ELECTROGENIC Na + /HCO 3 COTRANSPORTERS: Cloning and Physiology

Transcription

1 Annu. Rev. Physiol : Copyright c 1999 by Annual Reviews. All rights reserved ELECTROGENIC Na + /HCO 3 COTRANSPORTERS: Cloning and Physiology Michael F. Romero Department of Physiology and Biophysics and Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio ; mfr2@po.cwru.edu Walter F. Boron Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520; walter.boron@yale.edu KEY WORDS: homology cloning, Xenopus oocyte expression, intracellular ph, Na + transport, HCO 3 transport, rat ABSTRACT Bicarbonate and CO 2 comprise the major ph buffer of biological fluids. In the renal proximal tubule most of the filtered HCO 3 is reabsorbed by an electrogenic Na/HCO 3 cotransporter located at the basolateral membrane. This Na + bicarbonate cotransporter (NBC) was recently cloned. This review highlights the recent developments leading to and since the cloning of NBC: NBC expression cloning, protein features, clone physiology, isoforms and genes, mrna distribution, and protein distribution. With the NBC amino acid sequence 30 35% identical to the anion exchangers (AE1-3), a superfamily of HCO 3 transporters is emerging. Physiologically, NBC is electrogenic, Na + dependent, HCO 3 dependent, Cl independent, and inhibited by stilbenes (DIDS and SITS). NBC clones and proteins have been isolated from several tissues (other than kidney) thought to have physiologically distinct HCO 3 transporters. For example, NBC occurs in pancreas, prostate, brain, heart, small and large intestine, stomach, and epididymis. Finally, there are at least two genes that encode NBC proteins. Possible future directions of research are discussed /99/ $

2 700 ROMERO & BORON INTRODUCTION Bicarbonate, along with CO 2, is the major ph buffer of biological fluids. And HCO 3 is a special ion in that it coexists with CO 2 gas in solution. The reaction CO 2 + H 2 O H 2 CO 3 H + + HCO 3 is well known to every chemistry and biology student. This reaction is readily reversible. In biological systems, the interconversion of CO 2 to HCO 3 is greatly potentiated by the family of carbonic anhydrase enzymes (1, 2). Certain organ systems have evolved to be particularly good at HCO 3 transport. The pancreatic ducts move CO 2 and HCO 3 from the blood into pancreatic secretions. Humans can secrete a pancreatic fluid that is virtually isotonic NaHCO 3 (i.e. roughly 130 mm) and has a ph of about 8.1. Similar, although not as extreme, HCO 3 secretions seem to occur in several other exocrine organs, such as salivary glands and the prostate. The kidneys reabsorb massive amounts of HCO 3 daily. In humans, the glomeruli filter 180 liters of blood plasma each day containing more than 4 mol of NaHCO 3 or roughly the amount contained in a pound of baking soda. To prevent the development of a massive metabolic acidosis, the epithelial cells in the remainder of the nephron subsequently reabsorb virtually all of this NaHCO 3. The proximal tubule (PT) isotonically reabsorbs the bulk of the filtered HCO 3, about 80% 90%, as well as most of the filtered Na +,Cl, and H 2 O. How does the PT reabsorb the NaHCO 3? Luminal Na + enters the PT cell via an array of Na + -coupled transport systems (e.g. Na-H exchanger, Na/glucose cotransporter) and perhaps Na + channels as well. The PT cell then extrudes Na + across the basolateral membrane via basolateral Na-K pumps, maintaining a low intracellular [Na + ]. H + secreted into the lumen by Na-H exchangers and H + pumps titrates the filtered HCO 3 in the lumen, forming CO 2 in a reactionfacilitated carbonic anhydrase IV tethered to the luminal membrane via a GPI linkage. CO 2 and H 2 O then rapidly enter the PT cell, where soluble carbonic anhydrase II in effect regenerates H + and HCO 3. For early segments of the PT, the vast majority of the intracellular HCO 3 moves across the basolateral membrane into the blood via the electrogenic Na/HCO 3 cotransporter (3). CLONING OF THE ELECTROGENIC NA + /HCO 3 COTRANSPORTER: NBC The Physiological Family of HCO 3 Transporters Bicarbonate transporters are often the major ph regulators in animal cells and play vital roles in acid-based movement in a number of epithelia, including

3 Na + /HCO 3 COTRANSPORTERS 701 those in the stomach, pancreas, intestine, kidney, reproductive system, and central nervous system. The functional family of HCO 3 transporters includes Cl-HCO 3 exchangers, Na/HCO 3 cotransporters with as many as three different stoichiometries (3 5), a K/HCO 3 cotransporter (6, 7), and a Na + -driven Cl- HCO 3 exchanger (8, 9). Yet until 1997, there had been no molecular information on any HCO 3 transporter except for the Cl-HCO 3 exchangers [anion exchangers (AEs)], whose cdnas were cloned several years ago (10 13). Several groups, including one of our laboratories, unsuccessfully used a number of approaches in attempts to purify the proteins or clone the cdnas of Na + -coupled HCO 3 transporters. These approaches included binding of inhibitors, protein purification, and screening of cdna libraries based on homology to the AEs. The Expression Cloning Strategy Encouraged by the success of others, who have expression cloned several membrane proteins using the oocytes of Xenopus laevis (for review see 14), we attempted to clone one of these HCO 3 transporters, the renal electrogenic Na/HCO 3 cotransporter. This approach, although time consuming, allows functional evaluation of properties attributed to a single or functional group of proteins. Expression cloning has had a major impact on the cloning of integral membrane proteins because the technique relies on a functional property rather than requiring an antibody, cdna probe, or some other insight to the putative protein s structure. In cloning a HCO 3 transporter, the ability to monitor multiple features of the transporter to make the correct molecular diagnosis is important. All HCO 3 transporters produce a change in intracellular ph (ph i ) that can be followed easily by using microelectrodes. Moreover, with the exception of the K/HCO 3 cotransporter, members of the functional family outlined above are almost always blocked by stilbene derivatives such as DIDS (4,4 -diisothiocyano-2,2 - stilbene disulfonate). However, the Na/HCO 3 cotransporter activity is unique among HCO 3 transporters in that, for most tissues described, the cotransport is electrogenic, moving net negative charge in the direction of Na + and HCO 3 cotransport (see below for assay). It is easy to use microelectrodes to monitor the changes in membrane potential (V m ) or membrane current produced by such an electrogenic transporter. Because the activity of the electrogenic Na/HCO 3 cotransporter was first described in the kidney, and its electrophysiology there had been described in detail (3), we attempted cloning this cotransporter from renal tissue. Our general approach was to simultaneously monitor changes in ph i and V m and to devise an assay that would identify the cotransporter unambiguously, easily, and with great sensitivity.

4 702 ROMERO & BORON The Diagnostic Assay for Electrogenic Na + /HCO 3 Cotransport To test more precisely for electrogenic Na/HCO 3 cotransporter activity, we set several criteria that when simultaneously observed should be characteristic of the cotransporter. The expressed protein should be (a) electrogenic, (b) Na + dependent, (c) HCO 3 dependent, and (d ) blocked by stilbenes such as DIDS. Our basic assay was to monitor ph i and V m in Xenopus oocytes injected with renal poly(a) + RNA. Adding CO 2 /HCO 3 to the extracellular solution bathing an oocyte rapidly acidifies the cell because of the entry of CO 2 and the subsequent formation of HCO 3 and H+ inside the cell. In control oocytes, i.e. those injected either with nothing (native oocytes) or with H 2 O instead of RNA, ph i does not recover from the CO 2 -induced acidification (15). Removing permeant ions such as Cl and Na + does not unmask any native HCO 3 or other acid-base transporters (e.g. Na-H exchangers or H + pumps) (15, 16). It is interesting to note that the control Xenopus oocyte does have both a native Na-H exchanger and a native H + pump. However, both of these are silent unless the cell is osmotically shrunken (17 19). Although the ph i of control oocytes does not recover from the initial CO 2 -induced acidification, oocytes expressing NBC do exhibit a slow ph i recovery (i.e. alkalinization). Once the CO 2 /HCO 3 exposure has acidified the oocyte and ph i has either stabilized or begun to recover, bath Na + is removed. The expectation is that in aco 2 /HCO 3 -buffered solution, Na+ removal should cause the cotransporter to operate as it normally does in the proximal tubule. That is, Na + and HCO 3 should exit the cell and thereby elicit a simultaneous depolarization (due to the exit of Na + and more than one HCO 3 ) and fall of ph i (due to the exit of the HCO 3 ). This two-pronged signal should be reversible and blocked by stilbenes such as DIDS. Initially, we injected Xenopus oocytes with poly(a) + RNA isolated from rabbit renal cortex, but the expected signal did not occur. Reasoning that an amphibian cell might express amphibian mrna better than mammalian mrna, we isolated and injected poly(a) + RNA from the kidney of a salamander Ambystoma tigrinum, the preparation from which the cotransporter was first described (3). The oocytes injected with salamander RNA expressed an activity that was robust and fit all our criteria. However, the activity became apparent no sooner than 6 days after injection of the mrna (15). Characteristics of the NBC cdna and Protein BASIC PHYSIOLOGICAL CHARACTERISTICS OF anbc After developing the diagnostic assay, we size fractionated poly(a) + RNA from Ambystoma and constructed a directional cdna library from the 3.5- to 5.0-kb fraction, which

5 Na + /HCO 3 COTRANSPORTERS 703 yielded a positive assay. Four rounds of functional screening of this library yielded a single clone, 9h5g, named the renal electrogenic Na bicarbonate cotransporter (NBC). Because several species forms of NBC have now been cloned, we refer to the Ambystoma clone as anbc (GenBank #AF001958). When this anbc protein is expressed in Xenopus oocytes, it has all the hallmarks of the electrogenic Na/HCO 3 cotransporter: It is electrogenic, produces the expected ph i changes, is Na + dependent, is HCO 3 dependent, and is blocked by DIDS (15). The electrogenicity can be observed either as a shift in V m or a current (in voltage-clamped oocytes) that can be blocked by DIDS. The V m or current change can be evoked either by changing [HCO 3 ] o or [Na + ] o.asfar as the Na + dependence is concerned, the removal of extracellular Na + halts the ph i recovery in oocytes exposed to CO 2 /HCO 3 and instead produces a slow ph i decrease. In addition, Na + removal elicits either a depolarization (i.e. Na moves as if it were an anion) or an inward current. Demonstrating the HCO 3 dependence requires more effort. We applied a butyric acid/butyrate solution and acidified oocytes to the same extent as when applying a CO 2 /HCO 3 solution. In oocytes acidified with butyric acid, no activity was observed in either control or NBC-expressing oocytes. Only NBC-expressing oocytes acidified with CO 2 /HCO 3 showed NBC activity. ANALYSIS OF THE DEDUCED AMINO-ACID SEQUENCE OF anbc The anbc cdna encodes a protein of 1035 amino acids. Hydropathy analysis indicates that NBC should have at least ten membrane-spanning segments. Interestingly, NBC is 30% 35% identical at the protein level to the AE gene family; the homology is strongest to AE3 and AE2. Figure 1 shows a multiple sequence alignment of the cloned NBCs and the AEs. Deduced amino-acid sequences indicate that anbc and the AEs share many areas of identity, both in the putative membrane-spanning segments and in the putative cytoplasmic regions, especially in the NH 2 terminus. The hydropathy plots of these proteins are virtually superimposable, which suggests that the folding of the NBC and the AE proteins are likely to be similar. Figure 2A illustrates that several basic features of the AEs are preserved in anbc: (a) both the NH 2 and COOH termini are presumably intracellular; (b) there is a large, glycosylated, extracellular loop between the fifth and sixth transmembrane (TM) segments; and (c) lysine residues are conserved at predicted DIDS-reactive sites. Thus, NBC and the AEs appear to be the first two major families of HCO 3 transporters (Figure 2B), likely forming a HCO 3 transporter superfamily. OTHER NBC-RELATED cdnas Shortly after cloning anbc, we also isolated and characterized NBC from rat kidney (rknbc) (GenBank #AF004017). The rat clone is 86% identical to anbc at the amino acid level (16, 20). Within

6 704 ROMERO & BORON

7 Na + /HCO 3 COTRANSPORTERS 705 the last year, Burnham and coworkers (21) isolated a NBC-cDNA from human kidney (hnbc1 or hknbc) (GenBank #AF007216) that is 86% identical to anbc and 97% identical to rknbc. All these renal clones have 1035 amino acids and have many conserved putative phosphorylation sites (Figure 2A; see also Table 2). CHARACTERISTICS OF THE HCO 3 -TRANSPORTER SUPERFAMILY With rknbc added to the superfamily, two general sequence features become more apparent: (a) signature sequences of the superfamily (Table 1) and (b) an additional potential DIDS-reaction-site motif. As illustrated in Figure 1 and highlighted in Table 1, several areas of amino acids identity or high similarity seem characteristic of HCO 3 transporter superfamily members. In particular, ETARWIKFEE, AITFGGLLG, VREQRVTG, and FLYMGV are blocks of six or more amino acids that are almost absolutely conserved among members. Both LISxIFIY and PALLVxILIF are also long blocks of conserved amino acids encompassing only one variant amino acid. (T/S)GP(V/L)LVFE in the NH 2 terminus of the superfamily is noteworthy because in the AEs the sequence is SGPLLVFE whereas in the NBCs the invariant sequence is TGPVLVFE. Table 1 lists other examples of this sequence conservation within AE and conservation within NBC contained in a larger identity group. These sequences are particularly interesting because they appear invariant among amphibians and mammals. The DIDS motif KL(X)K (X = I,V,Y) was originally identified after the cloning of AE3 (12), aided by a biochemical analysis of AE1 with which DIDS had covalently reacted (22, 23). The cognate sequence in both Ambystoma and rat NBC is KMIK ( ). In addition, at a more C-terminal site, both NBC clones have the original AE consensus motif sequence KLKK ( ). Figure 1 Multiple amino acid sequence alignment of electrogenic Na bicarbonate cotransporters (NBCs) and anion exchangers (AEs). The cdnas for Ambystoma kidney NBC (anbc), rat kidney NBC (rknbc), and human kidney NBC (hknbc) encode proteins of 1035 amino acids (GenBank #AF001958, AF004017, AF007216, respectively), whereas human heart NBC (hhnbc) (#AF or #AF from pancreas) encodes a 1079 amino acid protein. Clones identical to hhnbc have been isolated from human kidney (76), pancreas (25, 76), and prostate (MF Romero, unpublished results). The multiple sequence alignment was generated using DNAstar (Lasergene, Madison, WI) and the alignment shaded and annotated using GeneDoc c (available free at ketchup/genedoc.shtml). The consensus of the seven amino acid sequences appears below the alignment. (Capital letter) Indicates identity in all seven; (number) indicates all seven have a member of the functional group; (small letter) indicates identity in six of seven. (Highlighting and reverse type) In the multiple sequence alignment, AE sequences (GenBank Accession numbers S03074, S21086, A42497) identical or similar in functional group across all seven sequences. Similar amino acids are defined in six groups: 1 (DN), 2 (EQ), 3 (ST), 4 (KR), 5 (FYW), and 6 (LIVM). (Shaded and normal type) Identity or similarity across at least four members. (Line over sequence) NBC-predicted TMs.

8 706 ROMERO & BORON Figure 2 HCO 3 transporter superfamily relationships. (A) Membrane model of electrogenic Na bicarbonate cotransporter (NBC) protein. (Numbered rectangles) Putative transmembranes. (Diamonds) DIDS-binding motifs. Of eight consensus N-linked glycosylation sites, only three are predicted to be extracellular (four amino acids in anbc). Ser 982, predicted to be intracellular, is the only consensus protein kinase A [PKA (triangle)] site; the hhnbc-like clones that have 1079 amino acids (dotted line: additional amino acids MEDE start; MSTE start and 1035 amino acids) have an additional PKA site (T49) at the NH 2 terminus. Of the many consensus sites for protein kinase C [PKC (circles)], not all are predicted to be intracellular (see Table 2 for details). (+, ) Charged residues closely associated with transmembranes, or those at a high density. (B) This dendrogram shows the percentage of divergence of the amino acid sequences of members from the HCO 3 transporter superfamily [the NBCs and the most homologous anion exchangers (AEs), as in Figure 1]. The divergence is indicated by the total length of the horizontal line segments from one label [e.g. anbc (see Figure 1 legend for abbreviations) to another. anbc is 35%, 33%, and 34% similar to AE3, AE2, and AE3, respectively. (Modified from Reference 15.) Thus, the NBC sequence data suggest a more generalized DIDS-binding motif: K-(Y)(X)-K, where Y = M,L and X = I,V,Y. The single-consensus PKA phosphorylation site (S982) is conserved in salamander, rat, and human kidney clones. Recently, a longer human NBC clone was found to have an additional PKA site at T49 (24, 25). Several groups have shown that PKA pathways can modulate the activity of Na/HCO 3 cotransporters. In tissue preparations, angiotensin II (26, 27) stimulates the cotransporter via inhibition of PKA signaling pathways. Measuring 22 Na uptake after direct stimulation of basolateral membrane vesicles (BLMV) with angiotensin II (28) or endothelin-1 (29) also stimulated Na/HCO 3 cotransport, whereas directly activating PKA in BLMVs is reported to inhibit Na/HCO 3 cotransport

9 Na + /HCO 3 COTRANSPORTERS 707 Table 1 Signature sequences of the HCO 3 -transporter superfamilya rknbc amino NBC topology location Conserved amino acid sequence acid location NH 2 -terminal ETARWIKFEE cytoplasmic tail NH 2 -terminal E(V/L)PVPxRFLFxxxLGP cytoplasmic tail TM-1 AITFGGLLG TM-3 (T/S)GP(V/L)LVFE TM-5 prior to KMIK LISxIFIY TM-7 PALLVxILIF Beginning of TM-8 KGxG(Y/F)HLDL Beginning of TM-9 V(R/K)EQRVTG Central TM-9 IPmpVL(F/Y)G(V/I)FLYMGV TM-10 R(V/M)HLFTx(L/I)Q(V/I)xC(L/I)AxLW a Identity sequences between the anion exchangers and the electrogenic Na bicarbonate cotransporters (NBCs). NBC topology corresponds to that illustrated in Figure 2A. TM, Transmembrane; KMIK, NBC s sequence that comprises the first DIDS-binding motif K(Y)(X)K. (30). Interestingly, in the pancreas, both secretin and forskolin stimulate pancreatic HCO 3 secretion and appear to increase Na/HCO 3 cotransporter activity in guinea pig pancreas (31). Perhaps T49 provides the crucial PKA phosphorylation site for NBC in the pancreas. All NBC clones have multiple consensus PKC- and casein kinase II phosphorylation sites. There are also either one or two predicted tyrosine phosphorylation sites (Y897 in rknbc). Though the pathway is currently unknown, norepinephrine decreases electrogenic Na/HCO 3 cotransporter activity in both Ambystoma (32) and rat proximal tubules (33). This attenuated activity presumably occurs via second messenger signaling. Other predicted modification sites include several consensus myristylation and N-linked glycosylation sites (Table 2). Additionally, based on the identity with the AEs, palmitoylation (AE1-C843(34)) of NBC-C916 (C960 in hhnbc) is possible. PHYSIOLOGY OF NBC Modes of Na/HCO 3 Cotransport The anbc clone encodes the renal electrogenic Na/HCO 3 cotransporter that normally mediates Na + and HCO 3 efflux across the basolateral membrane of the proximal tubule. This cotransporter presumably has a Na + :HCO 3 stoichiometry of 1:3 (35, 36), at least as it functions in the proximal tubule. A functionally related cotransporter, which mediates both HCO 3 influx and efflux and has a

10 708 ROMERO & BORON Table 2 Putative modification sites of NBC a Predicted type Consensus site Sites (hhnbc) Sites (hk-, rk-, and anbc) N-linked N-{P}-[ST]-{P} 541, 636, 641, 661 (590), 592, 597, 617 glycosylation PKA [RK](2)-x-[ST]. T49, S1026 S982 PKC [ST]-x-[RK] S38, S65, T128, T216, T249, T84, T172, T205, S218, S356, S262, S400, T439, T750, S854, T395, T706, S810, S995, and S1039, S1044, S1064 S1000, S1020 (not rknbc) Casein kinase II [ST]-x(2)-[DE]. S68, T110, S157, T163, S223, T66, S113, T119, S179, S195, S239, S257, S336, S408, T419, S213, S292, S364, T375, S951, S995, S1000, S1029, S1064, S956, S985, and T1027 T1071 Tyrosine [RK]-x(2,3)-[DE]-x(2,3)-Y Y941 Y897 phosphorylation Myristylation G-{EDRKHPFYW}-x(2)- G423, G427, G488, G500, G379, G383, G444, G456, G463, [STA GCN]-{P} G507, G512, G520, G558, G468, G476, G514, (G635, G679, G683, G702, G1025 G639), G658, (G759, G981) a [], Indicates that one of the listed amino acids is present; {}, indicates an optional amino acid may be present; (), indicates sites found in anbc or hknbc but not rknbc (see Figure 1 legend for abbreviations).

11 Na + /HCO 3 COTRANSPORTERS 709 stoichiometry of 1:3, also has been identified in retinal Müller cells (37, 38) and corneal endothelial cells (39, 40). Other electrogenic or electroneutral Na/HCO 3 cotransporters mediate net HCO 3 influx (i.e. acid extrusion mode) in their native cells. Generally, net HCO 3 influx requires a Na+ :HCO 3 stoichiometry of less than 1:3 (i.e. 1:2 or 1:1). Cotransporters with Na + :HCO 3 stoichiometries of 1:2 are found in invertebrate glia (4), mammalian astrocytes (41, 42), liver (43 45), pancreas (46) and colon (47), and parotid (48). However, electrogenicity of the cotransporter has not been explicitly demonstrated in colon, pancreas, and parotid. Vaughan-Jones and associates (5, 5a) have evidence for a cotransporter with a 1:1 stoichiometry in mammalian heart. Such a cotransporter would be electroneutral. On the other hand, Camilion de Hurtado et al (49) have provided evidence for an electrogenic Na + -dependent HCO 3 -uptake mechanism in cardiac cells. It is attractive to speculate that NBC-related clones may underlie these physiologically identified Na/HCO 3 cotransport activities. In fact, the possibility has not been ruled out that the same clone is capable of mediating Na/HCO 3 cotransport with two or more different stoichiometries. Indeed, two groups have suggested that the electrogenic Na/HCO 3 cotransporter is capable of functioning in either the 1:3 or 1:2 modes (33, 50). Stoichiometry of the Renal Electrogenic Na/HCO 3 Cotransporter In their original work on the electrogenic Na/HCO 3 cotransporter of the salamander proximal tubule, Boron & Boulpaep (3) demonstrated that the cotransporter moves more HCO 3 than Na+. Based on measurements of ph i, V m, and intracellular Na + activity, they made a thermodynamic argument that the Na + :HCO 3 stoichiometry had to be at least 1:2. However, they could not rule out the possibility that it was higher (e.g. 1:3). Subsequent work by Lopes et al (51) on Necturus proximal tubules suggested, again on thermodynamic grounds, that the Na + :HCO 3 stoichiometry had to be at least 1:3. Using rabbit renal BLMV, Soleimani et al (36) reasoned that the net transport direction depends on both the Na + :HCO 3 coupling ratio and the electrochemical gradients for Na + and HCO 3. By altering these gradients and measuring the direction of net transport in rabbit BLMV, these workers concluded that the renal electrogenic Na/HCO 3 cotransporter must have a stoichiometry of 1:3. Any of three models could account for this apparent 1:3 stoichiometry of the cotransporter: (a)na + plus 3 HCO 3,(b)Na+ plus HCO 3 plus CO= 3,or(c) the NaCO 3 ion pair and HCO 3. Two groups working with isolated proximal tubules have suggested that, under special conditions, the renal electrogenic Na/HCO 3 cotransporter can alter its stoichiometry from 1:3 to 1:2 and thereby change the net direction of net HCO 3 transport (50, 52).

12 710 ROMERO & BORON Even though the data obtained under physiological conditions, on native renal cells or native cell-derived materials point to a stoichiometry of 1:3, it should be pointed out that the Na + :HCO 3 coupling ratio has never been measured directly. Characterization of NBC Clones Both anbc and rknbc have been functionally characterized by monitoring intracellular ph (ph i ) and voltage (V m ) or current when crna is injected and expressed in Xenopus oocytes (15, 16). A human renal homolog (hknbc) has been studied using ph-sensitive dyes in HEK-293 cells transiently transfected (21). Both aknbc and rknbc are electrogenic, and recently hhnbc and hknbc were shown to be electrogenic (24). Figure 3 illustrates several of the fundamental properties of anbc and rknbc as expressed in Xenopus oocytes. The NBC proteins expressed in Xenopus Figure 3 Expression of electrogenic Na bicarbonate cotransporters (NBCs) in Xenopus oocytes. Expression of anbc and rknbc (see Figure 1 legend for abbreviations) (10 ng/50 nl of crna solution) was first obvious on day 3 after injection of crna and continued until at least day 13. (A D) anbc expressed in Xenopus oocytes; (E, F ) rknbc expressed in Xenopus oocytes. (A) DIDS sensitivity. The bathing solution was switched from ND96 solution (CO 2 /HCO 3 free) to 1.5% CO 2 /10 mm HCO 3 (ph 7.5). Na+ was then removed four times in CO 2 /HCO 3. The last two Na + removals were in the presence of 200 µm DIDS. With DIDS, the response to Na + removal was similar to that observed in water-injected oocytes in the absence of DIDS (not shown). (B) HCO 3 dependence. From a resting value of in ND96, ph i decreased to 7.0 within 10 min of adding either 1.5% CO 2 /10 mm HCO 3 or 10 mm butyrate (not shown). Na+ was removed five times, twice in CO 2 /HCO 3 and three times in butyrate. The hyperpolarizing response to Na + removal in butyrate is similar to that observed in water-injected oocytes in the presence of CO 2 /HCO 3 (not shown). (C ) Changing [HCO 3 ] o at a fixed [CO 2 ] by altering extracellular ph (ph o ). Maintaining a PCO 2 of 1.5%, we changed [HCO 3 ] o from 10 to 2 mm (ph o : 7.5 to 6.8). After two 0-Na + pulses, we changed [HCO 3 ] o from 10 to 32 mm (ph o : 7.5 to 8.0). (D) NBC currents. The oocyte was voltage clamped at 60 mv as we switched from ND96 to 1.5% CO 2 /10 mm HCO 3. The 50 na outward current elicited by CO 2/HCO 3 corresponds to a 50 mv hyperpolarization observed in unclamped cells; the 50 na inward current elicited by Na + removal corresponds to a 50 mv depolarization observed in unclamped cells. Hence, anbc expression does not significantly change the native oocyte resistance of 1 M. (E) The bath solution was switched from ND96 solution to 5% CO 2 /33 mm HCO 3 (ph 7.5). Na+ was removed two times in CO 2 /HCO 3 and once in Cl. In comparison with anbc, the rknbc clone expresses poorly in oocytes even when bathed with threefold increased HCO 3.(F) rnbc-ptln2 expression in oocytes. The rknbc-cdna was subcloned into a Xenopus expression vector, ptln2 (93), a derivative of psp6 (94, 95). The 5 - and 3 -untranslated regions of the Xenopus β-globin mrna flank the rknbc cdna. An experiment is shown in which the bath solution was switched from ND96 solution (CO 2 /HCO 3 free) to only 1.5% CO 2/10 mm HCO 3 (ph 7.5). Na+ was removed three times in CO 2 /HCO 3. Expression of rknbc is increased by 50-fold. Thus, placing the mammalian rknbc cdna within this Xenopus context is apparently beneficial for expression in Xenopus oocytes. (Modified from References 15, 16.)

13 Na + /HCO 3 COTRANSPORTERS 711

14 712 ROMERO & BORON oocytes are electrogenic, Na + dependent, and HCO 3 dependent and are blocked by extracellular DIDS. As shown in Figure 3A, bath addition of CO 2 /HCO 3 to NBC-expressing oocytes elicits an immediate hyperpolarization that slowly relaxes; this relaxation of the hyperpolarization presumably reflects the slow increase in [HCO 3 ] i produced as NBC transports HCO 3 into the cell. Figure 3B shows that although acidifying an oocyte with CO 2 /HCO 3 can produce a sizeable hyperpolarization, applying butyrate (which acidifies the cell to the same extent; not shown) has no effect on V m. In the presence of CO 2 /HCO 3, removing bath Na + (Figure 3A,E,F ) or lowering bath HCO 3 (Figure 3C ) depolarizes NBC-expressing oocytes. Readdition of bath Na + or HCO 3 reverses these effects. Expression of rknbc may be substantially increased by placing the rknbc-cdna into a Xenopus expression plasmid (compare Figure 3E and F ) (16). Finally, most if not all of these NBC-mediated transport processes are blocked by extracellular DIDS (Figure 3A). Na + Dependence In experiments using 22 Na uptake on BLMVs of rabbit kidney cortex, Li +,K +, and choline each appeared to partially support Na/HCO 3 cotransporter activity (53). Studying 22 Na uptake, Jentsch and coworkers (54) determined electrogenic, DIDS-inhibitable Na/HCO 3 cotransporter activity in BSC-1 cells. They found an apparent K m for Na + of mm at 28 mm HCO 3. These investigators also found that Na/HCO 3 cotransporter activity was specific for Na + ; neither Li + nor K + could substitute. Amlal and colleagues (55) recently reported that after transfecting hknbc into HEK-293 cells, a low affinity for Li + and a lesser affinity for K + (they conclude no K + substitution; however, their data show measureable transport) is measured when monitoring ph i using bis(carboxethyl)-5(and 6)-carboxy fluorescein (BCECF). When expressed in Xenopus oocytes and studied electrophysiologically, neither anbc nor rknbc seems to be able to transport Li + (56, 57). Moreover, voltage-clamp experiments using rknbc show that neither Li + nor K + could substitute for Na + (58). Both influx (outward current, as for anbc in Figure 3D) and efflux (inward current, as for anbc in Figure 3D) of NaHCO 3 depend on extracellular Na + and voltage (58). Regardless of extracellular [Na + ], influx (outward I increasing with depolarization) is always measured for V m more positive than 40 mv; and efflux (inward I increasing with hyperpolarization) is always measured for V m more negative than 100 mv. The apparent affinity for extracellular Na + is 30 mm between 160 and 100 mv (NaHCO 3 efflux) and also between 20 and +60 mv (NaHCO 3 influx). For more typically physiologic V m s( 40 to 100 mv) both influx and efflux transport modes may be measured, depending on both extracellular [Na + ] and V m. In general, reducing [Na + ] o in this physiologic V m range enables NBC to predominantly efflux NaHCO 3 from the cell.

15 Na + /HCO 3 COTRANSPORTERS 713 HCO 3 Dependence The NBC protein in the renal proximal tubule is probably the major, perhaps exclusive, mode of HCO 3 exit from the cell into the blood; it may mediate the reabsorption of as much as 90% of renal HCO 3 absorption (59). However, the chemical form of HCO 3 (i.e. HCO 3,CO= 3, or the NaCO 3 ion pair) transported by the NBC protein has not yet been elucidated. Soleimani & Aronson (60), working on BLMV from rabbit renal cortex, found that raising [CO = 3 ] o at constant [HCO 3 ] o stimulates the influx of 22 Na, consistent with the hypothesis that CO = 3 is a substrate of NBC. On the other hand, raising [CO = 3 ] o at constant [HCO 3 ] o under equilibrium conditions necessarily involves raising ph o. Therefore this result does not unambiguously prove that NBC transports CO = 3. In other experiments, Soleimani & Aronson (60) found that in the presence of low [HCO 3 ], adding SO= 3 stimulates 22 Na uptake, consistent with the idea that SO = 3 substitutes for CO= 3 ataco= 3 binding site on NBC. On the other hand, SO = 3 solutions at physiological ph also contain HSO 3. Thus, one could also hypothesize that HSO 3 substitutes for HCO 3 at a HCO 3 binding site. More recent work on rknbc expressed in Xenopus oocytes shows that at least as expressed in oocytes rknbc transports neither SO = 3 nor HSO 3 (61). Thus, there is currently no convincing evidence that NBC transports CO = 3. Recent experiments with the NBC clones have provided data on the specificity and affinity for HCO 3 transport. In contrast to the anion promiscuity of the AEs (62 65), neither anbc nor rknbc expressed in oocytes seems to accept other organic anions (15, 56) or inorganic anions (II Grichtchenko, MF Romero, WF Boron, unpublished observations). For example, when an oocyte is acidified by exposing it to butyrate/butyric acid rather than to CO 2 /HCO 3, there is no negative shift in V m, and removing extracellular Na + fails to elicit a depolarization (Figure 3B) (15, 56). A recent report indicates that hknbc expressed in HEK-293 cells may also have a Na + :OH cotransport mode (55). Grichtchenko and colleagues have preliminary data on the extracellular [HCO 3 ] dependence of anbc and rknbc expressed in Xenopus oocytes. They exposed oocytes briefly to ph 7.5 solutions containing a range of HCO 3 concentrations [they also varied (CO 2 ) to keep ph o constant] and assessed transport either from the hyperpolarization or outward current mediated by NBC. They found that the apparent K m for external HCO 3, with the cotransporter running in the inward direction, is 12 mm for both NBCs (61, 67). ph i Dependence Several acid-base transporters are ph dependent (62, 68, 69). In studies on BBMV studies using 22 Na uptake to access Na/HCO 3 cotransporter activity,

16 714 ROMERO & BORON Soleimani and coworkers (70) found that 22 Na uptake increased as the ph gradient increased (ph i constant while varying ph o ). This same study indicated that maintaining ph i = ph o or maintaining a constant ratio of ([Na + ] i /[Na + ] o ) had a bell-shaped uptake profile (low uptake at ph o = 6.8, maximal uptake at 7.4, and low uptake at 8.0) (70). Transfecting hknbc into HEK-293 cells, this same group has observed increases of ph i elicited by raising ph o from 7.4 to 7.8 (55). Voltage Dependence A priori, one would expect that transport via a membrane transport protein should be sensitive to the voltage difference across a membrane, i.e. membrane potential (V m ). Newman & Astion (37) used whole cell recordings of retinal glial cells and found a voltage-dependent electrogenic Na/HCO 3 cotransporter (outward I for V m > 37 mv, and inward for V m < 37 mv) with a stoichiometry of 1 Na + :3 HCO 3. Recently, by permeablizing the apical membrane of monolayers of proximal tubule cell-lines, Gross & Hopfer (71) found a linear voltage dependence on the DNDS-inhibitable short-circuit current across the epithelial basolateral membrane. Cotransporter currents of anbc expressed in oocytes, although measurable, are small (Figure 3D). Interestingly, both giant patch (72) and 2-electrode voltage-clamp experiments (58) of rknbc show not only a voltage dependence of both inward and outward NBC transport (i.e. larger outward I with depolarization, or larger inward I with hyperpolarization), butalsoana + :HCO 3 stoichiometry of less than 1:3. NBC CLONES Because electrogenic Na/HCO 3 cotransporters apparently have a variety of stoichiometries, we have tried to clone and characterize NBC from several tissues and organisms (Ambystoma, rats, and humans). Mammalian, Non-Human, NBC cdnas and Genes Presently, only the renal NBC clone (rknbc) is a full-length open reading frame (ORF) from the rat (16). Screening rat brain libraries, Bevensee and coworkers (73) identified at least two varieties of NBC in the brain. One clone is identical to rknbc. The second clone contains a 3 deletion that shifts the reading frame to result in a slightly larger encoded protein. Recently, using genespecific primers and polymerase chain reaction, Shepard & Rae (74) provided evidence that NBC-mRNA is present in rabbit corneal epithelium, rabbit corneal endothelium, and human lens epithelium. At the same time, Rimmer and colleagues (75) obtained partial NBC clones from rabbit corneal endothelium. These partial clones have not yet been functionally characterized.

17 Na + /HCO 3 COTRANSPORTERS 715 Human NBC cdnas and Genes KIDNEY Knowing that NBC is related to the AEs, Burnham and associates (21) identified and obtained expressed sequence tags encoding a human analog of NBC. After screening a kidney library, they assembled a full-length clone 97% identical to rknbc. This hknbc clone has been characterized by transient transfections of HEK-293 cells and monitoring BCECF fluorescence to measure ph i. Recent work of Choi et al (76, 77) has shown that this transporter is indeed electrogenic. These investigators also isolated a second NBC form from human kidney with an alternative NH 2 terminus (MEDE-start and 5 region) (76) and have shown it to be electrogenic (44, 77). PANCREAS Because the pancreas secretes HCO 3, one might expect that NBC in the pancreas should have different sequence than that of the kidney. Abuladze et al (25) recently reported cloning a variant of NBC from human pancreas (pnbc is identical to hhnbc). When these investigators expressed this human clone in Xenopus oocytes, they measured both HCO 3 -stimulated 22 Na-uptake and decreases of ph i upon extracellular Na + removal (monitored by BCECFfluorescence) (25). Working at the same time, Choi et al (24) cloned an identical cdna from human heart but found it to be identical to the dominant NBC in pancreas (see below). HEART As noted earlier, physiological studies of mammalian heart indicate that the cells express an electroneutral Na/HCO 3 cotransporter (5, 5a). On the other hand, work on cat papillary muscle suggests that there is an electrogenic Na/HCO 3 cotransporter in the heart, presumably with a Na + :HCO 3 stoichiometry of 1:2 (49). Choi et al recently cloned an NBC-related cdna from human heart (hhnbc). It is identical to the cdna cloned from human kidney except that the 41 NH 2 -terminal amino acids of hknbc are replaced by 85 in hhnbc. Thus, hhnbc has an open reading frame of 1079 amino acids. The 5 end includes the unique 5 MEDE start. hhnbc is identical to the pancreatic NBC. Electrophysiological studies on oocytes indicate that hhnbc is an electrogenic Na/HCO 3 cotransporter (44, 77). Thus, the NH 2 terminus of NBC does not appear responsible for conferring electrogenicity. An electroneutral Na/HCO 3 cotransporter has yet to be cloned and expressed from any mammalian tissue. PROSTATE Using high-stringency Northern analysis, we identified the NBC mrna in tissues for which there is little or no HCO 3 transporter physiology known. The prostate is one such tissue. This NBC cotransporter also contains the MEDE start and is virtually identical to the clone from other tissues (MF Romero, unpublished results; GenBank#AF053753).

18 716 ROMERO & BORON HUMAN NBC GENES Two groups have obtained Pl artificial chromosome clones and used fluorescent in situ hybridization (FISH) to localize the NBC gene(s) to human chromosome 4q (25, 78). Our analysis of several NBC P1-derived artificial chromosome clones indicates that there are two human NBC genes on chromosome 4q2 and two human NBC-related genes (78). Studies are under way to determine gene structure and more precise chromosomal localization. Currently, it is difficult to distinguish the encoded cdnas. Perhaps the genes contain tissue-specific response elements to direct these virtually identical clones to tissues of varying apparent Na/HCO 3 cotransporter activity. LOCALIZATION OF NBC mrna Localization of NBC mrnas by Northern Analysis By Northern analysis, two groups have observed an rknbc mrna at 7.5 kb (16, 79). The rknbc mrna is abundant in kidney but present in substantial amounts in liver and brain, and at lower levels in heart, lung, and spleen. The rknbc-orf encodes a protein of 1035 amino acids, indicating that the message has additional untranslated regions of more than 4 kb. In Ambystoma, the message was smaller (4.4 kb); there was no reactivity in the liver, lung, and spleen; and an even smaller message ( 2 kb) was in the heart (15). Similarly, several groups have observed with human Northern blots that NBC-like transcripts (7.5 to 9 kb) are abundant in pancreas, kidney, brain, and prostate but detectable in liver, lung, small intestine, large intestine, and heart (21, 24, 25, 76). Recently, similar results were reported with the refinement of using transcriptspecific probes to distinguish forms (25). These data indicate that the 1035 amino acid form of human NBC (or hknbc) is predominant in the kidney whereas the 1079 amino acid form of human NBC is prominent in the pancreas and prostate. Localization of NBC mrnas by In Situ Hybridization Examining NBC-mRNA distribution by in situ hybridization appears to give conflicting results. Rat kidney NBC-mRNA predominates in the straight portion of the S2 renal nephron with no mrna in S1 or S3 (16). The in situ localization of NBC is more restricted spatially than the well-documented cotransporter activity in isolated perfused tubules. This activity has been detected in S1 and convoluted segments of S2 (27, 35, 80 85), as well as in S3 (86, 87). In rabbit kidney, the expected distribution of NBC mrna was observed (88). The basis for the difference in the rat and rabbit localizations is unclear. One possibility is that the rat has other NBC isoforms not yet detected with existing probes. Alternatively, NBC mrna and NBC protein may be controlled differently along the proximal tubule (see below).

19 Na + /HCO 3 COTRANSPORTERS 717 Physiological experiments indicate the presence of a Na + -coupled HCO 3 transporter in acini (89) and on the basolateral membrane of pancreatic duct cells (46). In the mouse pancreas, NBC-mRNA has been recently reported to be in both ductal epithelial cells and in acini (25). In adult rat brain, NBC mrna expression has been shown by both Northern blotting and in situ hybridization (89a). NBC is expressed throughout the neuraxis, but prominently in olfactory bulb, hippocampus, and cerebellum. Interestingly, NBC was present not only in glial cells (e.g. hippocampal astrocytes and Bergmann glia) but was also found at similar levels in neurons (e.g. in cortex and hippocampus). Antibody Localization of NBC Protein Schmitt and coworkers (90) generated polyclonal antibodies directed against both the middle (amino acids ) and carboxy terminus (amino acids ) of rat kidney NBC. These epitopes are conserved from salamander to human. Additionally, the NBC-epitopes are distinct from AE sequences (see Figure 1). Thus, it was expected that the antibodies would recognize NBC in a variety of species and yet not show cross-reactivity to the AEs. These expectations have proven true. In immunoblot experiments, the NBC antibodies react with a rat- and rabbitkidney protein with a molecular weight of 130,000. Smaller bands are seen at molecular weights of 100,000 and 85,000. rknbc expressed in Xenopus oocyte also has an apparent molecular weight of 130,000. In the Ambystoma kidney, the antibodies recognize a band at a substantially higher molecular weight, 160,000. Because the predicted molecular weight for these NBCs is 116,000, it is likely that the NBC protein is partially glycosylated both in vivo and in vitro (91, 92). In immunocytochemistry studies, NBC immunoreactivity is observed exclusively in basolateral membranes of rat, rabbit, and Ambystoma kidneys (90, 91). In rabbit kidney, the immunoreactivity is limited to the S1 and S2 segments of the proximal tubule; no reactivity was detected in the more distal S3 segment. Differences between the immunocytochemistry data and the in situ hybridization data could reflect differences in the sensitivities of the methods, differences in the relative abundances of NBC mrna vs protein, or differences in isoforms expressed in different segments of the proximal tubule. In the salamander, immunoreactivity was intense in a portion of the distal nephron with high rates of H + secretion and was present but less intense in the proximal tubule. Preliminary work with these NBC antibodies has identified NBC-related proteins in the basolateral membranes of parietal cells, colonic crypts (not mucussecreting cells), ductuli efferentes, and caput and cauda epididymis of rats (92, 92a). These NBC antibodies do not recognize species or isoform differences.

20 718 ROMERO & BORON By immunoblotting and immunohistochemistry, Schmitt et al (89a) have demonstrated that NBC protein is widely expressed in the central nervous system, especially in olfactory bulb, hippocampus, and cerebellum. As with mrna, NBC protein was present in both glial cells and in neurons. Immunoblotting on primary cultured cells confirmed that the characteristic 130-kDa protein is present at similar levels in hippocampal astrocytes and cortical neurons from rat. A study by Douglas et al (92b) showed that NBC is expressed at early developmental stages and reaches adult levels soon after birth, with regional differences. Furthermore, exposing rats to chronic hypoxia in utero appeared to upregulate the NBC protein levels. By screening a rat brain cdna library using a portion of rat kidney NBC, Bevensee and coworkers isolated a partial-length NBC-like clone that is identical to rknbc except for a 97 base pair deletion near the 3 end (MO Bevensee, BM Schmitt, M Romero & WF Boron, unpublished observations). This novel clone encodes a protein that has 61 unique C-terminal amino acids instead of the 46 C-terminal amino acids of rknbc. These investigators have developed a pair of polyclonal antibodies that distinguishes rknbc from a variant brain cdna that has a unique NH 2 terminus. The rat brain NBC variant also has an apparent molecular weight of 130 when detected using an isoform-specific NBC antibody (73). SUMMARY AND CONCLUSIONS With the cloning of NBC from several species and tissues, interest in acid-base transporters and particularly HCO 3 transporters appears renewed. Although some of the elemental transport processes of NBC are beginnig to be characterized, there are still many avenues of transporter function and regulation to be elucidated. Future studies, we hope, will determine (a) what ions are transported, (b) whether voltage dependence and/or stoichiometry is changeable, and (c) whether there are complementary proteins interacting with NBC to alter measured properties. It is clear that there are NBC isoforms. Yet it is unclear whether these NBC-variants arise from the same or different NBC genes. What signals and/or binding proteins regulate the NBC genes, mrnas, and protein? Are there pathophysiologic stimuli such as acidosis, alkalosis, hypertension, hypotension, or cancers that influence NBC activity, mrna expression, protein production, or protein regulation? Finally, there are now two branches of what seems to be a HCO 3 transporter superfamily. Several physiologically important HCO 3 transporters have yet to be identified at the molecular level. We hope interest in HCO 3 transport will flourish. Perhaps with the aid of several genome sequencing projects, the ever-growing expressed sequence tags databases, cloning projects, and the

21 Na + /HCO 3 COTRANSPORTERS 719 fervent interest in functional genomics (physiology), many, if not all, putative superfamily members will be identified. ACKNOWLEDGMENTS We thank our colleagues and collaborators, in particular Emile L Boulpaep, Matthias A Hediger, Mark O Bevensee, Urs V Berger, Inyoung Choi, Bruce A Davis, Peying Fong, Irina I Grichtchenko, Nazih L Nakhoul, Chris M Sciortino, Caroline R Sussman, Bernhard M Schmitt, Patricia Bray-Ward, David Ward, and Duncan Wong who have been involved in many ways and in several phases of this work. This work was supported by National Institutes of Health grants DK30344, DK17433, and DK43171 to WFB. MFR was supported by grants from the National Kidney Foundation, National Institutes of Health grant NRSA DK09342, the American Heart Association, American Cancer Society Award IRg IRg and a HHMI-institutional grant to Case Western Reserve University. Visit the Annual Reviews home page at Literature Cited 1. Sly WS, Hu PY Human carbonic anhydrases and carbonic anhydrase deficiencies. Annu. Rev. Biochem. 64: Nakhoul NL, Romero MF, Waheed A, Davis BA, Mullins R, et al Processing and functional expression of carbonic anhydrase isoforms in Xenopus laevis oocytes. FASEB J. 10:A88 (Abstr.) 3. Boron WF, Boulpaep EL Intracellular ph regulation in the renal proximal tubule of the salamander. Basolateral HCO 3 transport. J. Gen. Physiol. 81: Deitmer JW, Schlue WR The regulation of intracellular ph by identified glial cells and neurones in the central nervous system of the leech. J. Physiol. 388: Dart C, Vaughan-Jones RD Na + - HCO 3 symport in the sheep cardiac Purkinje fibre. J. Physiol. 451: a. Lagadic-Gossmann D, Buckler KJ, Vaughn-Jones RD Role of bicarbonate in ph recovery from intracellular acidosis in the guinea pig ventricle myocyte. J. Physiol. 458: Hogan EM, Cohen MA, Boron WF K + - and HCO 3 -dependent acidbase transport in squid giant axons. I. Base efflux. J. Gen. Physiol. 106: Hogan EM, Cohen MA, Boron WF K + - and HCO 3 -dependent acid-base transport in squid giant axons. II. Base influx. J. Gen. Physiol. 106: Russell JM, Boron WF Role of chloride transport in regulation of intracellular ph. Nature 264: Thomas RC The role of bicarbonate, chloride and sodium ions in the regulation of intracellular ph in snail neurones. J. Physiol. 273: Kopito RR, Lodish HF Primary structure and transmembrane orientation of the murine anion exchange protein. Nature 316: Alper SL, Kopito RR, Libresco SM, Lodish HF Cloning and characterization of a murine band 3-related cdna from kidney and from a lymphoid cell line. J. Biol. Chem. 263: Kopito RR, Lee BS, Simmons DM, Lindsey AE, Morgans CW, Schneider K Regulation of intracellular ph by a neuronal homolog of the erythrocyte anion exchanger. Cell 59: Linn SC, Kudrycki KE, Shull GE The predicted translation product of a