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JOP. J Pancreas (Online) 2001; 2(4 Suppl):182-191.

The Electrogenic Na+/HCO3- Cotransporter, NBC

Michael F Romero

Department of Physiology and Biophysics, Department of Pharmacology, Case Western Reserve University School of Medicine. Cleveland, OH, USA

Summary

Electrogenic Na+/HCO3- (NBC) function has been characterized in many mammalian tissues including, kidney, pancreas, and brain. Cloning efforts identified a single cDNA, NBC/NBC1, that possesses all the functional attributes of the electrogenic Na+/HCO3- cotransporter. This NBC clone is related to the anion exchangers and thus forms a bicarbonate transporter superfamily. Presently two N-terminal and one C-terminal isoforms are known. All three isoforms appear to arise from the same gene and seem to have identical function. NBC antibodies have localized NBC isoforms in kidney, pancreas, brain, small intestine, colon, epididymis, eye, heart, liver, salivary glands, stomach, and testis. Functionally, NBC appears HCO3- and Na+ selective. NBC stoichiometry in Xenopus oocytes is 1 Na+ : 2 HCO3-, implicating a possible accessory protein interaction.


Background of the Electrogenic Na+/HCO3- Cotransporters

HCO3-, like other ions and nutrients in the blood, is filtered in the kidney at the glomerulus, then absorbed by transport processes in the renal nephron. The proximal tubule is responsible for 80-90% of renal HCO3- absorption. HCO3- in the luminal fluid combines with secreted H+ (mostly by Na+-H+ exchange [1]) to form CO2 and H2O, both of which easily enter the proximal tubule cell. Prior to the 1980’s the mode of HCO3- movement from the proximal tubule cells back into the blood was elusive. A basolateral HCO3- conductance pathway was hypothesized.

Boron and Boulpaep made the astonishing discovery that this HCO3- absorption process was coupled asymmetrically to Na+ transport [2]. This transport activity was called the "electrogenic Na+/HCO3- cotransporter." This electrogenic Na+/HCO3- cotransporter mediated a "fingerprint" transport [2]: Na+ transport, HCO3- transport, electrogenic (1 Na+ : at least 2 HCO3-), no Cl- transport/dependence, and stilbene inhibition. Later, a functionally similar cotransport activity was reported in mammals: bovine corneal endothelial cells [3], the basolateral membrane of rat proximal convoluted tubule [4], the basolateral membrane of rabbit proximal straight tubule [5], and many other preparations (for review see [6]).

Yet until 1995, the molecular nature of this protein(s) was unknown. This kidney of the salamander Ambystoma tigrinum was used to expression clone a renal electrogenic Na+/HCO3- cotransporter (NBC) [7, 8]. As the cotransport activity originally characterized in this tissue, NBC transported Na+ and HCO3-, and was electrogenic (1 Na+ : at least 2 HCO3-), Cl- independent, and inhibited by stilbenes (such as 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid: DIDS). Interestingly using amphibian kidney rather than mammalian tissue to clone NBC was the key to success [8, 9]. Surprisingly, this electrogenic NBC sequence was molecularly related to the electroneutral band-3 like proteins, i.e., the anion exchangers AE1, AE2, and AE3 [8, 9]. This homology revealed a probable bicarbonate transporter superfamily (BTS) [8] that now has many seemingly topologically related members (Figure 1). This relationship and NBC’s cloning has renewed interest in HCO3- transporters.

Figure 1. Predicted bicarbonate transporter superfamily relationships.
The a) panel is a dendrogram for representative member of the bicarbonate transporter superfamily (BTS) [54]. The open circle indicates the electrogenic Na+/HCO3- cotransporter isoforms collectively known as NBC or NBC1. AE1-3 are the mammalian anion exchangers. Other BTS members have been identified and cloned: ceNBC (AF004926), NDAE1 (AF47468), NBC3 (AF47033), NBCn1-D (AF80106), NCBE (AB033759), NDCBE1 (AF069512), AE4a and AE4b (AB038264), NBC4 (AF207661), and yeast yn1275w1. A second Drosophila protein (CG8177) and three Arabidopsis proteins (Arab-Chr1, Arab-Chr2, and Arab-Chr3) have been predicted from genome sequencing projects but not functionally evaluated. The b) panel is a working membrane topology model of NBC based largely on structural results of AE1 (see [55]). Both N- and C-termini are predicted as intracellular. A large, central extracellular loop uses one N-linked glycosylation site N617 [56] though seven are predicted. Three predicted DIDS-binding motifs are indicated by boxes.

NBC Clones, Proteins and Gene

The renal or "kidney" NBC orf (open reading frame) (kNBC) encodes 1035 amino acids (Figure 2, top) and predicts a protein of 116 kDa [8, 9, 10, 11]. The NBC-protein is predicted to have both the N- and C-termini intracellular (Figure 1), many potential phosphorylation sites, as well as several N-linked glycosylation sites.

A second N-terminal NBC isoform was cloned from pancreas (pNBC) [12] and heart (hhNBC) [13]. This clone has the first 41 amino acids replaced by a different 85 amino acids (Figure 2, middle). This pNBC encodes 1079 amino acids and predicts a protein of 120 kDa [12, 13, 14]. The longer NBC protein also encodes similar transport [12, 13] and is electrogenic [13].

Figure 2. Schematic of electrogenic Na+/HCO3- cotransporter isoforms and antibodies.
Diagram of the three NBC1 isoforms: two N-terminal (kNBC and pNBC) and one additional C-terminal (rb2NBC). All other sequence between clones is identical within a species. Sequence regions recognized by NBC antibodies are indicated by brackets and annotated with "
a".

A unique C-terminus accounts for the third NBC isoform (rb2NBC) (Figure 2, bottom). This rb2NBC was recently cloned and characterized from the rat brain [14]. The rb2NBC clone results from 61 unique COOH-terminal amino acids, the result of a 97-bp deletion and frame shift near the end of the open-reading. The encoded rat protein is 1094 amino acids and predicts a protein of about 130 kDa [14]. This C-terminal NBC isoform has not yet been identified in human. Again, rb2NBC was found to mediate apparently identical transport activity as rat kidney NBC (rkNBC) and human pancreatic/heart NBC (hpNBC/hhNBC) [14].

The human NBC1 gene (SLC4A4) resides at 4q21 [12, 15]. More recent data indicates that SLC4A4 is about 400-450 kb [16]. Both pNBC and kNBC are transcribed from the same gene, but kNBC is transcribed from an alternative internal promoter [16].

NBC clones and their corresponding proteins have been identified in several tissues other than the kidney, pancreas, and brain (Table 1). Interestingly, the kidney seems to express all of the identified NBC mRNAs and proteins. In keeping with this observation, renal disease is one of the major phenotypes of human NBC mutations [17, 18, 19]. That is, these affected patients have a permanent proximal renal tubular acidosis (type 2 RTA) manifest as blood pH about 7.05 and blood [HCO3-] about 5-8 mM, rather than the normal 7.4 and about 23-29 mM, respectively. The eye is also effected by these NBC point mutations, manifest as bilateral glaucoma, bilateral cataracts, and bandkeratopathy [18]. The effects on other tissues where NBC isoforms are expressed (Table 1) are not obvious. Whether the mutations cause a biophysical change in cotransport activity or result in a cellular protein processing problem, is not well understood.

Table 1. Tissue distribution of the electrogenic Na+/HCO3- cotransporters NBC (NBC-1).

Tissue

NBC Isoform

Identification Method

Reference

Brain

rb1NBC (pNBC)

Cloning

[14, 39]

Brain

rb2NBC

Cloning
IF localization (a)

[14]
[40, 41]

Colon

pNBC
kNBC/pNBC

Northern blot
Western blot

[12]
[14]

Duodenum
(Small Intestine)

kNBC and pNBC
kNBC/pNBC

RT-PCR / IF localization
Western blot

[42]
[14]

Epididymis

NBC

IF localization

[43]

Eye

kNBC and pNBC
pNBC
pNBC

RT-PCR
Cloning / RT-PCR
Western blot

[44]
[45]
[45]

Heart

hhNBC/pNBC
kNBC/pNBC

Cloning
Western blot

[13]
[14]

Kidney

kNBC

Cloning

IF localization

[8, 11]

[46, 47]

Kidney

pNBC

Cloning

[48]

Kidney

rb2NBC

Western blot

[14]

Liver

NBC
rb2NBC

Northern blot
Western blot

[9, 12]
[14]

Lung

NBC
kNBC/pNBC

Northern blot
Western blot

[9]
[14]

Pancreas

pNBC

Cloning

IF localization

[12, 49]

[49, 50]

Prostate

pNBC

RT-PCR / cloning
Northern

[51]
[12]

Salivary Glands

NBC

IF localization

[52]

Stomach

NBC

Cloning / RT-PCR
Northern

[53]
[11, 12]

Testis

kNBC/pNBC

Western blot

[14]

Thyroid

pNBC

Northern blot

[12]

IF is immunofluorescence. NBC-1 is SLC4A4. Note that "NBC" as an isoform designation indicates that the exact isoform of NBC is currently unknown or unpublished.
(a) The study by Bevensee et al. identifying rb2NBC in the brain [14] is the only study to date identifying NBC isoforms by immunohistochemistry.

NBC Expression in Oocytes

Xenopus oocytes were used to expression clone kNBC [7, 8]. Figure 3 illustrates the experimental arrangement with two or more microelectrodes. The experimental assay uses a bath perfusion system. Addition of CO2/HCO3- to the solutions causes a decrease of intracellular pH (pHi) because CO2 may traverse the oocyte plasma membrane, be hydrated intracellularly to form HCO3- and H+. If an oocyte is expressing NBC, this CO2/HCO3- addition will elicit an immediate hyperpolarization (Figure 4a) due to the coupled entrance of Na+ with multiple HCO3- ions ("reverse transport" in Figure 3). Once pHi achieves a steady-state, extracellular removal of Na+ (replacement by an impermeant cation such as choline or N-methyl-D-glucamine), depolarizes the oocyte and decreases pHi (Figure 4a) ("forward transport" as in the proximal tubule, Figure 3). Figure 4a illustrates that this electrogenic HCO3- transport activity is unaffected by extracellular Cl- removal. By contrast, Figure 4b shows that an oocyte expressing AE2 does not mediate electrogenic transport and increases pHi after extracellular Cl- removal, yet is unaffected by extracellular Na+ replacement.

Figure 3. Experimental arrangement-Xenopus oocyte electrophysiology.

Figure 4. Oocyte expression of NBC and AE2
A comparison of NBC expression (A) and AE2 expression (B) in Xenopus oocytes.
A. Rat kidney NBC expressed in a Xenopus oocyte. Addition of CO2/HCO3- elicits an immediate hyperpolarization (HCO3- influx) and a decrease of pHi which begins to recover as a result of the HCO3- influx. Na+ removal in CO2/HCO3-, depolarizes and acidifies the oocyte. However, bath Cl- removal does not change pHi or Vm.
B. Intracellular pH experiment on an oocyte expressing the AE2 anion exchanger. Addition of CO2/HCO3- acidifies the oocyte but does not elicit a hyperpolarization as with NBC. Removal of bath Cl- elicits an increase in pHi (ab and cd). However, Na+ removal in CO2/HCO3- does not change pHi in an oocyte expressing AE2.

Anions transported

The NBC protein in the renal proximal tubule is the major, perhaps exclusive, mode of "HCO3-" exit from the cell into the blood [18, 19, 20]. However, the chemical form of "HCO3-" (i.e., HCO3-, CO32- or the NaCO3- ion pair) transported by the NBC protein is still under investigation. Anions transported are indicated in Table 2.

Table 2. Ion specificity of the electrogenic Na+/HCO3- cotransporter NBC.

 

Transported

Apparent K0.5

Vm dependence

Reference

         

Cation

       

Na+

Yes

about 30 mM

Yes

[25]

Choline+

No

-

Yes, as 0Na+

[8, 9, 25]

NMDG+

No

-

Yes, as 0Na+

Romero and Boron, unpublished

Li+

Minor

-

Yes, as 0Na+

[25]

K+

No

-

ND

[25]

 

 

 

 

 

Anion

       

HCO3-

Yes

about 6.5 mM

Yes

[8, 9, 13, 14, 21, 25]

Cl-

No

-

No

[9, 14, 21, 25]

Butyrate-

No

-

No

[8, 9]

Propionate-

No

-

No

Sciortino and Romero, unpublished

SO42-

No

-

No

[21]

SO32-

No

-

No

[21]

CO32-

Unlikely

-

No

[21]

Vm is membrane voltage, apparent K0.5 is the ion concentration at the apparent half maximal transport rate, and ND is not determined. "Yes, as 0Na+" indicates that NBC exhibits Vm dependence when non-transported test ions are used for a Na+ removal maneuver (i.e., reverse transport).

Grichtchenko and coworkers have determined the extracellular [HCO3-] dependence of Ambystoma NBC (aNBC) and rkNBC expressed in Xenopus oocytes [21]. Exposing oocytes briefly to pH 7.5 solutions containing a range of HCO3- concentrations (also varying [CO2] to keep extracellular/outside pH (pHo) constant), they measured transport either from the hyperpolarization or outward current mediated by NBC. The apparent Km for external HCO3-, with the cotransporter running in the inward direction, was about 6-7 mM for both NBCs [21, 22, 23]. This same study revealed that SO42-, SO32-, and HSO3- are not transported by NBC [21].

Our initial expression experiments with NBC, indicated that organic anions could not substitute for the HCO3- ion [8, 9]. Similarly, total removal of Cl- does not effect the activity of NBC [9, 12, 13, 21]. In contrast to oocyte experiments, NBC activity assayed by 2’7’-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) pH measurements in transfected HEK-293 cells does not appear to require HCO3- presence, i.e., a Na+/(OH-)n cotransport mode [24]. HCO3- is absolutely required for electrogenic Na+/HCO3- cotransport in oocyte experiments [25].

Cations transported

In experiments using 22Na uptake on basolateral membrane vesicles of rabbit kidney cortex, Li+ , K+, and choline each appeared to partially support Na/HCO3 cotransporter activity [26]. Studying 22Na uptake, Jentsch and coworkers [27] determined electrogenic, DIDS-inhibitable Na+/HCO3- cotransporter activity in BSC-1 cells. They found an apparent Km for Na+ of 20-40 mM at 28 mM HCO3-. These investigators also found that Na+/HCO3- cotransporter activity was specific for Na+; neither Li+ or K+ could substitute. Amlal et al. have reported that after transfecting hkNBC into HEK-293 cells, a low affinity for Li+ and lesser affinity for K+ is measured when monitoring pHi using BCECF [24]. When expressed in Xenopus oocytes and studied electrophysiologically, Na+ transport is observed [25]. Neither aNBC nor rkNBC seem to be able to transport Li+ [25, 28, 29].

Voltage clamp experiments using rkNBC show that neither choline+, Li+, nor K+ could substitute for Na+ (Figure 5) [25, 30]. Cation transport by NBC is summarized in Table 2. Moreover, both influx (outward current) and efflux (inward current) of NaHCO3 depend on extracellular Na+ and voltage [25]. Regardless of extracellular [Na+], influx (outward I increasing with depolarization) is always measured for Vm more positive than –40 mV; and efflux (inward I increasing with hyperpolarization) is always measured for Vm more negative than –100 mV. The apparent affinity (K0.5) for extracellular Na+ is about 30 mM for all voltages between –160 and +60 mV [25]. In general, reducing [Na+]o in this physiologic Vm range enables NBC to mediate predominantly efflux of NaHCO3 from the cell.

Figure 5. Cation dependence of NBC
A. Solution pulse protocol. This solution protocol is used to test whether K+, Li+, or choline+ are capable of stimulating a HCO3- -dependent current from rkNBC oocytes. Oocytes were voltage clamped at –60 mV and bathed in ND96 for 5 min before switching to test cation/non-HCO3- ringer for 5 min. The Bath solution was then switched to the corresponding 1.5% CO2/10 mM HCO3- solution for 2 min, e.g., Li+-ND96 to 1.5% CO2/10 mM HCO3-/96 mM Li+) and returned to non-HCO3- Ringer for 2 min. An I-V relation was recorded before and after each solution change. HCO3- -stimulated current for each cation was taken as the difference between the non-HCO3- and HCO3- I-V responses. Cation solutions order was randomized.
B. rkNBC I-V response of cations. HCO3- subtracted I-V response curves from the current sweeps show that only Na+ (black) stimulates a strong HCO3- -dependent current. Extracellular K+ (red) and choline+ (green) have I-V relations that lie on the voltage axis, indicating no transport. Li+ (blue) shows only a slight current response of a maximal about 3% of the Na+ response over the voltage range tested. Modified from [25].

Stoichiometry

In their original work on the electrogenic Na+/HCO3- cotransporter of the salamander proximal tubule, Boron and Boulpaep demonstrated that the cotransporter moves more HCO3- than Na+ [2]. Based on measurements of pHi, Vm and intracellular Na+ activity, they made a thermodynamic argument that the Na+:HCO3- stoichiometry had to be at least 1:2. However, they could not rule out the possibility that it is higher (e.g., 1:3). Subsequent work by Lopes et al. [31] on proximal tubule suggested, again on thermodynamic grounds, that the Na+:HCO3- stoichiometry was at least 1:3.

Using rabbit renal basolateral membrane vesicles (BLMV), Soleimani and Aronson reasoned that the net transport direction depends on both the Na+:HCO3- coupling ratio and the electrochemical gradients for Na+ and HCO3- [32]. By altering these gradients and measuring the direction of net transport in rabbit BLMV, these workers concluded that the renal electrogenic Na+/HCO3- cotransporter must have a stoichiometry of 1:3. Any of three models could account for this apparent 1:3 stoichiometry of the cotransporter: (i) Na+ plus 3 HCO3-, (ii) Na+ plus HCO3- plus CO32-, or (iii) the NaCO3- ion pair and HCO3-. Two groups working with isolated proximal tubules have suggested that, under special conditions, the renal electrogenic Na+/HCO3- cotransporter can alter its stoichiometry from 1:3 to 1:2, and thus change the net direction of net HCO3- transport [33, 34].

Even though the data, obtained under "physiological" conditions, on native renal cells or native cell-derived materials points to a stoichiometry of 1:3, it should be pointed out that the Na+:HCO3- coupling ratio has not been measured directly. Recently, by permeabilizing the apical membrane of monolayers of proximal tubule cell-lines, Gross and Hopfer found a linear voltage dependence on the 4,4’-dinitrostilben-2,2’-disulfonic acid- (DNDS)-inhibitable short-circuit current across the epithelia basolateral membrane [35]. When expressed in Xenopus oocytes, both giant patch [36] and 2-electrode voltage clamp experiments [25] 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), but also a Na+:HCO3- stoichiometry of 1 Na+ : 2 HCO3-. This result is surprising, given that the human NBC mutations [18] imply that NBC is the major HCO3- exit pathway back to the blood for the proximal tubule and the kidney in general. That is, a putative accessory protein (Figure 6) or modification factor must modify NBC stoichiometry in the renal proximal tubule.

Figure 6. Biophysical modification of NBC-model
Left. Model voltage clamp experiment illustrating the predicted change in the NBC I-V plot after modification by "factor X" in the proximal tubule or eye. Changing from a 1:2 (solid line with squares) to 1:3 (dotted line with circles) Na+:HCO3- coupling will not only shift ERev, but would likely increase the current measured at the voltage extremes. The basal current at –60mV for 1:3 is also predicted to be much less than we typically measure for NBC, i.e., +200 nA to slight inward current (-50 to -100 nA).
Right. Diagram indicating "how" mechanistically a NBC-interacting protein could change ionic coupling. We envision a conformational change in NBC which exposes/opens an additional HCO3- binding pocket.

Future Directions and Summary

With the cloning of several genomes, one wonders the direction science will take. Recent emphasis on protein interactions, will undoubtedly lead to a better understanding of cellular processes and integrated cellular function. NBC is found throughout mammalian tissues. NBC like all of our "favorite proteins" will likely be found to have several protein partners mediating specialized cellular functions. For example, NBC is postulated to have an accessory role in facilitating CFTR’s role as a Cl- and HCO3- channel in CaLu-3 cells [37]. Another study implicated Na+-H+ exchange regulatory factor (NHERF) might also regulate NBC activity [38].

Molecular and immunologic reagents will enable investigators to study HCO3- transport processes more easily. Localization will be necessary to generate new cellular models for ion transport and acid-base homeostasis. And, the physiology of several tissues should be revisited to integrate the role of NBC.

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Key words Electrophysiology; Epithelium; Hydrogen-Ion Concentration; Ion Transport; Microelectrodes; Nervous System; Oocytes; Xenopus

Abbreviations AE: anion exchanger; aNBC: Ambystoma NBC; BCECF: 2’7’-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; BLMV: basolateral membrane vesicles; BTS: bicarbonate transporter superfamily; DIDS: 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid; DNDS: 4,4’-dinitrostilben-2,2’-disulfonic acid; hhNBC: human heart NBC: hpNBC: human pancreas NBC; kNBC: kidney NBC; NBC: electrogenic Na+/HCO3- cotransport; NHERF: Na+-H+ exchange regulatory factor; orf: open reading frame; pHi: intracellular pH; pHo: extracellular/outside pH; rb2NBC: C-terminal isoform of rat brain NBC; rkNBC electrogenic rat kidney NBC; RTA: renal tubular acidosis

Acknowledgements The author would like to thank collaborators and colleagues whose work was summarized here: Walter F. Boron, 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, Eleni Roussa, Chris M. Sciortino, Caroline R Sussman, Bernhard M Schmitt, Frank Thévenod, Patricia Bray-Ward, David Ward, and Duncan Wong This work was supported by a grant from the American Heart Association and a Howard Hughes Medical Institute grant to CWRU.

Correspondence
Michael F Romero
Department Physiology and Biophysics
Case Western Reserve University
School of Medicine
2119 Abington Rd, SOM-E545
Cleveland, OH 44106-4970
USA
Phone: +1-216.368.3180
Fax:+1-216.368.4952
E-mail address: mfr2@po.cwru.edu

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