Cloning and Functional Characterization of Novel Variants and Tissue-Specific Expression of Alternative Amino and Carboxyl Termini of Products of Slc4a10

Abstract

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.

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

“…Consistent with previous observations, 14 anti-MCDL recognizes a band of apparent molecular mass approximately , creatine, and other luminal buffers. In the cytosol of the proximal-tubule epithelium, the protons to be secreted across the apical membrane arise from three sources: (1) hydration of CO 2 reclaimed from the lumen (pathway 1); (2) metabolism of glutamine (pathway 2); and (3) hydration of CO 2 that enters from the blood (pathway 3).…”

“…Depending on the usage of different promoters, 15 the extreme Nt of these variants can begin either with MCDL (NBCn2-E/F/G/H) or MEIK (NBCn2-A/B/C/D). In the MCDL group, the rat kidney mainly expresses NBCn2-G, and in the MEIK group, it expresses mainly NBCn2-C. 14,15 Here, we find that MCDL-NBCn2 and MEIK-NBCn2 exhibit distinct tissue and subcellular distributions in rat kidney, with MCDL-NBCn2 being localized in the apical membrane of PTs, and MEIK-NBCn2 localized in the BLM of mTALs and IMCDs.…”

“…Rabbit polyclonal antibodies against NBCn2, including anti-MCDL and anti-MEIK, were generated by Genscript (Nanjing, China). Anti-MCDL directed against SGNRKVMQPGTCEHC in the unique Nt of MCDL-NBCn2 has been described previously, 14 and is further characterized in this study (see Supplemental Figures 1, A-C and 2, A-C). Anti-MEIK was directed against MEIKDQGAQMEPLLPTC, an epitope in the unique portion of the Nt of MEIK-NBCn2 that has been well characterized previously.…”

“…Anti-MEIK recognizes bands of apparent molecular mass approximately 180 kD from the brain and approximately 190 kD from the kidney ( Figure 2B). The difference between the apparent and predicted molecular masses (approximately 125 kD) reflects N-glycosylation of NBCn2 proteins (see Liu et al 14 and Chen et al 16 , and Supplemental Material). The difference in the apparent molecular mass of MEIK-NBCn2 in the brain and kidney presumably reflects differences in MEIK-NBCn2 variants and glycosylation in these tissues (Supplemental Material).…”

“…Previous studies have shown that the rat kidney expresses two groups of full-length NBCn2 (Slc4a10) variants, differing in the amino termini (Nts), one starting with residue MCDL (mainly NBCn2-G), and the other with MEIK (mainly NBCn2-C). 14,15 Here, we report that MCDL-NBCn2 is expressed at the apical membrane of the PT, whereas MEIK-NBCn2 is expressed at the BLM of the medullary thick ascending limb (mTAL) and inner medullary collecting ducts (IMCDs). Systemic acid-base disturbances reciprocally regulate the abundances of MCDL-NBCn2 and NHE3.…”