Molecular insight into coordination sites for substrates and their coupling kinetics in Na+ /HCO3- cotransporter NBCe1.

The secondary active transporter NBCe1 couples the transmembrane movement of Na+ and carbonate species with an apparent stoichiometry of 1Na+ :2HCO3- (the 'influx' mode) or 1Na+ :3HCO3- (the 'efflux' mode). Here, we employed molecular biology, electrophysiology and structural biology approaches to investigate the molecular mechanism for the transport coupling of Na+ and HCO3- in NBCe1. In Xenopus oocytes, decreasing extracellular [HCO3- ] from 66 to 4 mm progressively decreases the Na+ affinity of NBCe1. However, decreasing [Na+ ] from 96 to 35 mm has little effect on the HCO3- affinity. The residues responsible for the coordination of Na+ and HCO3- in the substrate pocket of NBCe1 were respectively determined by mutational and molecular simulation studies. Mutation to the residues for HCO3- coordination decreased the affinities of NBCe1 for both Na+ and HCO3- . However, mutation to the residues for Na+ coordination decreased the affinity for Na+ but had little effect on the affinity for HCO3- . Molecular simulation showed that NBCe1 has the capacity to coordinate only two ions of HCO3- or CO32- . We propose that (1) NBCe1 has an ordered substrate-binding kinetics with the binding of HCO3- preceding that of Na+ ; (2) NBCe1 operating in the influx mode moves 1Na+  + 2HCO3- , whereas NBCe1 in the efflux mode moves 1Na+  + 1HCO3-  + 1CO32- . The substrate-binding kinetics of NBCe1 is distinct from the known kinetics models of many other Na+ -coupled transporters with Na+ binding preceding the driven solute. KEY POINTS: Under physiological conditions, the secondary active transporter NBCe1 can operate in the 'influx' mode with an apparent stoichiometry of 1Na+ :2HCO3- or in the 'efflux' mode with an apparent stoichiometry of 1Na+ :3HCO3- . NBCe1 has an ordered substrate-binding kinetics with HCO3- preceding the binding of Na+ . The kinetics of NBCe1 is distinct from the known kinetics of many other Na+ -driven cotransporters for which the binding of Na+ usually precedes the driven substrate. The residues responsible for the coordination of Na+ and those for carbonate species in the substrate-binding pocket of NBCe1 were determined by mutation and molecular simulation studies. The substrate-binding pocket of NBCe1 contains just two coordination sites for HCO3- or CO32- . It is proposed that NBCe1 in the influx mode moves 1Na+  + 2HCO3- across the plasma membrane, whereas NBCe1 in the efflux mode moves 1Na+ +1HCO3- +1CO32- .

The Balance of [Formula: see text] Secretion vs. Reabsorption in the Endometrial Epithelium Regulates Uterine Fluid pH.

Uterine fluid contains a high concentration of [Formula: see text] which plays an essential role in sperm capacitation and fertilization. In addition, the [Formula: see text] concentration in uterine fluid changes periodically during the estrous cycle. It is well-known that the endometrial epithelium contains machineries involving the apical SLC26 family anion exchangers for secreting [Formula: see text] into the uterine fluid. In the present study, we find for the first time that the electroneutral Na+/[Formula: see text] cotransporter NBCn1 is expressed at the apical membrane of the endometrial epithelium. The protein abundance of the apical NBCn1 and that of the apical SLC26A4 and SLC26A6 are reciprocally regulated during the estrous cycle in the uterus. NBCn1 is most abundant at diestrus, whereas SLC26A4/A6 are most abundant at proestrus/estrus. In the ovariectomized mice, the expression of uterine NBCn1 is inhibited by ß-estradiol, but stimulated by progesterone, whereas that of uterine SLC26A4/A6 is stimulated by ß-estradiol. In vivo perfusion studies show that the endometrial epithelium is capable of both secreting and reabsorbing [Formula: see text]. Moreover, the activity for [Formula: see text] secretion by the endometrial epithelium is significantly higher at estrus than it is at diestrus. The opposite is true for [Formula: see text] reabsorption. We conclude that the endometrial epithelium simultaneously contains the activity for [Formula: see text] secretion involving the apical SLC26A4/A6 and the activity for [Formula: see text] reabsorption involving the apical NBCn1, and that the acid-base homeostasis in the uterine fluid is regulated by the finely-tuned balance of the two activities.

Na+/HCO3- Cotransporter NBCn2 Mediates HCO3- Reclamation in the Apical Membrane of Renal Proximal Tubules.

The kidney maintains systemic acid-base balance by reclaiming from the renal tubule lumen virtually all HCO3- filtered in glomeruli and by secreting additional H+ to titrate luminal buffers. For proximal tubules, which are responsible for about 80% of this activity, it is believed that HCO3- reclamation depends solely on H+ secretion, mediated by the apical Na+/H+ exchanger NHE3 and the vacuolar proton pump. However, NHE3 and the proton pump cannot account for all HCO3- reclamation. Here, we investigated the potential contribution of two variants of the electroneutral Na+/HCO3- cotransporter NBCn2, the amino termini of which start with the amino acids MCDL (MCDL-NBCn2) and MEIK (MEIK-NBCn2). Western blot analysis and immunocytochemistry revealed that MEIK-NBCn2 predominantly localizes at the basolateral membrane of medullary thick ascending limbs in the rat kidney, whereas MCDL-NBCn2 localizes at the apical membrane of proximal tubules. Notably, NH4Cl-induced systemic metabolic acidosis or hypokalemic alkalosis downregulated the abundance of MCDL-NBCn2 and reciprocally upregulated NHE3 Conversely, NaHCO3-induced metabolic alkalosis upregulated MCDL-NBCn2 and reciprocally downregulated NHE3 We propose that the apical membrane of the proximal tubules has two distinct strategies for HCO3- reclamation: the conventional indirect pathway, in which NHE3 and the proton pump secrete H+ to titrate luminal HCO3-, and the novel direct pathway, in which NBCn2 removes HCO3- from the lumen. The reciprocal regulation of NBCn2 and NHE3 under different physiologic conditions is consistent with our mathematical simulations, which suggest that HCO3- uptake and H+ secretion have reciprocal efficiencies for HCO3- reclamation versus titration of luminal buffers.

Effects of Nt-truncation and coexpression of isolated Nt domains on the membrane trafficking of electroneutral Na+/HCO3- cotransporters.

The SLC4 genes are all capable of producing multiple variants by alternative splicing or using alternative promoters. The physiological consequences of such diversity are of great interest to investigators. Here, we identified two novel variants of the electroneutral Na(+)/HCO3- cotransporter NBCn1, one full-length starting with "MIPL" and the other Nt-truncated starting with "MDEL". Moreover, we identified a new promoter of Slc4a10 encoding NBCn2 and a novel type of Nt-truncated NBCn2 starting with "MHAN". When heterologously expressed, the new NBCn1 variants were well localized to the plasma membrane and exhibited characteristic NBCn1 activity. However, MHAN-NBCn2 was poorly localized on the plasma membrane. By deletion mutations, we identified the Nt regions important for the surface localization of NBCn2. Interestingly, coexpressing the full-length NBCn2 greatly enhances the surface abundance of the Nt-truncated NBCn2. Co-immunoprecipitation and bimolecular fluorescence complementation studies showed that the full-length and Nt-truncated NBCn2 interact with each other to form heterodimers in neuro-2A cells. Finally, we showed that the isolated Nt domain interacts with and enhances the surface abundance of the Nt-truncated NBCn2. The present study expands our knowledge of the NBCn1 and NBCn2 transcriptome, and provides insights into how the Nt domain could affect transporter function by regulating its membrane trafficking.

Cloning and functional characterization of novel variants and tissue-specific expression of alternative amino and carboxyl termini of products of slc4a10.

Previous studies have shown that the electroneutral Na(+)/HCO(3) (-) cotransporter NBCn2 (SLC4A10) is predominantly expressed in the central nervous system (CNS). The physiological and pathological significances of NBCn2 have been well recognized. However, little is known about the tissue specificity of expression of different NBCn2 variants. Moreover, little is known about the expression of NBCn2 proteins in systems other than CNS. Here, we identified a set of novel Slc4a10 variants differing from the originally described ones by containing a distinct 5' untranslated region encoding a new extreme amino-terminus (Nt). Electrophysiology measurements showed that both NBCn2 variants with alternative Nt contain typical electroneutral Na(+)-coupled HCO(3) (-) transport activity in Xenopus oocytes. Luciferase reporter assay showed that Slc4a10 contains two alternative promoters responsible for expression of the two types of NBCn2 with distinct extreme Nt. Western blotting showed that NBCn2 proteins with the original Nt are primarily expressed in CNS, whereas those with the novel Nt are predominantly expressed in the kidney and to a lesser extent in the small intestine. Due to alternative splicing, the known NBCn2 variants contain two types of carboxyl-termini (CT) differing in the optional inclusion of a PDZ-binding motif. cDNA cloning showed that virtually all NBCn2 variants expressed in epithelial tissues contain, but the vast majority of those from the neural tissues lack the PDZ-binding motif. We conclude that alternative transcription and splicing of Slc4a10 products are regulated in a tissue-specific manner. Our findings provide critical insights that will greatly influence the study of the physiology of NBCn2.