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

Ying Liu,Zhang-Dong Xie,De-Zhi Jiang,Mugen Liu,Li-Ming Chen,Xue Qin,Deng-Ke Wang,Qing K Wang,Robert A Fenton

doi:10.1371/journal.pone.0055974

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.

Highlights

NBCn2, encoded by SLC4A10, is an electroneutral Na+-coupled HCO32 transporter (NCBT) of the solute carrier 4 (SLC4) family
The protein abundance of NBCn2—as well as NBCn1 (Slc4a7) and NDCBE (Slc4a8) is greatly reduced in the brains of mice subjected to chronic continuous hypoxia, suggesting that these electroneutral NCBTs are involved in the adaptive response to low-oxygen stress in central nervous system (CNS) [8,9]
Cloning of novel NBCn2 variants By 59-rapid amplification of complementary DNA (cDNA) ends (59-Rapid amplification of cDNA ends (RACE)) with mouse tissues, we identified a new exon of Slc4a10

Summary

Introduction

NBCn2 (aka NCBE), encoded by SLC4A10, is an electroneutral Na+-coupled HCO32 transporter (NCBT) of the solute carrier 4 (SLC4) family. Knockout of Slc4a10 causes an increase in the seizure threshold in mice [5], an observation that, taken at face value, appears to be in conflict with the above reports in humans. The reason for this apparent inconsistency remains mystic. Two subsequent studies with the Slc4a10-null mice have shown that loss of NBCn2 expression in mice alters the protein abundance as well as the cellular targeting of a series of membrane transporters/channels as well as cytoskeleton components in the choroid plexus [6,7]. The protein abundance of NBCn2—as well as NBCn1 (Slc4a7) and NDCBE (Slc4a8) is greatly reduced in the brains of mice subjected to chronic continuous hypoxia, suggesting that these electroneutral NCBTs are involved in the adaptive response to low-oxygen stress in CNS [8,9]

Methods

Results

Discussion

Conclusion