Cloning, Identification And Expressional Analysis Of Splice Variants Of Na⁺-Coupled HCO_3<sup>- Transporters

It is critically important to maintain the acid-base homoestasis in organism. Acid-base balance or pH affects a broad range of physiological processes/systems, such as the central nervous system (CNS) as well as mammalian reproduction.The five known Na⁺-coupled HCO₃^- transporters (NCBTs) of SLC4 family (solute carrier 4 family)—including electrogenic NBCe1 (SLC4A4), NBCe2 (SLC4A5) and electroneutral NBCn1 (SLC4A7), NBCn2 (SLC4A10), NDCBE (SLC4A8)—are major HCO₃^- carriers across plasma membrane, thus play critical roles in intracellular pH regulation as well as transepithelial HCO₃^- transport.Due to transcription under the control of distinct promoters and/or alternative splicing, virtually all NCBT genes are able to produce multiple splice variants. For example, the SLC4A4 gene contains two distinct promoters as well as one known alternative splicing unit. So far, three NBCe1 variants, i.e., NBCe1-A/B/C have been identified. Compared to NBCe1-B/C, NBCe1-A has a unique short amino terminus (Nt). Compared to NBCe1-A/B, NBCe1-C has a unique long carboxyl terminus (Ct). Considering SLC4A10 encoding NBCn2, it contains two known alternative splicing units, enabling SLC4A10 to express four splice variants: NBCn2-A/B/C/D. The cDNAs encoding the first three NBCn2 variants have been identified. Although the expression of the fourth variant NBCn2-D has been demonstrated by western blotting, the cDNA encoding this variant has never been cloned.In the present study, using reverse transcription polymerase chain reaction (RT-PCR), we systematically examined the expression of NBCe1 variants in the reproductive tract tissues of mice. We identified, from the mouse reproductive tract tissues, five NBCe1 variants: NBCe1-A/B/C/D/E. Cloning of NBCe1-D and -E reveals a novel alternative splicing unit—designated as cassetteⅠ—in mouse slc4a4.In addition, we cloned for the first time the cDNA encoding NBCn2-D from mouse subcortex (SCX) and medulla (MD). By colony PCR, we examined the relative abundance of the NBCn2 -A/B/C/D in MD and SCX. Our data shows that the four NBCn2 variants have characteristic distribution pattern in MD and SCX, and that NBCn2-D is least abundant in both MD and SCX. Alternagtive splicing often affect the function and/or protein interactions of NCBTs. Our study made great contributions to our knowledge about the molecular physiology of NCBTs.