontal lines linking shaded bars represent Bonferroni post hoc comparisons from the two-way paired ANOVA, and dashed horizontal lines represent unpaired t-tests. *P < 0.05; ***P < 0.001. D: mean CO2/HCO3–-dependent I-V relationship in 24 oocytes expressing sqNBCe. For each oocyte, we subtracted the steady-state current in ND96 from the steady-state current observed 5 min after switching to CO2/HCO3–. The filled squares represent means (±SE). For clarity, error bars are not drawn when they underlie the symbol.
), using the voltage-clamp protocol described in MATERIALS AND METHODS. pHo was 7.50 throughout. B: steady-state current-voltage relationships from a representative H2O-injected (control) oocyte. C: slope-conductance summary for sqNBCe (shaded bars) and control (open bars) oocytes. The bars represent the mean slope conductances (±SE) measured at 0 mV, with number of oocytes in parentheses. Solid horizontal lines linking shaded bars represent Bonferroni post hoc comparisons from the two-way paired ANOVA, and dashed horizontal lines represent unpaired t-tests. *P < 0.05; ***P < 0.001.
