Activity of the renal Na⁺-K⁺-2Cl⁻cotransporter is reduced by mutagenesis ofN-glycosylation sites: role for protein surface charge in Cl⁻transport

Abstract

The renal-specific Na(+)-K(+)-2Cl(-) cotransporter NKCC2 belongs to the SLC12 gene family; it 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 (86)Rb(+) influx, Western blotting, and confocal microscopy. Inhibition of glycosylation with tunicamycin in wild-type NKCC2-injected oocytes resulted in an 80% reduction of NKCC2 activity. Immunoblot of injected oocytes revealed that glycosylation of NKCC2 was completely prevented in N442,452Q-injected oocytes. Functional activity was reduced by 50% in N442Q- and N452Q-injected oocytes and by 80% in oocytes injected with N442,452Q, whereas confocal microscopy of oocytes injected with wild-type or mutant enhanced green fluorescent protein-tagged NKCC2 clones revealed that surface fluorescence intensity was reduced approximately 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 N442Q 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 insertion into the plasma membrane and the intrinsic activity of the cotransporter.

Regulation of the NKCC2 ion cotransporter by SPAK-OSR1-dependent and -independent pathways

“…We calculated that bumetanide inhibited NKCC2 isoform B in HEK-293 cells with an IC 50 value of 0.54 mM (supplementary material Fig. S4), broadly consistent with previously reported values obtained employing Xenopus laevis oocytes as an heterologous expression system (Carota et al, 2010;Paredes et al, 2006;Plata et al, 2002). Individual mutation of Ser91, Thr95 or Thr100 did not significantly affect NKCC2 activity (Fig.…”

Membrane-associated aquaporin-1 facilitates osmotically driven water flux across the basolateral membrane of the thick ascending limb

“…In addition, it remains unclear why our data showed an approximately threefold difference in dDAVP-stimulated expression of glycosylated vs. nonglycosylated AQP1; however, it could be that glycosylation determines where AQP1 is located within cell compartments, as with other membrane-associated proteins (13,21,40). All these possibilities are beyond the scope of this manuscript but merit further investigation.…”

NKCC2 Surface Expression in Mammalian Cells

“…As negative controls, these bands were not detected neither in mock-transfected cells nor when immunoprecipitation was carried out using anti-V5 or anti-HA antibodies indicating that the observed signal is specific to Myc-NKCC2 fusion protein. Cells treatment with peptide:N-glycosidase and endoglycosidase H (data not shown) revealed that these bands correspond to the complex-glycosylated and core-glycosylated forms of the NKCC2 protein (2,43). To further confirm this, we mutated the two predicted glycosylation sites, Asn-442 and Asn-452 to glutamine, and assessed the effect of these mutations on NKCC2 glycosylation by immunoblot.…”