Identification of a Carboxyl-terminal Motif Essential for the Targeting of Na -HCO3 Cotransporter NBC1 to the Basolateral Membrane*

The Na -HCO3 cotransporter NBC1 is located exclusively on the basolateral membrane and mediates vectorial transport of bicarbonate in a number of epithelia, including kidney and pancreas. To identify the motifs that direct the targeting of kidney NBC1 to basolateral membrane, wild type and various carboxyl-terminally truncated kidney NBC1 mutants were generated, fused translationally in-frame to GFP, and transiently expressed in kidney epithelial cells. GFP was linked to the NH2 terminus of NBC1, and labeling was examined by confocal microscopy. Full-length (1035 aa) and mutants with the deletion of 3 or 20 amino acids from the COOHterminal end of NBC1 (lengths 1032 and 1015 aa, respectively) showed strong and exclusive targeting on the basolateral membrane. However, the deletion of 26 amino acid residues from the COOH-terminal end (length 1010 aa) resulted in retargeting of NBC1 to the apical membrane. Expression studies in oocytes demonstrated that the NBC1 mutant with the deletion of 26 amino acid residues from the COOH-terminal end is functional. Additionally, the deletion of the last 23 amino acids or mutation in the conserved residue Phe at position 1013 on the COOH-terminal end demonstrated retargeting to the apical membrane. We propose that a carboxyl-terminal motif with the sequence QQPFLS, which spans amino acid residues 1010–1015, and specifically the amino acid residue Phe (position 1013) are essential for the exclusive targeting of NBC1 to the basolateral membrane.


Introduction
The Na + :HCO 3 -contransporter NBC1 mediated bicarbonate transport and is located on the basolateral membrane of kidney proximal tubule and a number of other epithelia including pancreatic duct, gastric parietal cells, small intestine, and pulmonary alveolar cells (1)(2)(3)(4)(5)(6).NBC1 functions in tandem with apical Na + /H + exchanger 3 (NHE3) and is essential for bicarbonate reabsorption in kidney proximal tubule (4,7,8).In pancreas, NBC1 is responsible for bicarbonate transport into pancreatic duct for eventual secretion into the pancreatic duct (4,9,10).Two well known variants of NBC1 are expressed in various epithelial tissues.These are the kidney (kNBC1) and pancreatic (pNBC1) variants, which differ only in their N-terminal sequence, where amino acids 1 to 41 in kNBC1 has been replaced with 85 amino acids in pNBC1 (3).The carboxyl terminal end of NBC1 is identical in kidney and pancreatic variants (1)(2)(3).Kidney NBC1 has a stoichiometry of 3 equivalent of bicarbonate for each sodium whereas pancreatic NBC1 has a stochiometry of 2 equivalents of bicarbonate per each sodium (11,12).This alteration in stochiometry allows for the kidney NBC1 to function in the absorptive mode and for the pancreatic NBC1 to function in the secretory mode (4,(9)(10)(11)(12).
In all tissues examined to date NBC1 is found to be located on the basolateral membrane of epithelial cells (4-6) strongly suggesting the presence of specific amino acid motif(s) that direct its targeting to the basolateral membrane.To identify possible motifs that are responsible for the targeting of NBC1 to the basolateral membrane, various Cterminally truncated NBC1 mutants were generated, fused translationally in-frame to GFP and transiently expressed in cultured kidney epithelial cells.The results demonstrate that deletion of amino acid residues 1010 to 1015, with the sequence QQPFLS, results in the retargeting of NBC1 preferentially to the apical membrane.Site directed mutagenesis of the conserved amino acid residue F (phenylalanine) at 1013 within this domain recapitulates the retargeting of NBC1 to the basolateral membrane.The sequence QQPFLS is preserved in NBC1 from various species and also in a number of transporters, especially the F (phenylalanine) among the sequence is very conservative in NBC1 protein from species to species and they may play an important role in basolateral targeting of these transport proteins.

Functional expression of full length GFP-NBC1 and GFP-NBC1 mutants in oocytes.
Stage IV-V oocytes were isolated as previously described and used for expression studies according to established methods (15).The capped GFP-NBC1 (full length), GFP-NBC1

Results
In the first series of experiments we examined the expression and targeting of GFP with or without the NBC1 insert in MDCK cells.The next series of experiments examined the expression of the GFP-NBC1 mutant that lacked the last 26 amino acids of NBC1 carboxyl terminal end (CD26).Transfection studies were performed as in the above.The Z-stack analysis (front view) of the expression of GFP-NBC1CD26, co-labeled with phalloidin, shows strong membrane as well as sub-membrane labeling with the GFP (Fig. 4a).The Z-line (side view) image analysis of the results show the surprising and unexpected finding of apical membrane targeting of NBC1 (Fig. 4b and 4c).Experiments were performed with both phalloidin (4b) and PNA-lectin binding (4c).Both methods clearly confirm that deletion of the last 26 amino acids from the C-terminal end causes the mistargeting of NBC1 to the apical membrane (4b and 4c).Some images show residual labeling of the basolateral membrane (4b and 4c) but clearly the majority of the labeling is localized on the apical membrane with little cytoplasmic expression.
To determine whether further truncation of the carboxyl terminal end affects its membrane localization, NBC1 mutants with 50 amino acid residues deletion were generated, coupled to GFP and transiently expressed in MDCK cells.Figure 5a shows transfection of MDCK cells with the NBC1-CD50 mutant and indicates that GFP-NBC1 CD50 localized on the cell membrane as well as in the cytoplasm.As demonstrated in In the next series of experiments we examined the functional activity of NBC1-GFP CD26 mutant in oocytes by membrane potential measurement (1,2,14).For comparison, oocytes injected with GFP-NBC1 full length or GFP only (no NBC insert) cRNA were utilized.We were specifically interested in NBC1-CD26, as this is the mutant with the shortest truncation, which shows mistargeting to the apical membrane (Fig. 4).As demonstrated (Fig. 6, bottom panel, representative tracing), exposure to CO 2 /HCO 3 -in oocytes expressing the full length GFP-NBC1 (2-4 days after cRNA injection, 0.5 µg/µl) resulted in an immediate membrane hyperpolarization, which reached a peak within 1 min.The hyperpolarization showed very little decay during the time of exposure to CO 2 /HCO 3 -and was reversible upon returning to the CO 2 /HCO 3 --free perfusion solution.This is consistent with published reports and indicates that NBC1 is highly electrogenic (1,2,15).

Discussion
The targeting of NBC1 to the basolateral membrane was examined using a series of mutants with progressive truncation of the carboxyl terminal end that were fused in frame to GFP and visualized by confocal microscope.The results demonstrated that the full length NBC1 and mutants with up to 20 amino acid deletion from the carboxyl terminal end are targeted to the basolateral membrane (Figs.1-3).However, the NBC1 mutant with 26 amino acids deletion from its C-terminal end showed mistargeting to the apical membrane domain, with some labeling observed on the basolateral membrane (Fig. 4).
Additional truncations for up to 67 amino acid residues showed persistence of labeling on the apical membrane but also resulted in accumulation of mutant proteins in the cytoplasm (Figs.NBC1 is located basolaterally in various epithelia and mediates vectroial transport of bicarbonate.In kidney, NBC1 is located on the basolateral membrane of proximal tubules and mediates the exit of bicarbonate from cell to blood (4-6, 17, 18).In pancreas, NBC1 is located on the basolateral membrane of ducts and mediates the entry of bicarbonate from blood to cell (4-6, 19, 20).Studies on NBC1 have demonstrated that the kidney variant (kNBC1) has a stoichiometry of 3 equivalent of bicarbonate per sodium whereas the pancreatic variant (pNBC1) has a stoichiometry of 2 bicarbonate per sodium (11,12).
The difference in the stoichiometry allows for the change in the direction of NBC1 movement from absorptive (i.e. in kidney) to secretory (i.e. in pancreatic duct).
The most salient feature of the current study is the identification of a carboxyl terminal motif and residues that are essential for the targeting of NBC1 to the basolateral membrane.It is noteworthy that in the absence of this motif (CD-23 or CD-26 amino acid deletion) or in mutants in which the conserved residue F (phenylalanine at position 1013) is mutated, NBC1 is predominantly mistargeted to the apical membrane rather than being accumulated in the cytoplasm.This observation raises the possibility that other existing motifs may be responsible for the targeting of NBC1 to the apical membrane.
Alternatively it is plausible that the mistargeting of NBC1 to the apical membrane occurs by default (as a result of the deletion of basolateral targeting domain).Further deletion of the carboxyl terminal end beyond 26 amino acids (up to 67 a.a.residues) resulted in more intra-cytoplasmic accumulation of NBC1 (Fig. 5 and personal observations), indicating that the carboxyl terminal end may play important role in membrane targeting of NBC1.
The apically targeted NBC1 is functionally active (Fig. 6), raising the likelihood that possible mutations or deletions in the identified motif (QQPFLS) in human, do not impair its functional activity.However such a mutation or deletion may cause significant impairment in bicarbonate reabsorption due to the absence of an exit pathway for bicarbonate transport in the basolateral membrane.In other words, the mistargeting of NBC1 to the apical membrane may impair the vectroial transport of bicarbonate and lead to proximal tubular acidosis.Whether motifs on the amino terminal end of NBC1 play any role in basolateral membrane targeting of NBC1 remains speculative at the present and warrants detailed examination.
Recent studies have identified mutations in the Cl -/HCO 3 -exchanger AE1, which are associated with distal renal tubular acidosis, a bicarbonate wasting condition.AE1 is normally located on the basolateral membrane of alpha(A) intercalated cells in cortical and outer medullary collecting duct and, in tandem with apical H+-ATPase, is responsible for majority of bicarbonate reabsorption in these nephron segments (21,22).
Bicarbonate wasting in these subjects raised the possibility that AE1 mutants either became functionally inactive or were retained intracellularly.However, studies with AE1 mutants have been less conclusive (23)(24)(25)(26) casting doubt on the explanations regarding bicarbonate wasting in humans with AE1 mutations.Very recent studies have deciphered this puzzle (16,27).In studies in cultured polarized kidney epithelial cells, Dennovald et al and Rungroj et al showed that AE1 mutants tagged with GFP are mistargeted to the apical membrane (16,27).Given the functionality of AE1 mutants in non epithelial cells, Denovald et al and Rungroj et al concluded that the mistargeting of AE1 to the apical membrane may actually worsen the bicarbonate wasting, as the apically located AE1 secretes bicarbonate into the lumen in exchange for luminal chloride (16,27).This should lead to renal bicarbonate wasting and worsen distal renal tubular acidosis.It is worth mentioning that other mutations cause the retention of AE1 in the ER or in late endosomes/lysosomes therefore causing distal renal tubular acidosis by reducing bicarbonate exit across the basolateral membrane of the collecting duct (25,28).
A Genbank search of other members of NBC/AE superfamily demonstrated that a motif with high homology to the one identified in our studies is also expressed in NBC2, NBC4, NCBE, AE1 and AE2, all known bicarbonate transporters located on the basolateral membrane of epithelia.An analysis of other known chloride/bicarbonate or anion exchangers from SLC26 family which share little homology with NBC/AE superfamily demonstrates that those anion exchangers that are targeted to the basolateral membrane (SLC26A1 and SLC26A7) contain motifs with high homology to the current motif (Genbank NM_022042 for A1, from residue 675 to 678, with a.a.sequence QLFL, with full length of 701 a.a. ; Genbank NM_134266 for A7, from residue 343 to346, with a.a.sequence QEFL, with full length of 663 a.a.).However, SLC26 exchangers that are targeted to the apical membrane (SLC26A3 and SLC26A4) do not express any motif with high homology to the current motif.
In conclusion, NBC1 mutants lacking the last carboxyl terminal 23 or mutant in which the conserved residue F1013 mutated into A1013 show mistargeting to the apical membrane.We propose that a carboxyl terminal motif, with the sequence QQPFLS, which spans amino acid residues 1010 to 1015, and specifically the amino acid residue F(phenylalanine) at position 1013, are essential for the exclusive targeting of NBC1 to the basolateral membrane.Future studies should clarify the underlying mechanism of this targeting rearrangement and attempt to identify the molecules that interact with NBC1 through this motif.
Material.Construction of GFP-NBC1 full length and mutants.The full length and various Cterminally truncated NBC1 mutants were generated by PCR, using the human full length NBC1 DNA (3257bp and 1035 a.a residues) as a template (AF007216).The truncated NBC1 proteins have C-terminal deletions of 3, 20, 26, or 50 amino acid residues and are designated as NBC1 CD3, CD20, CD26, or CD50, respectively.The resulting wild type and various C-terminally truncated NBC1 mutants were amplified and fused translationally in-frame to GFP by cloning into pcDNA3.1/NT-GFP-TOPOvector (Invitrogen, Carlsbad, California).Full length kNBC1 was amplified using the following primers: t986730: 5' GAATTCGAGATGGCACTGAAAATGTGGAAGGG (sense) and t968120: 5' AGCGGCCGCCTCAGCATGATGTGTGGCGTTCAAGGAATGT (antisense).These primers were used to generate full length NBC1 cDNA from PCI-NEO NBC1 plasmid(1).This resulted in the expression of GFP-NBC1 fusion protein with the GFP fusing to the N-terminus of kNBC1 (GFP-NBC1).Using a similar approach, primer 1 (sense, (antisense), were used to generate the NBC1-CD3 cDNA.This resulted in the expression of the GFP-NBC1 fusion protein, which coded the truncated NBC1 (CD3), with deletion of the last 3 amino acid residues on the carboxyl terminal end.For CD20, CD26, and CD50 (deletion of the last 20, 26, and 50 amino acid residues, respectively) the following (antisense) and CD50 3' primer---5'TCAGTC ACTGTCCAGACTTCCCTTCTT3'.In separate experiments we also generated NBC1 CD67 construct (67 amino acid deletion) using 3' primer : 5' CTATTTCTTGTCCTTTTCTGGAATGAC3'.For the last series of experiments we generated NBC1 CD23 construct (23 amino acid deletion) using 3' primer : 5' GCGGCCGCTCAAGGTTGCTGTTCCATGATGTCCATTGGAAT 3'.The sense primer in all mutants was the same as in full length NBC1 (above).The diagram shown in Fig.1depicts the schematic constructs of human NBC1 truncated mutants.
Figure1a(left and right panels) shows that transfection of cultured cells with GFP vector alone (no NBC1 insert) results in the accumulation of GFP in the cytoplasm (Z-line images in left panel) with no localization on the membrane (Z.stack images in right panel).However, transfection with GFP-NBC1 full-length cDNA shows exclusive localization of the GFP in the membrane (as visualized by Z-stack, front view) in cells co-labeled with phalloidin (Fig.1b).The specific membrane labeling (apical vs. basolateral) was examined using the Z-line image (side view) analysis.The results are demonstrated in Fig.1c.Similar studies were performed with the GFP-NBC1 construct in MDCK cells co-labeled with PNA-lectin dye, a very specific marker of apical membrane labeling in polarized cells(16).The Zline (side view) images of the results (NBC1-GFP and PNA-lectin double labeling) are depicted in Fig.1d.As demonstrated, the full length NBC1 is targeted exclusively to the basolateral membrane domain (Fig.1c and 1d).These results are consistent with published reports on the basolateral localization of NBC1 in epithelial cells (4-6).In the next series of experiments we examined the expression of GFP-NBC1 mutants with 3 (CD-3) or 20 (CD-20) amino acid deletion from their carboxyl terminal end (Method).Figure2ais a Z-stack (front view) image analysis of the expression of GFP-NBC1 CD3 co-labeled with phalloidin in MDCK cells, showing GFP-NBC1 CD3 localization on the cell membrane.The Z-line (side view) image analysis of the expression studies doubly labeled with phalloidin or PNA-lectin demonstrate that NBC1-CD3 mutant is targeted to the basolateral membranes (2b and 2c).
Figure 3 depicts similar results with NBC1 mutant lacking the last 20 amino acids from the C-terminal end.The Z-stack (front view) image analysis of the expression of GFP-NBC1 CD20 colabeled with phalloidin (3a) is shown in MDCK cells and demonstrates exclusive localization on the cell membrane.The Z-line image analysis (side view) of the expression studies demonstrates that NBC1-CD20 mutant is targeted to the basolateral membrane (3b and 3c).

( 2 -
4 days after cRNA injection, 0.5 µg/µl) also caused significant membrane hyperpolarization (Fig.6, middle panel, representative tracing).Control oocytes(GFP   only)  showed no alteration in membrane potential measurement in response to CO 2 /HCO 3 -exposure (Fig. 6, top panel, representative tracing).The summary results of the effect of CO 2 /HCO 3 -on membrane potential were -114.6 ± 6.0 mV in full length GFP-NBC1-injected oocytes (n=5), -106.1 ± 5.6 mV in GFP-NBC1 CD26-injected oocytes (n=6) and -62 ± 3.8 mV in GFP alone injected oocytes (n=5).The baseline membrane potentials (before exposure to CO 2 /HCO 3 -) were -49.4 ± 3.2 mV in full length GFP-NBC1-injected oocytes, -53.2 ± 4.1 mV in GFP-NBC1 CD26 -injected oocytes, and -50.9 ± 4.2 mV in GFP-only injected oocytes.As indicated, oocytes injected with either the full length or the mutant NBC1 showed significant hyperpolarization (p<0.01 vs. no CO 2 /HCO 3 -) whereas GFP-only injected oocytes did not show any alteration in membrane potential in response to CO 2 /HCO 3 -exposure.The magnitude of hyperpolarization in response to CO 2 /HCO 3 -exposure was not different between the full length and the CD26 truncated mutant (p>0.05).Taken together, these results demonstrate that the GFP-NBC1 mutant with the deletion of the last 26 carboxyl terminal amino acid residues is functionally active.In the last series of experiments we examined the membrane targeting of NBC1-CD23 and mutants in which the conserved residue at position 1013 on the C-terminal end was mutated (F1013A) by site directed mutagenesis.As demonstrated in Figure 7 (panel A), deletion of the last 23 amino acid residues causes the retargeting of NBC1 to the apical membrane, indicating that the sequence FLS (residues 1013-1015) is essential for the targeting of NBC1 to the basolateral membrane.To identify the amino acid residue(s) responsible for the exclusive targeting of NBC1 to the basolateral membrane, mutants in which the conserved residue F was mutated (F1013A) were used for transfection in MDCK cells.As demonstrated in Fig. 7 (panel B), the F1013A mutation caused the retargeting of NBC1 to the apical membrane.
5 and personal observation).Membrane potential recording in oocytes demonstrated that the NBC1 mutant with 26 amino acid residues deletion mediates Na + :HCO 3 -cotransport (Fig. 6), indicating that the apically targeted molecule is functionally active.Additionally, truncation of the last 23 amino acids or mutants in which the conserved residue F (phenylalanine) at position 1013 on the C-terminal end was mutated by site directed mutagenesis (F1013A) demonstrated retargeting to the apical membrane.We further find other truncated mutants (upto 67 amino acid deletion) are functional, though at a much smaller rate, when expressed in non-GFP forms and assayed by BCECF (data not shown).

Figure 6 . 3 -.
Figure 6.Membrane potential recordings in oocytes injected with full length or