The kidney maintains systemic acid-base balance by reclaiming from the renal tubule lumen virtually all HCO 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 HCO reclamation depends solely on H secretion, mediated by the apical Na/H exchanger NHE and the vacuolar proton pump. However, and the proton pump cannot account for all HCO reclamation. Here, we investigated the potential contribution of two variants of the electroneutral Na/HCO 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, NHCl-induced systemic metabolic acidosis or hypokalemic alkalosis downregulated the abundance of MCDL-NBCn2 and reciprocally upregulated NHE Conversely, NaHCO-induced metabolic alkalosis upregulated MCDL-NBCn2 and reciprocally downregulated NHE We propose that the apical membrane of the proximal tubules has two distinct strategies for HCO reclamation: the conventional indirect pathway, in which NHE and the proton pump secrete H to titrate luminal HCO, and the novel direct pathway, in which NBCn2 removes HCO from the lumen. The reciprocal regulation of NBCn2 and NHE under different physiologic conditions is consistent with our mathematical simulations, which suggest that HCO uptake and H secretion have reciprocal efficiencies for HCO reclamation versus titration of luminal buffers.
Key points• The human SLC4A7 gene and the mouse Slc4a7 gene each have alternative promoters that can yield two groups of NBCn1 variants, one in which the extreme N terminus begins with MEAD (representing the first four residues of the N-terminal domain (Nt)) and the other in which it begins with MERF.• The mouse Slc4a7 gene contains, and the human SLC4A7 gene is predicted to contain, a novel exon that encodes an alternatively spliced cassette IV of 20 aa in the cytoplasmic Nt domain of NBCn1. This new cassette IV is in a position homologous to that of a previously described cassette in the Nt of NBCn2.• From combinations of known optional structural elements (OSEs), SLC4A7 is theoretically able to produce 32 major variants, of which 16 have now been identified, 10 for the first time in the present study.• With heterologous expression in Xenopus oocytes, the OSEs have strong effects on surface abundance and intrinsic HCO 3 − transport activity. Cassettes II, III and the novel cassette IV have stimulatory effects on the intrinsic HCO 3 − transport activity of NBCn1.Abstract The SLC4A7 gene encodes the electroneutral sodium/HCO 3 cotransporter NBCn1, which plays important physiological and pathophysiological roles in many cell types. Previous work identified six NBCn1 variants differing in the sequence of the extreme N terminus -MEAD in rat only, MERF in human only -as well as in the optional inclusion of cassettes I, II, and III. Earlier work also left open the question of whether optional structural elements (OSEs) affect surface abundance or intrinsic (per-molecule) transport activity. Here, we demonstrate for the first time that SLC4A7 from one species can express both MEAD-and MERF-NBCn1. We also identify a novel cassette IV of 20 aa, and extend by 10 the number of full-length NBCn1 variants. The alternative N termini and four cassettes could theoretically produce 32 major variants. Moreover, we identify a group of cDNAs predicted to encode just the cytosolic N-terminal domain (Nt) of NBCn1. A combination of electrophysiology and biotinylation shows that the OSEs can affect surface abundance and intrinsic HCO 3 − transport activity of NBCn1, as expressed in Xenopus oocytes. Specifically, MEAD tends to increase whereas novel cassette IV reduces surface Y. Liu and X. Qin contributed equally to this work. abundance. Cassettes II, III and novel cassette IV all appear to increase the intrinsic activity of NBCn1.
Previous studies have shown that the electroneutral Na+/HCO3 − 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 HCO3 − 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.
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