Cloning, Identification, And Tissue Specificity Of Expression Products Of Na⁺-HCO₃^-Cotransporter Slc4a10

Acid-base balance has profound effects on virtually all pH-sensitive biologicalprocesses, such as the modulation of excitation of neurons and myocardial cells, thegeneration of heart rate, muscle contraction, digestion, fertilization and so on. Thus, it is offundamental significance to maintain acid-base homeostasis in organisms. HCO₃^–is a keycomponent of the most important pH buffer system CO2/HCO₃^–in the body.HCO₃^–transporters are a major class of pH regulators and play vital roles in the body.Members of the SLC4family are major such transporters for HCO₃^–movement across theplasma membrane. One member of the family, SLC4A10, encodes an electroneutralsodium-coupled bicarbonate cotransporter NBCn2. Previous studies have demonstratedthat NBCn2is highly expressed in the central nervous system. The transporter plays acritical role in intracellular pH regulation of neurons as well as in the production ofcerebrospinal fluid. In humans, genetic disruption of SLC4A10is associated with epilepsyand mental retardation. In mice, knocking out Slc4a10results in an increase in epilepsythreshold.Although the expression, physiological as well as pathological significance ofNBCn2in the central nervous system have been well recognized, little is known about itsexpression and physiological roles in other systems. Moreover, it is not clear about themechanism involved in the regulation of Slc4a10expression. In the present study, in ourattempts to address the above questions, we made the following important findings:（1） By5’-RACE, we identified two novel exons of Slc4a10, encoding a novelamino-terminus （Nt） of NBCn2distinct from that of the originally described NBCn2products.（2） By cDNA cloning, we identified from rats for the first time a group of full-lengthNBCn2variants with the novel Nt （starting with MCDL, the first four amino acidresidues of Nt） differing from the previously described NBCn2variants （starting withMEIK）.（3） Surprisingly, we found that the corresponding variants from mouse—orthologs of ratMCDL-NBCn2—starts with MQPG. Analysis with genomic DNA sequence indicates that this species-specific difference is derived from a single nucleotide variation inmouse Slc4a10.（4） By western blotting using polyclonal antibodies, we demonstrated that the expressionof NBCn2with different extreme Nt exhibits distinct tissue specificity. In rat,MEIK-NBCn2is predominantly expressed in the central nervous system, whereasMCDL-NBCn2is predominantly expressed in the kidney, and to lesser extent in thesmall intestine.（5） Finally, we demonstrated the tissue-specificity of the expression of alternativecarboxyl-termini （Ct） of NBCn2. Due to alternative splicing of mRNA, NBCn2variants have two types of Ct: a short one ending with SPSS as well as a long oneending with “ETCL”, a typical PDZ-binding motif. By RT-PCR and cDNA cloning,we found that NBCn2variants with the short Ct are expressed only in neural tissues.In contrast, those with the long Ct are specifically expressed in epithelial tissues.In summary, we conclude that the expression of alternative NBCn2Nts—likelyderived from usage of alternative promoters—as well as the alternative Cts derived fromalternative splicing are tissue specific. Our findings shed important light on the study ofphysiology of NBCn2...