Abstract
The cDNA coding for a rabbit renal Na+/dicarboxylate cotransporter, designated NaDC-1, was isolated by functional expression in Xenopus oocytes. NaDC-1 cDNA is approximately 2.3 kilobases in length and codes for a protein of 593 amino acids. NaDC-1 protein contains eight putative transmembrane domains, and the sequence and secondary structure are related to the renal Na+/sulfate transporter, NaSi-1. Northern analysis shows that the NaDC-1 message is abundant in kidney and small intestine, and related transporters may be found in liver, lung, and adrenal. The transport of succinate by NaDC-1 was sodium-dependent, sensitive to inhibition by lithium, and inhibited by a range of di- and tricarboxylic acids. This transporter also carries citrate, but it does not transport lactate. In kinetic experiments, the Km for succinate was around 0.4 mM and the Vmax was 15 nmol/oocyte/h, while the Hill coefficient of Na+ activation of succinate transport was 1.9. The transport of succinate by NaDC-1 was insensitive to changes in pH, whereas the transport of citrate increased with decreasing pH, in parallel with the concentration of divalent citrate in the medium. The results of the functional characterization indicate that NaDC-1 likely corresponds to the renal brush-border Na+/dicarboxylate cotransporter.
Highlights
The low affinity Na+/dicarboxylate cotransporter of the mammalian kidney has been functionally characterized in isolated brush-border membrane vesicles (3) and intact proximal tubules (4)
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM IEMBL Data Bank with accession number(s) U12120
EDNA Library Construction and Screening-The size fraction of rabbit renal cortex mRNA that induced the highest uptake of succinate transport when injected into Xenopus oocytes {1.5-3 kb, Fig. 1) was
Summary
Xenopus Oocytes-Stage V and VI oocytes from Xenopus laevis (NASCO) were dissected and defolliculated as described previously (9, 11) They were injected with 50 nl of RNA (0.5 p,g/p,l) 1 day following isolation. EDNA Library Construction and Screening-The size fraction of rabbit renal cortex mRNA that induced the highest uptake of succinate transport when injected into Xenopus oocytes {1.5-3 kb, Fig. 1) was. Oocytes were injected with 50 nl of RNA, and transport of250 JLM succinate was measured 6 days later in sodium-containing buffer. The blot was probed (50% formamide, 42 °C) and washed (0.1 X SSC, 0.1% SDS, 55 °C) at high stringency
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
