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

Abstract

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.

Functional characterization of NBC4: a new electrogenic sodium-bicarbonate cotransporter

“…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 ϩ -HCO 3 Ϫ cotransporters are required to address the question as to why in the mammalian genome there are two distinct genes that encode electrogenic Na ϩ -HCO 3 Ϫ cotransporters.…”

“…kNBC1 mediates the majority of basolateral HCO 3 Ϫ 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 Ϫ -independent and stilbene-insensitive Na ϩ -HCO 3 Ϫ cotransport in various tissues (10,20,22,25).…”

Topological Location and Structural Importance of the NBCe1-A Residues Mutated in Proximal Renal Tubular Acidosis

“…Insoluble material was removed by centrifugation at 20,000 ϫ g for 15 min at 4°C. The supernatant was pre-cleared with preimmune rabbit serum and protein A-Sepharose resin, and then NBCe1-A protein was immunoprecipitated by a rabbit anti-human NBCe1-A N terminus polyclonal antibody (22). For detailed protocols see Ref.…”

A Novel Missense Mutation in the Sodium Bicarbonate Cotransporter (NBCe1/SLC4A4) Causes Proximal Tubular Acidosis and Glaucoma through Ion Transport Defects

“…Ocular defects with NBCe1 mutations (S427L, R298S, R510H) indicate that NBCe1 is critical for fluid homeostasis (glaucoma) and transparency (cataracts) in the eye. Two groups have localized eye NBCe1 isoforms (30,31). Bok and co-workers (31) found that NBCe1-B (pNBCe1) expressed throughout the rat eye (ciliary body, conjunctiva surface cells, cornea, lens epithelium, and retina), whereas NBCe1-A (kNBCe1) was only present in the conjunctiva basal cells.…”

“…Two groups have localized eye NBCe1 isoforms (30,31). Bok and co-workers (31) found that NBCe1-B (pNBCe1) expressed throughout the rat eye (ciliary body, conjunctiva surface cells, cornea, lens epithelium, and retina), whereas NBCe1-A (kNBCe1) was only present in the conjunctiva basal cells. These investigators postulated that all of the eye pathologies are associated with mutations in pNBCe1.…”