Dynamic localization of the Na+-HCO3- co-transporter NBCn1 to the plasma membrane, centrosomes, spindle and primary cilia.

Finely tuned regulation of transport protein localization is vital for epithelial function. The Na+-HCO3- co-transporter NBCn1 (also known as SLC4A7) is a key contributor to epithelial pH homeostasis, yet the regulation of its subcellular localization is not understood. Here, we show that a predicted N-terminal ß-sheet and short C-terminal α-helical motif are essential for NBCn1 plasma membrane localization in epithelial cells. This localization was abolished by cell-cell contact disruption, and co-immunoprecipitation (co-IP) and proximity ligation (PLA) revealed NBCn1 interaction with E-cadherin and DLG1, linking it to adherens junctions and the Scribble complex. NBCn1 also interacted with RhoA and localized to lamellipodia and filopodia in migrating cells. Finally, analysis of native and GFP-tagged NBCn1 localization, subcellular fractionation, co-IP with Arl13B and CEP164, and PLA of NBCn1 and tubulin in mitotic spindles led to the surprising conclusion that NBCn1 additionally localizes to centrosomes and primary cilia in non-dividing, polarized epithelial cells, and to the spindle, centrosomes and midbodies during mitosis. We propose that NBCn1 traffics between lateral junctions, the leading edge and cell division machinery in Rab11 endosomes, adding new insight to the role of NBCn1 in cell cycle progression.

Expression of sodium-dependent dicarboxylate transporter 1 (NaDC1/SLC13A2) in normal and neoplastic human kidney.

Regulated dicarboxylate transport is critical for acid-base homeostasis, prevention of calcium nephrolithiasis, regulation of collecting duct sodium chloride transport, and the regulation of blood pressure. Although luminal dicarboxylate reabsorption via NaDC1 (SLC13A2) is believed to be the primary mechanism regulating renal dicarboxylate transport, the specific localization of NaDC1 in the human kidney is currently unknown. This study's purpose was to determine NaDC1's expression in normal and neoplastic human kidneys. Immunoblot analysis demonstrated NaDC1 expression with an apparent molecular weight of ~61 kDa. Immunohistochemistry showed apical NaDC1 immunolabel in the proximal tubule of normal human kidney tissue; well-preserved proximal tubule brush border was clearly labeled. Apical NaDC1 expression was evident throughout the entire proximal tubule, including the initial proximal convoluted tubule, as identified by origination from the glomerular tuft, and extending through the terminal of the proximal tubule, the proximal straight tubule in the outer medulla. We confirmed proximal tubule localization by colocalization with the proximal tubule specific protein, NBCe1. NaDC1 immunolabel was not detected other than in the proximal tubule. In addition, NaDC1 immunolabel was not detected in tumors of presumed proximal tubule origin, clear cell and papillary renal cell carcinoma, or in tumors of nonproximal tubule origin, oncocytoma and chromophobe carcinoma. In summary, 1) in the human kidney, apical NaDC1 immunolabel is present throughout the entire proximal tubule, and is not detectable in other renal cells; and 2) NaDC1 immunolabel is not present in renal tumors. These studies provide important information regarding NaDC1's role in human dicarboxylate metabolism.

Immunocytochemical identification of electroneutral Na⁺-coupled HCO3⁻ transporters in freshly dissociated mouse medullary raphé neurons.

The medullary raphé (MR) of the medulla oblongata contains chemosensitive neurons that respond to increases in arterial [CO2], by altering firing rate, with increases being associated with serotonergic (5-hydroxytryptamine [5HT]) neurons and decreases, with GABAergic neurons. Both types of neurons contribute to increased alveolar ventilation. Decreases in intracellular pH are thought to link the rise in [CO2] to increased ventilation. Because electroneutral Na(+)-coupled HCO3(-) transporters (nNCBTs), which help protect cells from intracellular acidosis, are expressed robustly in the neurons of the central nervous system, a key question is whether these transporters are present in chemosensitive neurons. Therefore, we used an immunocytochemistry approach to identify neurons (using a microtubule associated protein-2 monoclonal antibody) and specifically 5HT neurons (TPH monoclonal antibody) or GABAergic neurons (GAD2 monoclonal antibody) in freshly dissociated cells from the mouse MR. We also co-labeled with polyclonal antibodies against the three nNCBTs: NBCn1, NDCBE, and NBCn2. We exploited ePet-EYFP (enhanced yellow fluorescent protein) mice (with EYFP-labeled 5HT neurons) as well as mice genetically deficient in each of the three nNCBTs. Quantitative image analysis distinguished positively stained cells from background signals. We found that >80% of GAD2(+) cells also were positive for NDCBE, and >90% of the TPH(+) and GAD2(+) cells were positive for the other nNCBTs. Assuming that the transporters are independently distributed among neurons, we can conclude that virtually all chemosensitive MR neurons contain at least one nNCBT.

Clock genes show circadian rhythms in salivary glands.

Circadian rhythms are endogenous self-sustained oscillations with 24-hour periods that regulate diverse physiological and metabolic processes through complex gene regulation by "clock" transcription factors. The oral cavity is bathed by saliva, and its amount and content are modified within regular daily intervals. The clock mechanisms that control salivary production remain unclear. Our objective was to evaluate the expression and periodicity of clock genes in salivary glands. Real-time quantitative RT-PCR, in situ hybridization, and immunohistochemistry were performed to show circadian mRNA and protein expression and localization of key clock genes (Bmal1, Clock, Per1, and Per2), ion and aqua channel genes (Ae2a, Car2, and Aqp5), and salivary gland markers. Clock gene mRNAs and clock proteins were found differentially expressed in the serous acini and duct cells of all major salivary glands. The expression levels of clock genes and Aqp5 showed regular oscillatory patterns under both light/dark and complete-dark conditions. Bmla1 overexpression resulted in increased Aqp5 expression levels. Analysis of our data suggests that salivary glands have a peripheral clock mechanism that functions both in normal light/dark conditions and in the absence of light. This finding may increase our understanding of the control mechanisms of salivary content and flow.

Responses of gill mitochondria-rich cells in Mozambique tilapia exposed to acidic environments (pH 4.0) in combination with different salinities.

On exposure to hyposmotic acidic water, teleost fish suffer from decreases in blood osmolality and pH, and consequently activate osmoregulatory and acid-base regulatory mechanisms to restore disturbed ion and acid-base balances. In Mozambique tilapia Oreochromis mossambicus exposed to acidic (pH 4.0) or neutral (pH 7.4-7.7) freshwater in combination with 0mM or 50mM NaCl, we examined functional and morphological changes in gill mitochondria-rich (MR) cells. We assessed gene expression of Na(+)/H(+) exchanger-3 (NHE3), Na(+)/Cl(-) cotransporter (NCC), vacuolar-type H(+)-ATPase (V-ATPase) and Na(+)/HCO(3)(-) cotransporter-1 (NBC1) in the gills. The mRNA expression of NHE3 and NCC in tilapia gills were higher in acidic freshwater than in that supplemented with 50mM NaCl, while there was no significant difference in mRNA levels of V-ATPase and NBC1. In addition, immunocytochemical observations showed that apical-NHE3 MR cells were enlarged, and frequently formed multicellular complexes with developed deep apical openings in acidic freshwater with 0mM and 50mM NaCl. These findings suggest that gill MR cells respond to external salinity and pH treatments, by parallel manipulation of osmoregulatory and acid-base regulatory mechanisms.

Variation in a bicarbonate co-transporter gene family member SLC4A7 is associated with propensity to addictions: a study using fine-mapping and three samples.

AIMS: Classical genetic studies consistently reveal substantial heritability for addictions. However, the genes that harbour the variations providing these genetic influences remain largely unknown. We have focused attention on 'reproducible substance abuse vulnerability' (rSA) genomic regions, where linkage and association studies performed in several population provide evidence for such variations. DESIGN: We nominated rSA1 on human chromosome 3p23 within a 5 Mb region. We sought to replicate this finding and identify variations within this region. SETTING: We examine the role of allelic variations in the SLC4A7 gene, a member of the bicarbonate co-transporter family that is expressed in tissues including brain and kidney. PARTICIPANTS: A total of 1158 unrelated individuals with informed consent about the genetic study were recruited from three independent populations. MEASUREMENTS: The single nucleotide polymorphism (SNP) markers in the SLC4A7 gene were analysed by case-control study. FINDINGS: The rs3278 is associated reliably with substance abuse vulnerability in (1) a European American sample selected from pedigrees within the Collaborative Study on the Genetics of Alcoholism (COGA; nominal P = 0.03); (2) an African American sample recruited by the National Institute on Drug Abuse (NIDA; nominal P = 0.008); and (3) a NIDA European American sample (P = 0.001). CONCLUSIONS: While the current results do not exclude additional roles for allelic variants in nearby genes, they do suggest that SLC4A7 allelic variants might alter dispositions and/or excretion of drugs and neurotransmitters in brain and periphery in ways that could contribute to differential vulnerabilities to addictions. SLC4A7 is thus a novel candidate in the contribution to vulnerability to addictions.

Molecular and functional expression of anion exchangers in cultured normal human nasal epithelial cells.

AIMS: Anions have an important role in the regulation of airway surface liquid (ASL) volume, viscosity and pH. However, functional localization and regulation of anion exchangers (AEs) have not been clearly described. The aim of this study was to investigate the regulation of AE mRNA expression level in accordance with mucociliary differentiation and the functional expression of AEs cultured normal human nasal epithelial (NHNE) cells. METHODS: Nasal mucosal specimens from three patients are obtained and serially cultured cells are subjected to morphological examinations, RT-PCR, Western blot analysis and immunocytochemistry. AE activity is assessed by pHi measurements. RESULTS: Expression of ciliated cells on the apical membrane and expression of MUC5AC, a marker of mucous differentiation, increased with time. AE2 and SLC26A4 mRNA expression decreased as mucociliary differentiation progressed, and AE4, SLC26A7 and SLC26A8 mRNA expression increased on the 14th and 28th day after confluence. Accordingly, AE4 protein expression also progressively increased. AE activity in 100 mM K(+) buffer solutions was nearly twofold higher than that in 5 mM K(+) buffer solutions. Moreover, only luminal AE activity increased about fourfold over the control in the presence of 5 microM forskolin. In the presence of 100 microM adenosine-5'-triphosphate (ATP) which evokes intracellular calcium signalling through activation of purinergic receptors, only luminal AE activity was again significantly increased. On the other hand, 500 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of most SLC4 and SLC26AE isoforms, nearly abolished AE activity in both luminal and basolateral membranes. We found that AE activity was affected by intracellular cAMP and calcium signalling in the luminal membrane and was DIDS-sensitive in both membranes of cultured NHNE cells. CONCLUSION: Our findings through molecular and functional studies using cultured NHNE cells suggest that AEs may have an important role in the regulation of ASL.

Vacuolar H+-ATPase expression is increased in acid-secreting intercalated cells in kidneys of rats with hypercalcaemia-induced alkalosis.

AIMS: Hypercalcaemia is known to be associated with systemic metabolic alkalosis, although the underlying mechanism is uncertain. Therefore, we aimed to examine whether hypercalcaemia was associated with changes in the expression of acid-base transporters in the kidney. METHODS: Rats were infused with human parathyroid hormone (PTH, 15 microg kg(-1) day(-1)), or vehicle for 48 h using osmotic minipumps. RESULTS: The rats treated with PTH developed hypercalcaemia and exhibited metabolic alkalosis (arterial HCO: 31.1 +/- 0.8 vs. 28.1 +/- 0.8 mmol L(-1) in controls, P < 0.05, n = 6), whereas the urine pH of 6.85 +/- 0.1 was significantly decreased compared with the pH of 7.38 +/- 0.1 in controls (P < 0.05, n = 12). The observed alkalosis was associated with a significantly increased expression of the B1-subunit of the H(+)-ATPase in kidney inner medulla (IM, 233 +/- 45% of the control level). In contrast, electroneutral Na(+)-HCO cotransporter NBCn1 and Cl(-)/HCO anion exchanger AE2 expression was markedly reduced in the inner stripe of the outer medulla (to 26 +/- 9% and 65 +/- 6%, respectively). These findings were verified by immunohistochemistry. CONCLUSIONS: (1) hypercalcaemia-induced metabolic alkalosis was associated with increased urinary excretion of H(+); (2) the increased H(+)-ATPase expression in IM may partly explain the enhanced urinary acidification, which is speculated to prevent stone formation because of hypercalciuria and (3) the decreased expression of outer medullary AE2 suggests a compensatory reduction of the transepithelial bicarbonate transport.

[Water- and electrolyte secretion by salivary glands. ]. / Víz- es elektrolitszekréció a nyálmirigyekben.

INTRODUCTION: In salivary glands, fluid transport is thought to be driven osmotically in response to transepithelial salt gradients. According to the classical two-stage hypothesis of salivary secretion, an isotonic primary fluid is generated by the acinar cells and the fluid is subsequently modified by solute reabsorption and secretion as it passes along the ductal system resulted in the final, hypotonic solution. AIM: Very little is known about the molecular and functional nature of the transporters involved in salivary secretion, especially in human salivary glands. Therefore a systematic investigation of membrane transporters expressed also in the kidney, has been undertaken in healthy human salivary glands. METHODS: RT-PCR and immunohistochemical analysis of different membrane transport proteins was used in rat and human salivary glands. RESULTS: Clear evidence for the expression of aquaporin water channels in human salivary glands was found. AQP1 in the myoepithelial cells, AQP3 in the basolateral, AQP5 in the apical membrane of the acini is localized. The electroneutral NBC3 Na(+)-HCO3(-)-cotransporter is present in the apical membrane of the serous acini and of the ducts, while the NBCn1 only in the basolateral membrane of the striated duct is localized. The NHE1 Na+/H+ exchanger is present in the basolateral membrane of the acini and ducts. The vacuolar H(+)-ATPase is localized apically in the ducts, except for the intercalated duct. CONCLUSION: Aquaporin water channels are likely to be involved in water secretion. The NBC3 and NBCn1 electroneutral Na(+)-HCO3(-)-cotransporters, the NHE1 Na+/H+ exchanger and the vacuolar H(+)-ATPase may play an important role in the pH regulation of salivary acinar and duct cells.

NBCn1 (slc4a7) mediates the Na+-dependent bicarbonate transport important for regulation of intracellular pH in mouse vascular smooth muscle cells.

The contribution of sodium-dependent bicarbonate transport to intracellular pH (pH(i)) regulation in vascular smooth muscle cells is controversial, partly because the molecular identity of the transporter(s) responsible has not been identified. Here, using the pH-sensitive fluorophore 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), we show that smooth muscle cells of intact mouse mesenteric, coronary, and cerebral small arteries all display a sodium- and bicarbonate-dependent pH(i) recovery after an NH4+-prepulse. The sodium-dependent bicarbonate flux was largely 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) sensitive (56% to 91%) and of a magnitude similar to the amiloride-sensitive flux. Additionally, steady-state pH(i) was lower (0.2 to 0.4 pH units magnitude) in all 3 vascular beds when CO2/bicarbonate was omitted. RT-PCR analyses showed that NBCn1 (slc4a7) is the only Na+-dependent bicarbonate transporter of the slc4 family detectable at the mRNA level in all 3 vascular beds investigated. Whole-mount immunolabeling and immunogold electron microscopy confirmed the presence of NBCn1 protein in the sarcolemma of mouse mesenteric small arterial smooth muscle cells. Intact mouse mesenteric small arteries were electropermeated to facilitate transfection with small interfering RNA targeting NBCn1, which resulted in an approximate 43% decrease in the ratio of NBCn1 to glyceraldehyde-3-phosphate dehydrogenase mRNA. After knock-down, we found a decreased steady-state pH(i) (0.21+/-0.08 pH units) as well as a 68+/-10% decrease in the net Na+-dependent, amiloride-insensitive base influx after acid load. Finally, omission of CO2/bicarbonate resulted in a decreased contractile response to norepinephrine after sustained exposure to the agonist, underlining the importance of CO2/bicarbonate for vascular contractility. We conclude that NBCn1 mediates the Na+-dependent bicarbonate transport important for pH(i) regulation in smooth muscle cells of mouse mesenteric, coronary, and cerebral small arteries.

Expression of Na+/HCO3- co-transporter proteins (NBCs) in rat and human skeletal muscle.

AIM: Sodium/bicarbonate co-transport (NBC) has been suggested to have a role in muscle pH regulation. We investigated the presence of NBC proteins in rat and human muscle samples and the fibre type distribution of the identified NBCs. METHODS AND RESULTS: Western blotting of muscle homogenates and sarcolemmal membranes (sarcolemmal giant vesicles) were used to screen for the presence of NBCs. Immunohistochemistry was used for the subcellular localization. The functional test revealed that approximately half of the pH recovery in sarcolemmal vesicles produced from rat muscle is mediated by bicarbonate-dependent transport. This indicates that the NBCs are preserved in the vesicles. The western blotting experiments demonstrated the existence of at least two NBC proteins in skeletal muscle. One NBC protein (approximately 150 kDa) seems to be related to the kidney/pancreas/heart isoform NBC1, whereas the other protein (approximately 200 kDa) is related to the NBC4 isoform. The two NBC proteins represent the electrogenic isoforms named NBCe1 and NBCe2. Membrane fractionation and immunofluorescence techniques confirmed that the two NBCs are located in the sarcolemmal membrane as well as in some internal membranes, probably the T-tubules. The two NBCs localized in muscle have distinct fibre type distributions. CONCLUSIONS: Skeletal muscle possesses two variants of the sodium/bicarbonate co-transporter (NBC) isoforms, which have been called NBCe1 and NBCe2.

Identification and cloning of the Na/HCO(3-) cotransporter (NBC) in human corneal endothelium.

Fluid secretion by the corneal endothelium is associated with the net flux of HCO(3)(-) from basolateral (stromal) to apical (anterior chamber) sides of the tissue. In this study we asked if Na(+)/HCO(3)(-) cotransporter (NBC-1) protein expression and functional activity are present in freshly isolated human corneal endothelium. Immunoblot analysis using a polyclonal antibody to NBC-1 showed a single band at approximately 130 kDa. Indirect immunofluorescence indicated that NBC-1 is expressed on the basolateral, but not apical side of human corneal endothelium. RT-PCR was used to determine whether the kidney or pancreatic isoform of NBC-1 is expressed. Using the specific primers for pNBC and kNBC isoforms, RT-PCR showed that only pNBC could be detected in human corneal endothelium. The product was cloned and confirmed by sequencing. Full-length NBC-1 was also cloned from human corneal endothelium. This clone (hcNBC) is 100% identical to the longer, more common form of NBC [pNBC; 1079 amino acids (aa); 122 kDa in human heart, pancreas and prostate]. To test for functional activity of NBC-1, freshly isolated endothelium was loaded with the pH sensitive fluorescent dye BCECF and HCO(3)(-) fluxes were measured. HCO(3)(-) fluxes were Na(+)-dependent, electrogenic and H(2)-DIDS sensitive. We conclude that the long isoform of the sodium bicarbonate cotransporter (pNBC-1) is expressed on the basolateral side of fresh human corneal endothelium (hcNBC). The shorter form, kNBC, could not be detected. As in bovine corneal endothelium, hcNBC is instrumental in loading HCO(3)(-) into endothelial cells from the basolateral membrane.

Cellular bicarbonate protects rat duodenal mucosa from acid-induced injury.

Secretion of bicarbonate from epithelial cells is considered to be the primary mechanism by which the duodenal mucosa is protected from acid-related injury. Against this view is the finding that patients with cystic fibrosis, who have impaired duodenal bicarbonate secretion, are paradoxically protected from developing duodenal ulcers. Therefore, we hypothesized that epithelial cell intracellular pH regulation, rather than secreted extracellular bicarbonate, was the principal means by which duodenal epithelial cells are protected from acidification and injury. Using a novel in vivo microscopic method, we have measured bicarbonate secretion and epithelial cell intracellular pH (pH(i)), and we have followed cell injury in the presence of the anion transport inhibitor DIDS and the Cl(-) channel inhibitor, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). DIDS and NPPB abolished the increase of duodenal bicarbonate secretion following luminal acid perfusion. DIDS decreased basal pH(i), whereas NPPB increased pH(i); DIDS further decreased pH(i) during acid challenge and abolished the pH(i) overshoot over baseline observed after acid challenge, whereas NPPB attenuated the fall of pH(i) and exaggerated the overshoot. Finally, acid-induced epithelial injury was enhanced by DIDS and decreased by NPPB. The results support the role of intracellular bicarbonate in the protection of duodenal epithelial cells from luminal gastric acid.

Coordinated down-regulation of NBC-1 and NHE-3 in sodium and bicarbonate loading.

BACKGROUND: Bicarbonate reabsorption in the kidney proximal tubule is predominantly mediated via the apical Na+/H+ exchanger (NHE-3) and basolateral Na+: HCO(-3) cotransporter (NBC-1). The purpose of these studies was to examine the effects of Na+ load and altered acid-base status on the expression of NHE-3 and NBC-1 in the kidney. METHODS: Rats were placed on 280 mmol/L of NaHCO(3), NaCl, or NH(4)Cl added to their drinking water for 5 days and examined for the expression of NHE-3 and NBC-1 in the kidney. RESULTS: Serum [HCO(-3)] was unchanged in NaHCO(-3) and NaCl-loaded animals versus control (P> 0.05). However, a significant hyperchloremic metabolic acidosis was developed in NH4Cl-loaded animals. A specific polyclonal antibody against NBC-1 recognized a 130 kD band, which was exclusively expressed in the basolateral membrane of proximal tubules. Immunoblot studies indicated that the protein abundance of NBC-1 and NHE-3 in the cortex decreased by 74% (P < 0.04) and 66% (P < 0.03), respectively, in NaHCO(3) loading and by 72% (P < 0.003) and 55% (P < 0.04), respectively, in NaCl loading. Switching from NaHCO(3) to distilled water resulted in rapid recovery of NHE-3 and NBC-1 protein expression toward normal levels. Metabolic acidosis increased the abundance of NHE-3 (P < 0.0001) but not NBC-1 (P> 0.05). CONCLUSIONS: NaHCO(-3) or NaCl loading coordinately down-regulates the apical NHE-3 and basolateral NBC-1 in rat kidney proximal tubule, presumably due to increased Na+ load. We propose that the down-regulation of these two Na+- and HCO(3)-absorbing transporters is, to a large degree, responsible for enhanced excretion of excess of Na+ and alkaline load and prevention of metabolic alkalosis in rats subjected to NaHCO(-3) loading.

Immunolocalization of electrogenic sodium-bicarbonate cotransporters pNBC1 and kNBC1 in the rat eye.

The human NBC1 gene encodes two electrogenic sodium-bicarbonate cotransport proteins, pNBC1 and kNBC1, which are candidate proteins for mediating electrogenic sodium-bicarbonate cotransport in ocular cells. Mutations in the coding region of the human NBC1 gene in exons common to both pNBC1 and kNBC1 result in a syndrome with a severe ocular and renal phenotype (blindness, band keratopathy, glaucoma, cataracts, and proximal renal tubular acidosis). In the present study, we determined the pattern of electrogenic sodium-bicarbonate cotransporter protein expression in rat eye. For this purpose, pNBC1- and kNBC1-specific antibodies were generated and used to detect these NBC1 protein variants by immunoblotting and immunocytochemistry. pNBC1 is expressed in cornea, conjunctiva, lens, ciliary body, and retina, whereas the expression of kNBC1 is restricted to the conjunctiva. These results provide the first evidence for extrarenal kNBC1 protein expression. The data in this study will serve as a basis for understanding the molecular mechanisms responsible for abnormalities in ocular electrogenic sodium-bicarbonate cotransport in patients with mutations in the NBC1 gene.

Essential role for NHERF in cAMP-mediated inhibition of the Na+-HCO3- co-transporter in BSC-1 cells.

Prior studies have indicated a requirement for the PDZ domain-containing protein, Na(+)/H(+) Exchanger Regulatory Factor (NHERF), for protein kinase A (PKA)-mediated inhibition of the renal basolateral Na(+)-HCO(3)(-) co-transporter (NBC). The present studies explore the potential mechanisms by which NHERF transduces cAMP signals to inhibit NBC. In BSC-1 cells, cells that express NBC but lack NHERF, 8-bromo-cAMP (100 microm for 15 min) failed to inhibit transport until wild-type mNHERF-(1-355) was expressed. mNHERF-(116-355) containing PDZ II and C-terminal ezrin-binding sequences or a mutant unphosphorylated form of rabbit NHERF effectively transduced the cAMP signals that inhibited NBC. By contrast, mNHERF-(1-126) encompassing N-terminal PDZ I and mNHERF-(1-325), which lacks ezrin-binding, failed to support cAMP inhibition of NBC activity. NBC and NHERF did not associate with each other in yeast two-hybrid or co-immunoprecipitation assays, and confocal microscopy indicated distinct subcellular localization of the two proteins. NBC was phosphorylated in BSC-1 cells, but its phosphorylation was not increased by cAMP nor was immunoprecipitated NBC phosphorylated by PKA in vitro. Acute exposure of mNHERF-(1-355)-expressing BSC-1 cells to cAMP did not change cell surface expression of NBC. Although these results established an essential role for NHERF in cAMP-mediated inhibition of NBC in BSC-1 cells, they also suggest a novel mechanism for NHERF-mediated signal transduction distinct from that previously characterized from studies of other NHERF targets.