Renal Cortical Basolateral Na + /HCO − 3 Cotransporter: IV Characterization and Localization with Polyclonal Antibodies

Abstract

We have previously partially purified the basolateral Na+/HCO3- cotransporter from rabbit renal cortex and this resulted in a 400-fold purification, and an SDS-PAGE analysis showed an enhancement of a protein band with a MW of approximately 56 kDa. We developed polyclonal antibodies against the Na+/HCO3- cotransporter by immunizing Dutch-belted rabbits with a partially purified protein fraction enriched in cotransporter activity. Western blot analysis of renal cortical basolateral membranes and of solubilized basolateral membrane proteins showed that the antibodies recognized a protein with a MW of approximately 56 kDa. The specificity of the purified antibodies against the Na+/ HCO3- cotransporter was tested by immunoprecipitation. Solubilized basolateral membrane proteins enriched in Na+/HCO3- cotransporter activity were incubated with the purified antibody or with the preimmune IgG and then reconstituted in proteoliposomes. The purified antibody fraction caused a concentration-dependent inhibition of the Na+/HCO3- cotransporter activity, while the preimmune IgG failed to elicit any change. The inhibitory effect of the antibody was of the same magnitude whether it was added prior to (inside) or after (outside) reconstitution in proteoliposomes. In the presence of the substrates (NaHCO3 or Na2CO3) for the cotransporter, the inhibitory effect of the antibody on cotransporter activity was significantly blunted as compared with the inhibition observed in the absence of substrates. Western blot analysis of rabbit kidneys showed that the antibodies recognized strongly a 56 kDa protein band in microsomes of the inner stripe of outer medulla and inner medulla, but not in the outer stripe of outer medulla. A 56 kDa protein band was recognized in microsomes of the stomach, liver, esophagus, and small intestine but was not detected in red blood cell membranes. Localization of the Na+/HCO3- cotransporter protein by immunogold technique revealed specific labeling of the cotransporter on the basolateral membranes of the proximal tubules, but not in the brush border membranes. These results demonstrate that the polyclonal antibodies against the 56 kDa basolateral protein inhibit the activity of the Na+/HCO3- cotransporter suggesting that the 56 kDa protein represents the cotransporter or a component thereof. These antibodies interact at or near the substrate binding sites. The Na+/HCO3- cotransporter protein is expressed in different regions of the kidneys and in other tissues.

CO2 chemosensing in rat oesophagus

“…The expression of HCO 3 − transporters, such as Cl − / HCO 3 − exchangers and the Na + : HCO 3 − co-transporters in oesophageal epithelial cells, 5051 suggests that HCO 3 − is transported from the interstitium to the lumen. In the stratified layers of gland-free oesophageal mucosa, H + may traverse a few layers from the lumen before being neutralised by extracellular HCO 3 − secreted from underlying epithelial cells, producing CO 2 .…”

The Divergence, Actions, Roles, and Relatives of Sodium-Coupled Bicarbonate Transporters

“…We now appreciate that the "NBC3" reported by Pushkin et al (765) is an Slc4a7 product (139), whereas the "NBC-3" reported Amlal et al (35) is a partial human Slc4a8 product. 74 In fact, the report of the partial sequence postdated the depositing of a full-length human SLC4A8 product by Grichtchenko et al 75 With the physiological characterization of the SLC4A8 product as a Na ϩ -driven Cl-HCO 3 exchanger, the product was renamed NDCBE1 (337). With the assumption that the SLC4A8 gene encodes the sole human Na ϩ -driven Cl-HCO 3 exchanger, we propose to drop the numerical suffix, and refer to the transporter as NDCBE.…”

“…The next critical step is to demonstrate net 74 This partial human clone (AF107099) is identical along its length to subsequently cloned human SLC4A8 products. A full-length mouse Slc4a8 product (now called NDCBE-A) was subsequently cloned in its entirety by a group that included the same authors (1029).…”

“…In a third study, conducted prior to the cloning of NBCe1, rabbit proximal tubule vesicles enriched for Na/HCO 3 cotransporter activity were used to raise an anti-Na/HCO 3 cotransporter antibody (74). The antibody reacted on western blots with a 56-kDa protein and apparently blocked NBCe1 activity (74 (1066).…”

“…In two studies, the action of NBCe1 was inhibited using antibodies raised against an epitope in the third (i.e., longest) extracellular loop of NBCe1 (225,481). In a third study, conducted prior to the cloning of NBCe1, rabbit proximal tubule vesicles enriched for Na/HCO 3 cotransporter activity were used to raise an anti-Na/HCO 3 cotransporter antibody (74). The antibody reacted on western blots with a 56-kDa protein and apparently blocked NBCe1 activity (74 (1066).…”