Expression of the sodium-myo-inositol cotransporter SMIT2 at the apical membrane of Madin-Darby canine kidney cells.
Myo-inositol is a compatible osmolyte used by cells which are challenged by variations in extracellular osmolarity, as in the renal medulla. In order to accumulate large quantities of this polyol, cells rely on Na(+)-dependent transporters such as SMIT1. We have recently identified a second Na(+)-myo-inositol cotransporter, SMIT2, which presents transport characteristics corresponding to those recently described for the apical membrane of renal proximal tubules. In order to further characterize this transport system, we transfected Madin-Darby canine kidney (MDCK) cells with rabbit SMIT2 cDNA and selected a stable clone with a high expression level. The accumulation of radiolabelled myo-inositol by this cell line is 20-fold larger than that seen in native MDCK cells. The affinity for myo-inositol of MDCK cells transfected with SMIT2 is slightly lower (K(m)= 334 microm) than that found in voltage-clamped Xenopus laevis oocytes expressing SMIT2 (K(m)= 120 microm). Transport studies performed using semipermeable filters showed complete apical targeting of the SMIT2 transporter. This apical localization of SMIT2 was confirmed by transport studies on purified rabbit renal brush border membrane vesicles (BBMVs). Using a purified antibody against SMIT2, we were also able to detect the SMIT2 protein (molecular mass = 66 kDa) in Western blots of BBMVs purified from SMIT2-transfected MDCK cells. SMIT2 activity was also shown to be stimulated 5-fold when submitted to 24 h hypertonic treatment (+200 mosmol l(-1)). The SMIT2-MDCK cell line thus appears to be a promising model for studying SMIT2 biochemistry and regulation.
Bissonnette
Pierre
P
Dép. physiologie, Université de Montréal, C.P. 6128, Succ. Centre-Ville, Montréal, Québec, Canada, H3C3J7. pierre.bissonnette@umontreal.ca
Coady
Michael J
MJ
Lapointe
Jean-Yves
JY
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D008871:Microvilli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011817:Rabbits; D027981:Symporters / Q000096:biosynthesis* / Q000235:genetics; D014982:Xenopus laevis
D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D027981:Symporters; C073884:SLC5A3 protein, human