Liqun Bai

The substrate recognition domain in the Na+/dicarboxylate and Na+/sulfate cotransporters is located in the carboxy-terminal portion of the protein

The Na+/dicarboxylate cotransporter, NaDC-1, and the Na+/sulfate cotransporter, NaSi-1, share 43% sequence identity, but they exhibit no overlap in substrate specificity. A functional chimera, SiDC-4, was prepared from NaDC-1 and NaSi-1 by homologous recombination and expressed in Xenopus oocytes. SiDC-4 contains putative transmembrane domains 1-4 of NaSi-1 (amino acids 1-139) and putative transmembrane domains 5-11 of NaDC-1 (amino acids 141-593). SiDC-4 retains the substrate specificity of NaDC-1, which suggests that the substrate recognition domain is found in the carboxy-terminal portion of the protein, past amino acid 141. However, residues that affect substrate affinity and inhibition by furosemide and flufenamate are found in the amino terminal third of the protein. The cation binding properties of SiDC-4, including a stimulation of transport by lithium, differed from both parental transporters, suggesting that cation binding is determined by interactions between the amino- and carboxy-terminal portions of the protein. We conclude that the substrate recognition site of NaDC-1 and NaSi-1 is found in the carboxy-terminal portion of the protein, past amino acid 141, but residues in the amino terminus can affect substrate affinity, inhibitor sensitivity, and cation selectivity.

Molecular cloning of a murine type III sodium-dependent phosphate cotransporter (Pit-2) gene promoter

We report the novel cloning and preliminary characterization of a murine type III sodium-dependent phosphate cotransporter (Pit-2) gene promoter. Five promoter/luciferase reporter gene constructs, -1816/+61, -1620/+61, -1223/+61, -600/+61 and -225/+61, showed significant luciferase activity (6-14-fold over background) when transfected into human colon carcinoma (Caco-2) and opossum kidney (OKP) cells.

Molecular cloning of murine sodium-phosphate cotransporter type IIb (Na/P(i)-IIb) gene promoter and characterization of gene structure.

We report the cloning of the murine Na/P(i)-IIb cotransporter gene, which spans more than 18 kilobases and consists of 12 introns and 13 exons. Three promoter/reporter gene constructs, -159/+73, -429/+73 and -954/+73, showed significant luciferase activity (22-82-fold over background) when transfected into in rat intestinal epithelial (RIE-1) cells.

sp1-mediated transcriptional regulation of the rat sodium hydrogen exchanger isoform 2 (NHE-2) gene promoter

The sodium hydrogen exchanger isoform 2 (NHE-2) is an apical membrane transporter which is highly expressed in the intestine. The regulation of NHE-2 under physiological and pathophysiological conditions is important in maintenance of electrolyte and acid-base balance. We have recently cloned the promoter of rat NHE-2 and identified two cis-elements required for osmotic response of rat NHE-2 gene in mIMCD-3 cells (Bai, et al., Am. J. Physiol. 46:RII12-9, 1999). To further investigate the mechanism controlling the activity of NHE-2 promoter, its regulatory elements and transcription factors were characterized in mIMCD-3 cells and Drosophila Schneider cells. Transient transfection of luciferase reporter gene constructs containing different lengths of NHE-2 promoter indicated that the minimal promoter was localized to the region of (-)21 to (+ )101 (as related to transcription initial site). Within this putative promoter region, two Sp1 elements were found to overlap in the (-)21 to (+)1 bp region. The introduction of mutations within these SpI elements and the use of SpI antisera in electrophoretic mobility shift assays demonstrated that Spl bound to this region of the NHE-2 promoter. Moreover, these in vitro studies indicated that both putative SpI response elements were utilized. Transient transfection assays using NHE-2 promoter constructs that incorporated mutations of the SpI elements clearly demonstrated that binding of Spl to the NHE-2 promoter was absolutely required for optimal levels of reporter gene expression. Expression of luciferase reporter gene linked to the (-)21 and (-)289 NHE-2 promoter regions was up-regulated by coexpression of Sp I expression vector in Drosophila Schneider cells, which lack the native SpI transcription factor. Therefore, these studies provide strong evidence that Sp I plays a central role in regulating basal levels of NHE-2 transcription. This study was supported by NIDDK Grant 2ROIDK41274-1O. 3054