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Pflugers Arch
2019 Jan 01;4711:7-14. doi: 10.1007/s00424-018-2198-9.
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Expression cloning human and rat renal cortex Na/Pi cotransporters: behind the scenes in the Murer laboratory.
Magagnin S
,
Werner A
.
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In the pre-genomic era, the cloning of a cDNA represented a significant achievement, particularly if the gene of interest encoded a membrane protein. At the time, molecular probes such as partial peptide sequences, suitable nucleic acid sequences, or antibodies were unavailable for most proteins and the "sodium-phosphate transporter" was no exception. In contrast, brush-border membrane vesicles and epithelial cell culture experiments had established a reliable set of functional hallmarks that described Na-dependent phosphate transport activity in some detail. Moreover, aspects of hormonal regulation of phosphate homeostasis could be recapitulated in these model systems. Expression cloning elegantly combined functional protein expression in Xenopus laevis oocytes with molecular biology to overcome the lack of molecular probes.
Fig. 1. Schematic representation of the expression cloning strategy. a Summary of the molecular biology arm of the cloning strategy. Reverse transcription was primed using an oligo-dT-NotI adaptor primer. After second strand synthesis, SalI linkers were added, followed by NotI restriction digest. The two different “sticky” ends enabled efficient directional cloning—but excluded the functional cloning of cDNAs that contain an internal NotI site (as, for example, human NaPi-IIc). b Outline of the screening strategy using functional expression in Xenopus oocytes. The left panel explains the steps to successively narrow down the number of clones. Each of the initial plates contained 600–1000 individual bacterial colonies. Sib selection, as exemplified in the second screening step (middle panel), was applied to reduce the number of injections. The right panel shows the uptake results obtained with the RNA synthesized from the related plasmid pools
Fig. 2. Characterization of the human cDNA. a Functional test of in vitro-transcribed RNA expressed in oocytes. The expressed transporter is specific for Pi and does not accept neither sulfate nor arginine. b Northern blot including RNA from different human tissues (top) or RNA isolated from renal cortex of different species (bottom). The faint band of approximately 5 kb in human lung is likely to reflect the highly expressed SLC34A2 (intestinal) isoform. Mouse appears to express two different renal isoforms derived from alternative polyadenylation. The figure is composed of original material published by Magagnin et al. [16]
Fig. 3. Functional characterization of NaPi-3, the human Na-Pi cotransporter. In vitro-transcribed RNA was expressed in Xenopus oocytes and transport activity measured by radiotracer flux under different experimental conditions. a Varying concentrations of Pi were included and transport showed hyperbolic saturation kinetics indicating that one Pi molecule was transported per cycle (apparent Km 0.170 mM). b Na kinetics showed a sigmoidal shape pointing to cooperative binding of at least 2 Na+ ions per transport cycle. c Transport showed pronounced pH dependence with maximal transport rates at neutral pH and inhibition in acidic conditions. Figure from original material published by Magagnin et al. [16]
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