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1 Molecular Physiology Unit, Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico and Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubrian, Tlalpan, Mexico City, Mexico
2 Departamento de Bioquimica, Facultad de Quimica, Universidad Nacional Autonoma de Mexico, Coyoacan, Mexico City, Mexico
3 Department of Cellular and Molecular Physiology, Yale University Medical School, New Haven, Connecticut, USA
* To whom correspondence should be addressed. E-mail: gamba{at}biomedicas.unam.mx.
The renal-specific Na+:K+:2Cl- cotransporter NKCC2 belongs to the SLC12 gene family, is the target for loop diuretics and the cause of type I Bartter's syndrome. Because the NKCC2 sequence contains two putative N-linked glycosylation sites, one of which is conserved with the renal Na+:Cl- cotransporter in which glycosylation affects thiazide affinity, we assessed the role of glycosylation on NKCC2 functional properties. One (N442Q or N452Q) or both (N442,452Q) N-glycosylation sites were eliminated by site-directed mutagenesis. Wild type NKCC2 and mutant clones were expressed in Xenopus laevis oocytes and analyzed by 86Rb+ influx, Western blot and confocal microscopy. Inhibition of glycosylation with tunicamycin in the wild type NKCC2-injected oocytes resulted in 80% reduction of NKCC2 activity. Immunoblot of injected oocytes revealed that glycosylation of NKCC2 was completely prevented in the N442,452Q-injected oocytes. Functional activity was reduced by 50% in N442Q and N452Q, and 80% in N442,452Q, while confocal microscopy of injected oocytes with wild type or mutant EGFP-tagged NKCC2 clones revealed that surface fluorescence intensity was reduced ~20% in single mutants and 50% in the double mutant. Ion transport kinetic analyses revealed no changes in cation affinity and a small increase in Cl- affinity by N442A and N442,452Q. However, a slight decrease in bumetanide affinity was observed. Our data demonstrate that NKCC2 is glycosylated and suggest that prevention of glycosylation reduces its functional expression by affecting both the insertion into plasma membrane and the intrinsic activity of the cotransporter.
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