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Characterization of the endogenous carnitine transport and expression of a rat renal Na(+)-dependent carnitine transport system in Xenopus laevis oocytes.

ABSTRACT: L-Carnitine transport was characterized in Xenopus laevis oocytes before and after injection of mRNA isolated from rat renal cortex. Non-injected oocytes revealed endogenous Na(+)-dependent transport of L-carnitine. After injection of 15 ng of rat kidney mRNA, the Na(+)-dependent L-carnitine transport increased 2-3-fold, reaching maximal activity after 5-6 days. The expressed carnitine transport was maximal at pH 7.5, whereas the endogenous transport showed no clear maximum between pH 6.0 and 8.5. Kinetic analysis revealed apparent Km values for L-carnitine of 66 microM for the endogenous and 149 microM for the expressed transport. Trimethyl-lysine and D-carnitine inhibited both the endogenous and the expressed transport. In contrast, L-acetylcarnitine, L-isovalerylcarnitine, L-palmitoylcarnitine and butyrobetaine inhibited predominantly the expressed transport, whereas glycinebetaine had no inhibitory effect on either transport system. Size-fractionated rat renal-cortex mRNA (median size 2 kb) induced a 3-fold higher L-carnitine transport than did unfractionated mRNA. These studies demonstrate that Xenopus laevis oocytes exhibit Na(+)-dependent L-carnitine transport and provide the basis for expression-cloning of a rat renal Na(+)-dependent L-carnitine transport system.

SUBMITTER: Berardi S

PROVIDER: S-EPMC1135744 | biostudies-other | 1995 Jul

REPOSITORIES: biostudies-other

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Project description:BackgroundA growing number of proteins, including ion transporters, have been shown to interact with Cystic Fibrosis Transmembrane conductance Regulator (CFTR). CFTR is an epithelial chloride channel that is involved in Cystic Fibrosis (CF) when mutated; thus a better knowledge of its functional interactome may help to understand the pathophysiology of this complex disease. In the present study, we investigated if CFTR and the sodium-phosphate co-transporter type 2a (NPT2a) functionally interact after heterologous expression of both proteins in Xenopus laevis oocytes.Methodology/findingsNPT2a was expressed alone or in combination with CFTR in X. laevis oocytes. Using the two-electrode voltage-clamp technique, the inorganic phosphate-induced current (IPi) was measured and taken as an index of NPT2a activity. The maximal IPi for NPT2a substrates was reduced when CFTR was co-expressed with NPT2a, suggesting a decrease in its expression at the oolemna. This was consistent with Western blot analysis showing reduced NPT2a plasma membrane expression in oocytes co-expressing both proteins, whereas NPT2a protein level in total cell lysate was the same in NPT2a- and NPT2a+CFTR-oocytes. In NPT2a+CFTR- but not in NPT2a-oocytes, IPi and NPT2a surface expression were increased upon PKA stimulation, whereas stimulation of Exchange Protein directly Activated by cAMP (EPAC) had no effect. When NPT2a-oocytes were injected with NEG2, a short amino-acid sequence from the CFTR regulatory domain that regulates PKA-dependent CFTR trafficking to the plasma membrane, IPi values and NPT2a membrane expression were diminished, and could be enhanced by PKA stimulation, thereby mimicking the effects of CFTR co-expression.Conclusion/perspectivesWe conclude that when both CFTR and NPT2a are expressed in X. laevis oocytes, CFTR confers to NPT2a a cAMPi-dependent trafficking to the membrane. This functional interaction raises the hypothesis that CFTR may play a role in phosphate homeostasis.

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Characterization of the endogenous carnitine transport and expression of a rat renal Na(+)-dependent carnitine transport system in Xenopus laevis oocytes.

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