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.

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.

The electrogenic sodium bicarbonate cotransporter NBCe1-A mediates the basolateral absorption of sodium and bicarbonate in the proximal tubule. In this study the oligomeric state and minimal functional unit of NBCe1-A were investigated. Wildtype (wt) NBCe1-A isolated from mouse kidney or heterologously expressed in HEK293 cells was predominantly in a dimeric state as was shown using fluorescence energy transfer, pulldown, immunoprecipitation, cross-linking experiments, and nondenaturing perfluorooctanoate-PAGE. NBCe1-A monomers were found to be covalently linked by S-S bonds. When each of the 15 native cysteine residues were individually removed on a wt-NBCe1-A backbone, dimerization of the cotransporter was not affected. In experiments involving multiple native cysteine residue removal, both Cys 630 and Cys 642 in extracellular loop 3 were shown to mediate S-S bond formation between NBCe1-A monomers. When native NBCe1-A cysteine residues were individually reintroduced into a cysteineless NBCe1-A mutant backbone, the finding that a Cys 992 construct that lacked S-S bonds functioned normally indicated that stable covalent linkage of NBCe1-A monomers was not a necessary requirement for functional activity of the cotransporter. Studies using concatameric constructs of wt-NBCe1-A, whose activity is resistant to methanesulfonate reagents, and an NBCe1-A T442C mutant, whose activity is completely inhibited by methanesulfonate reagents, confirmed that NBCe1-A monomers are functional. Our results demonstrate that wt-NBCe1-A is predominantly a homodimer, dependent on S-S bond formation that is composed of functionally active monomers.

ABSTRACT:Trichloroethylene (TCE) and other halogenated alkenes are known environmental contaminants with cytotoxic and nephrotoxic effects, and are potential carcinogens. Their metabolism via the mercapturate metabolic pathway was shown to lead to their detoxification. The final products of this pathway, mercapturic acids or N-acetyl-L-cysteine S-conjugates, are secreted into the lumen in the renal proximal tubule. The proximal tubule may also deacetylate mercapturic acids, and the resulting cysteine S-conjugates are transformed by cysteine S-conjugate ␤-lyases to nephrotoxic reactive thiols. The specificity and rate of mercapturic acid deacetylation may determine the toxicity of certain mercapturic acids; however, the exact enzymologic processes involved are not known in detail. In the present study we characterized the kinetics of the recently cloned mouse aminoacylase III (AAIII) toward a wide spectrum of halogenated mercapturic acids and N-acetylated amino acids. In general, the V max value of AAIII was significantly larger with chlorinated and brominated mercapturic acids, whereas fluorination significantly decreased it. The enzyme deacetylated mercapturic acids derived from the TCE metabolism including N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (NA-1,2-DCVC) and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine (NA-2,2-DCVC). Both mercapturic acids induced cytotoxicity in mouse proximal tubule mPCT cells expressing AAIII, which was decreased by an inhibitor of ␤-lyase, aminooxyacetate. The toxic effect of NA-2,2-DCVC was smaller than that of NA-1,2-DCVC, indicating that factors other than the intracellular activity of AAIII mediate the cytotoxicity of these mercapturic acids. Our results indicate that in proximal tubule cells, AAIII plays an important role in deacetylating several halogenated mercapturic acids, and this process may be involved in their cyto-and nephrotoxicity.Trichloroethylene (TCE) and other industrial solvents are shown to cause nephrotoxicity and hepatotoxicity, and are probable human carcinogens (Maltoni et al., 1988;Cummings and Lash, 2000). One of the TCE toxicity mechanisms involves the conjugation with glutathione, which is transformed by ␥-glutamyltransferase and dipeptidase to cysteine S-conjugates Berrnauer et al., 1996;Anders and Dekant, 1998). The formation of cysteine S-conjugates takes place mainly in liver and, to some extent, also in kidney. Cysteine S-conjugates may be acetylated in the liver and kidney into corresponding N-acetyl S-conjugates (mercapturic acids) and transported from the liver into the kidney. In the kidney proximal tubules, mercapturic acids may be secreted or deacetylated by aminoacylases into cysteine S-conjugates Dekant, 1994, 1998). The deacetylation reaction may play an important role in the renal nephrotoxicity since cysteine S-conjugates, but not mercapturates, are transformed by cysteine conjugate ␤-lyases into toxic metabolites (Hayden and Stevens, 1990;Commandeur et al., 1995). Cysteine S-conjugates also may be transformed by flavoprotein monooxygenase...