Sodium-bicarbonate cotransporters are homologous membrane proteins mediating the electrogenic or electroneutral transport of sodium and bicarbonate. Of the functionally characterized sodium-bicarbonate cotransporters (NBC), NBC1proteins are known to be electrogenic. Here we report the cloning and functional characterization of NBC4c, a new splice variant of the NBC4 gene. At the amino acid level, NBC4c is 56% identical to NBC1 protein variants and 40% identical to electroneutral NBC3. When expressed in mammalian cells, NBC4c mediates electrogenic sodium-bicarbonate cotransport. The transport of sodium and bicarbonate is chloride independent and is completely inhibited by DIDS. NBC4c transcripts were detected in several tissues including brain, heart, kidney, testis, pancreas, muscle, and peripheral blood leukocytes. The data indicate that NBC4c is an electrogenic sodium-bicarbonate cotransporter. The finding that both NBC1 and NBC4c proteins function as electrogenic sodium-bicarbonate cotransporters will aid in determining the structural motifs responsible for this unique functional property, which distinguishes these transporters from other members of the bicarbonate transporter superfamily.
sodium-bicarbonate cotransport (NBC) proteins contribute to intracellular pH (pHi) regulation and transepithelial HCO transport in several tissues (1, 2, 6, 8-10, 20-26,29) by mediating electrogenic or electroneutral Na+-HCO cotransport in the absence of Cl−. NBC transporters are homologous proteins belonging to the bicarbonate transporter superfamily (BTS), which also includes the Cl−/HCO exchanger proteins AE1–AE4 (5, 31) and Na+-driven Cl−/HCO exchangers (12, 27,32). NBC1 is the only currently known gene in mammals encoding electrogenic Na+-HCO cotransport proteins (3). In humans, NBC1encodes two electrogenic Na+-HCO cotransport variants, kNBC1 and pNBC1, which differ in their NH2 terminus (3). kNBC1 mediates the majority of basolateral HCO efflux in the renal proximal tubule (2, 9, 26, 29). pNBC1 is highly expressed in pancreas and various other tissues (1, 8, 14, 20, 21). An electroneutral NBC, NBC3 in humans (rat ortholog NBCn1) mediates Cl−-independent and stilbene-insensitive Na+-HCO cotransport in various tissues (10, 20, 22, 25).
We now report the cloning and functional characterization of NBC4c, a new splice variant of the NBC4 gene (23, 24). The results indicate that NBC4c is an electrogenic Na+-HCO cotransporter. There is currently little information regarding the basis for the ion specificity and transport stoichiometry of members of the BTS. The finding that NBC1 and NBC4c proteins are functionally similar will provide a basis for additional studies addressing the structural requirements for electrogenic Na+-HCO cotransport.
Cloning and sequencing of NBC4c.
An arrayed human cDNA library (Origene Technologies, Rockville, MD) was screened by a PCR-based approach according to the manufacturer's instructions. PCR primers with sequence common to the previously reported NBC4 splice variants (23, 24) were used to identify new NBC4 clones: sense, 5′-CAGACCAGCCACAGCAGGAACTG-3′ (535); antisense, 5′-GTGCTGCTGGATTTGGACAGTGG-3′ (867). The primer positions refer to NBC4c. Vector-derived 5′ and 3′ primers were used to identify clones with the longest inserts. Positive clones were verified by sequencing. An ∼6.3 kb-clone was obtained from human testes that contained the coding region of a new splice variant of NBC4, which was named NBC4c. The predicted amino acid sequence of NBC4c is shown in Fig.1. The NBC4c nucleotide sequence has been submitted to GenBank/EMBL Data Bank with the accession number AF293337. The 5′ end of the coding sequence for this clone was confirmed by 5′ rapid amplification of cDNA ends (RACE) PCR amplification. Furthermore, to confirm that the NBC4c amino acid sequence was derived from a bona fide transcript, we amplified the entire open reading frame of human NBC4c by RT-PCR with Marathon Ready cDNA prepared from human testes (Clontech, Palo Alto, CA) as a template. Nucleotide sequences were determined bidirectionally by automated sequencing (ABI 310, Perkin-Elmer, Foster City, CA) using Taq polymerase (Ampli-Taq FS, Perkin-Elmer). Sequence assembly and analysis were carried out with Geneworks software (Oxford, UK). The alignment of human NBC4c with human NBC1 and NBC3 is shown in Fig. 1.
RT-PCR amplification of NBC4c.
In initial experiments using Northern blot analysis of human tissues and various probes specific to the 3′-untranslated region (UTR) of NBC4c, we were unable to detect specific labeling of NBC4c because of either low abundance or mRNA instability. Therefore, a PCR approach was used in which primers specific for NBC4c were used for amplification of cDNA obtained from various tissues. The following primers used in the PCR reactions were specific for the 3′-UTR of NBC4c and did not have any sequence homology to other NBC transporters: sense, 5′-CACCTTGCACTTCAAAATATCCTGTCCAG-3′ (3750–3778); antisense, 5′-GTTCAAACTTTTCATATAACCCTTAGGAAATTG-3′ (4402–4434). Controls were negative, and all PCR products were confirmed by sequencing.
Functional characterization of NBC4c.
HEK293-T cells were grown on fibronectin-coated coverslips and were transiently transfected by the calcium phosphate precipitation method with a pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA) containing the coding region for NBC4c. Mock-transfected cells were transfected with the vector alone. The plasmids were purified with the Endofree plasmid purification kit (Qiagen, Valencia, CA) before their use. Functional studies were performed 24 h after transfection. pHiwas monitored with the fluorescent probe 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and a microfluorometer coupled to the microscope (19). Data was obtained from ∼20 cells per coverslip. Calibration of intracellular BCECF was performed at the end of every experiment by monitoring the 500- to 440-nm fluorescence excitation ratio at various values of pHi in the presence of high-K+nigericin standards (30). The following experimental protocols were performed. 1) For Na+addition/removal in Cl−-containing HCO -buffered solutions, the cells were initially bathed for 25 min in a Na+-free Cl−-containing HEPES-buffered solution (solution A, Table1). The cells were acutely acidified to ∼pHi 6.5 by exposure to HCO -buffered Na+-free Cl−-containing solution (solution B, Table 1). The cells were then exposed to a HCO -buffered Na+- and Cl−-containing solution (solution C, Table 1) followed by subsequent Na+ removal (solution B). Additional experiments were performed in cells bathed in DIDS (1 mM) or ethylisopropylamiloride (EIPA; 50 μM). 2) For Na+ addition/removal in Na+-free and Cl−-free HCO -buffered solutions, the cells were bathed in a Na+-containing Cl−-free HEPES-buffered solution (solution D, Table 1) for 1 h before experimentation. The cells were then exposed to a Na+- and Cl−-free HEPES-buffered solution (solution E, Table 1) for 25 min. The cells were acutely acidified to ∼pHi 6.5 by exposure to a HCO -buffered Na+- and Cl-−free solution (solution F, Table 1). A Na+-containing Cl−-free solution was then added (solution G, Table 1), followed by the removal of Na+ (solution F). 3) For Na+ removal/addition in Cl−-containing HEPES-buffered solutions, the cells were initially bathed in a Na+-free Cl−-containing HEPES-buffered solution (solution A) for 25 min. The cells were acutely acidified to ∼pHi 6.5 by a brief exposure to and subsequent removal of a solution containing 30 mM NH4Cl (replacing 30 mM tetramethylammonium chloride; solution H, Table 1). A HEPES-buffered Na+- and Cl−-containing solution was then added (solution I, Table 1), followed by the subsequent removal of Na+(solution A).
Electrogenicity of NBC4c.
The methodology to measure the current through electrogenic Na+-HCO cotransporters in mammalian cell monolayers with an Ussing chamber was described previously (13,14). HEK293-T cells were not suitable for these measurements because they do not form a high-resistance monolayer. The mPCT renal cell line (13), which lacks endogenous electrogenic Na+-HCO cotransport function and forms a high-resistance confluent monolayer (transepithelial resistance ≥ 1,000 Ωcm2) when grown on filter inserts, was used to characterize the electrogenicity of NBC4. The cells were grown on filter inserts in mRTE medium (1:1 mixture of DMEM and Ham's F-12 medium and the following additives: 10 ng/ml epidermal growth factor, 5 μg/ml insulin, 5 μg/ml transferrin, 4 μg/ml dexamethasone, 10 U/ml interferon γ, 2 mM glutamine, and 5% fetal bovine serum). The cells were then transiently transfected with a pcDNA3.1 plasmid (Invitrogen) containing the coding region for NBC4c with Effectene (Qiagen) per the manufacturer's protocol. Control (mock-transfected cells) were transfected with the vector alone. All plasmids were purified with the Endofree plasmid purification kit (Qiagen) before use. Confluent cells grown on permeable filter supports (0.4 μm; Millipore, Bedford, MA) were mounted vertically in a thermostated Ussing chamber equipped with gas inlets for CO2 bubbling. Functional studies were performed 48 h after transfection. The cells were permeabilized apically with 10 μM amphotericin B to remove the electrical resistance of the apical membrane (4, 7, 16-18). The application of a Na+ gradient across the epithelial cell monolayer in cells expressing NBC4c in the presence of HCO generates two currents with opposite signs. The absolute sign of each current in the Ussing chamber depends on which compartment, basolateral or apical, is connected to the ground electrode. In the experiments shown in Fig.5, the apical chamber was connected to ground. In this case, the short-circuit current generated by the flux of Na+ through the paracellular shunt pathway (I Na) has a negative sign. In NBC4c-expressing cells, but not in mock-transfected cells, an additional positive short-circuit current generated by the flux of Na+ and HCO through NBC4c is also present (I NaHCO3). The resultant net baseline short-circuit current in NBC4c expressing cells is the vectorial sum of the two short-circuit currents: INaHCO3 + I Na. The net baseline short-circuit current before the addition of DIDS depends on both the magnitude of the paracellular pathway Na+conductance and NBC4c conductance. The net baseline short-circuit current is not identical in each monolayer because of slight differences in the paracellular Na+ conductance between monolayers and the level of expression of NBC4c. We therefore offset the net baseline short-circuit current to zero. When DIDS is added to the basolateral solution of NBC4c-expressing cells in the presence of both a Na+ gradient and HCO , the net short-circuit current becomes negative because the positive short-circuit current generated by the flux of Na+ and HCO through NBC4c is blocked by basolateral DIDS, leaving only the negative short-circuit current generated by the flux of Na+ through the paracellular shunt pathway (I Na). The DIDS-sensitive short-circuit current, i.e., the net current through NBC4c, is thus obtained by subtracting the short-circuit current measured in the presence of DIDS from that measured in the absence of DIDS Equation 1Only transfected cells for which the DIDS-sensitive current was at least 10-fold larger then that of the corresponding mock-transfected cells were used; 30% of cell monolayers met this criterion. The stoichiometry of the cotransporter was determined from its reversal potential (E rev) and Eq. 2 as described previously (13, 14, 16) Equation 2where n is the number of bicarbonate anions cotransported with each sodium cation, the subscripts i and o represent intra- and extracellular concentrations, respectively, of the indicated ion, R is the gas constant, T is temperature, andF is the Faraday constant. For a symmetrical HCO concentration, the ratio [HCO ] /[HCO ] equals 1 and E rev depends logarithmically only on the magnitude and direction of the Na+ concentration gradient. E rev of the cotransporter was measured for several different Na+ concentration gradients while keeping HCO concentrations symmetrical across the basolateral membrane. Initially, a fivefold Na+concentration gradient was applied across the monolayer by perfusing the basolateral compartment with a Cl−-free HCO -buffered solution containing 50 mM Na+ (solution J, Table 1) and the apical compartment with a Cl−-free HCO -buffered solution containing 10 mM Na+ (solution K, Table 1). In separate experiments, a twofold Na+ concentration gradient was used by replacing solution J in the basolateral compartment with a solution containing 20 mM Na+ (solution L, Table 1). All solutions were Cl− free. To measureE rev
Results are reported as means ± SE. Unpaired Student'st-test and linear regression analyses were used as required. Dunnett's t-test was used when more than one experimental group was compared with a control group.
Sequence alignment and tissue distribution.
The amino acid sequence of the NBC4c clone used for functional analysis in this study and its alignment with human NBC1 and NBC3 are shown in Fig. 1. NBC4c is 56% identical to electrogenic NBC1 protein variants and 40% identical to electroneutral NBC3 at the amino acid level. Therefore, NBC4c has a higher homology with electrogenic than with electroneutral Na+-HCO cotransporters. NBC4c transcripts are found in several tissues (Fig.2).
In Na+- and Cl−-containing HCO -buffered solutions, resting pHi was 6.88 ± 0.06 (n = 6) in mock-transfected control HEK293-T cells and 6.87 ± 0.04 (n = 7) in NBC4c-transfected cells [P = not significant (NS)]. The cells were initially bathed in Na+-free HEPES-buffered solutions (pH 7.4). When the cells were acutely acidified by the addition of Na+-free CO2/HCO -buffered solutions (pH 7.4), pHi failed to recover in the absence of external Na+ (Fig. 3). After the addition of Na+ (140 mM), pHi recovered slowly at a rate of 0.22 ± 0.03 pH units/min (n = 5) in mock-transfected HEK293-T cells (Fig. 3 A). In NBC4c-transfected cells, pHi recovered at a significantly faster rate of 1.00 ± 0.08 pH units/min (n = 8;P < 0.001; Fig. 3 B). Further experiments were done to determine the Cl− dependence of the Na+-induced H+/base flux in NBC4c-transfected cells. As shown in a typical experiment in Fig. 3 C, the Na+-induced pHi recovery was Cl−independent. To determine the HCO dependence of NBC4c-mediated transport, separate studies were done in the nominal absence of HCO in HEPES-buffered solutions utilizing the NH4 + prepulse technique to acidify the cells acutely. After the addition of Na+ (140 mM) in mock-transfected HEK293-T cells, pHi recovered slowly at a rate of 0.17 ± 0.02 pH units/min (n = 5). In NBC4c-transfected cells (Fig. 3 D), pHi recovered at a rate that was not significantly different from that in mock-transfected cells [0.19 ± 0.02 pH units/min (n = 6); P = NS]. These results indicate that NBC4c has an absolute requirement for Na+ and HCO . In separate experiments, the effect of DIDS (Fig. 3 E) and EIPA (Fig. 3 F) on NBC4c-mediated transport was studied. In NBC4c-transfected cells, in HCO -buffered solutions DIDS (1 mM) significantly inhibited the Na+-induced HCO -dependent pHi transients [0.13 ± 0.03 pH units/min (n = 5); P < 0.001]. In HCO -buffered solutions, EIPA (50 μM) decreased the rate of the Na+-dependent pHi recovery in mock-transfected cells from 0.22 ± 0.03 (n = 5) to 0.06 ± 0.01 (n = 7) pH units/min (P < 0.01). In NBC4c-transfected cells, the magnitude of the inhibition was similar in that EIPA (50 μM) decreased the rate of pHi recovery from 1.00 ± 0.08 (n = 8) to 0.75 ± 0.06 (n = 10) pH units/min (P < 0.02). Figure 4depicts the calculated Na+-dependent equivalent base fluxes in mock-transfected and NBC4c-transfected cells.
Electrogenicity of NBC4c.
To determine whether NBC4c is electrogenic, we measured the stilbene-inhibitable Na+- and HCO -dependent short-circuit current in mPCT cells tranfected with the transporter. The short-circuit current generated by application of a Na+ concentration gradient across apically permeabilized mPCT cells transfected with NBC4c was measured in an Ussing chamber. In the presence of HCO (25 mM), a fivefold Na+ concentration gradient generated an average DIDS-sensitive short-circuit current of 3.4 ± 0.6 μA/cm2 (n = 6; Fig.5 A) compared with 0.4 ± 0.2 μA/cm2 (n = 6) for mock-transfected monolayers (not shown). In the nominal absence of CO2/HCO , application of a fivefold Na+ gradient across monolayers of NBC4c-transfected cells generated an average DIDS-sensitive short-circuit current of 0.2 ± 0.1 μA/cm2 (n = 6; Fig.5 B). These results suggested that the electrogenic flux of Na+ through NBC4c is coupled to that of HCO and that the coupling ratio is at least 2 HCO per 1 Na+. To determine the coupling ratio (i.e, stoichiometry), we measured E rev in NBC4c-transfected cells from which the stoichiometry (n) was calculated as described in methods (see Eq. 2). Current-voltage relationships of NBC4c were measured at two different Na+ concentration gradients as shown in Fig.6. The E rev data indicate that the HCO :Na+ stoichiometry of NBC4c was ∼3:1 (Table 2).
In the present study, we have determined the transport properties and electrogenicity of a new member of the NBC family, NBC4. We have shown that the NBC4c splice variant is an electrogenic Na+-HCO cotransporter. The transport of Na+ and HCO is Cl−independent and is completely stilbene sensitive. NBC4c transcripts were detected in several tissues including brain, heart, kidney, testis, pancreas, muscle, and peripheral blood leukocytes, indicating a potentially widespread and functionally important role for this transporter in mediating electrogenic Na+-HCO cotransport.
Before this study, products of NBC1 were the only proteins known to mediate electrogenic Na+-HCO cotransport (3). Comparison of the amino acid sequence of NBC4c with other members of the BTS demonstrates that NBC4c has the highest similarity to the NBC1 electrogenic Na+-HCO cotransporter proteins. However, a detailed comparison of the amino acid sequence of NBC1 and NBC4c does not reveal obvious structural motifs unique to these proteins that may be responsible for conferring electrogenic Na+-HCO cotransport. These findings suggest that the three-dimensional structure of these transporters and/or regulatory factors may determine the electrogenic nature and transport stoichiometry of NBC proteins. Indeed, we have recently shown that the phosphorylation of Ser984 in kNBC1 shifts the HCO :Na+ coupling ratio from 3:1 to 2:1 (15). Whether the HCO :Na+coupling ratio of NBC4c and other NBC proteins can be similarly regulated is currently unknown. There is presently a paucity of information regarding the basis for the ion specificity and transport stoichiometry of members of the BTS. The finding that NBC1 and NBC4c proteins are functionally similar provides an additional basis for addressing the minimum structural requirements, which are necessary for a protein to mediate electrogenic Na+-HCO cotransport.
Although NBC1 and NBC4c proteins function as electrogenic Na+-HCO cotransporters, our results demonstrate that NBC4c-mediated transport is strictly HCO dependent. NBC4c had an absolute requirement for HCO because in its absence, the magnitude of the Na+-dependent H+/base fluxes in NBC4c-transfected and control mock-transfected cells were identical and there was no detectable stilbene-inhibitable current through the transporter. When expressed in mammalian cells, kNBC1 has been reported to function in the absence of HCO (6), although not all studies concur with this finding (13). Whereas NBC4c and NBC1 proteins may differ in their HCO dependence, they share the property of being stilbene inhibitable. In addition to these electrogenic NBC proteins, other known members of the BTS are stilbene sensitive with the exception of NBC3 and AE4 (22, 31). The putative stilbene binding motif KLFD (amino acids 742–745) in NBC3 and KMLN (amino acids 518–521) in AE4 may alter their affinity for stilbenes and account for their DIDS insensitivity (22, 31). A comparison of the amino acid sequence of NBC4 with NBC1 and other members of the BTS reveals that the putative stilbene binding motif [K(M/L)(X)K] is lacking in NBC4, which has the sequence KMIG (amino acids 655–658). Therefore, replacement of the highly conserved lysine at position 658 by glycine does not appear to appreciably alter the binding of negatively charged stilbene disulfonates.
Our finding that NBC4c transcripts are expressed in several tissues including brain, heart, kidney, testis, pancreas, muscle, and peripheral blood leukocytes suggests that the transporter may have a housekeeping function in regulating pHi. pNBC1 transcripts have also been detected in many organs, indicating the ubiquitous expression of this transporter (1). The second known NBC1 protein variant, kNBC1, is more restricted in its tissue expression and has thus far been detected in kidney and eye (8, 29). Additional studies of the cellular/subcellular localization in various tissues, transport kinetics, and regulation of these electrogenic Na+-HCO cotransporters are required to address the question as to why in the mammalian genome there are two distinct genes that encode electrogenic Na+-HCO cotransporters. Examples of other proteins in the BTS in which functionally similar transporters are encoded by separate genes are the AE1–4 Na+-independent Cl−-HCO exchangers (5, 31).
The functional characterization of NBC4 is of additional importance given that the disease gene in Alstrom syndrome, a rare multisystemic autosomal recessive disorder characterized by infantile cardiomyopathy, hepatic dysfunction, progressive sensorineural hearing loss, retinopathy, truncal obesity, asthma, diabetes mellitus, and hypogonadism, has been mapped to a 6.1-cM region of chromosome2p13 containing the coding region of NBC4 (11). Given that NBC4 is therefore a candidate gene for this syndrome, further studies are ongoing in patients with this syndrome to determine whether mutations in the coding region of NBC4 exist.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46976, DK-58563, and DK-07789, the Iris and B. Gerald Cantor Foundation, the Max Factor Family Foundation, the Richard and Hinda Rosenthal Foundation, the Fredericka Taubitz Foundation, a Cystic Fibrosis Foundation grant Gross01G0, and American Heart Association Grant 9706507. A. N. Abuladze is supported by National Kidney Foundation of Southern California Training Grant J891002.
Address for reprint requests and other correspondence: I. Kurtz, UCLA Division of Nephrology, 10833 Le Conte Ave., Rm. 7–155 Factor Bldg., Los Angeles, CA 90095-1689 (E-mail:).
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- Copyright © 2002 the American Physiological Society