Cloning of a Na/Pi cotransporter from opossum kidney cells.

V Sorribas,D Markovich,G Hayes,G Stange,J Forgo,J Biber,H Murer

doi:10.1016/s0021-9258(17)37417-3

Abstract

Opossum kidney (OK) cells have been extensively used to study cellular mechanisms of renal proximal tubular Na/P(i) cotransport. We have cloned a cDNA (NaPi-4) most likely encoding an apical Na/P(i) cotransporter from OK cells. The cloning strategy was based on homology to the recently cloned human renal (NaPi-3) Na/P(i) cotransporter (Magagnin, S., Werner, A., Markovich, D., Sorribas, V., Stange, G., Biber, J., and Murer, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5979-5983). Kinetic characterization (P(i) interaction, sodium interaction, and pH dependence) of NaPi-4-induced Na/P(i) uptake showed high similarity to apical Pi transport in OK cell monolayers. The NaPi-4 cDNA is 2548 base pairs long and encodes a protein of 70.5 kDa, containing at least 8 predicted transmembrane domains. Northern blot analysis with OK cell mRNA shows a NaPi-4-related signal (2.5 kilobases) in cells grown on impermeant and permeant supports. Hybrid depletion with NaPi-4 antisense oligonucleotides abolished the mRNA-induced Na/P(i) cotransport in oocytes. Similarly, NaPi-4 antisense oligonucleotides inhibited (up to 70%) Na/P(i) cotransport in OK cell monolayers. We presume that NaPi-4 is closely related to the OK cell apical Na/P(i) cotransporter.

Highlights

NaPi-4 antisense oligonucleo-OK cDNA Library Screening-Poly(A)+ RNA isolated from OK cells tides inhibited Na/Pi cotransport in OK cell grown on permeant collagen-coatedfilters [6]was fractionated through monolayers.We presume that NaPi-i4s closely the OK cell apical Na/Pi cotransporter
The mRNAinduced Na/Pi cotransport in oocytes was abolished by both antisense ODNs(located at 5' and 3' ends within the open reading frame), witnho effect provoked by sense ODNs (Fig.6)
These experiments suggest thaat NaPi-4-related transcript is responsible for the OK cell mRNA-induced N O i cotransport in oocytes

Summary

RESULTS

Cloning and Substrate Specificity-By homology screening, using NaPi-3 as a probe, we identified 4 positive clones from a n. 20 and 21) reveals at least eight putative transmembrane segments (MlM8; Fig. 2 B ) , in close agreement to predicted profiles for recently cloned NaPi-2/3 systems [1].Assuming that the N H 2 terminus is located at the cytoplasmic surface and that 3 potential N-glycosylation sites (positions 300, 325, and 332) are used (positioned extracellularly), there would be 3 transmembrane domains NHz-terminally located to the above glycosylation sites In such a model, six putative protein kinaseC sites would be exposed intracellularly. The mRNAinduced Na/Pi cotransport in oocytes was abolished by both antisense ODNs(located at 5' and 3' ends within the open reading frame), witnho effect provoked by sense ODNs (Fig.6) These experiments suggest thaat NaPi-4-related transcript is responsible for the OK cell mRNA-induced N O i cotransport in oocytes (see Ref. 13). Maximal inhibitionwas observed with 20 p~PT-ODNs (63% inhibition after days of incubation; Fig. 7B)

DISCUSSION

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