Activity of the renal Na+-K+-2Cl- cotransporter is reduced by mutagenesis of N-glycosylation sites: role for protein surface charge in Cl- transport.
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.
Paredes
Anahí
A
Molecular Physiology Unit, Vasco de Quiroga No. 15, Tlalpan 14000, México City, Mexico.
Plata
Consuelo
C
Rivera
Manuel
M
Moreno
Erika
E
Vázquez
Norma
N
Muñoz-Clares
Rosario
R
Hebert
Steven C
SC
Gamba
Gerardo
G
D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000502:physiology; D018118:Chloride Channels / Q000502:physiology*; D003001:Cloning, Molecular; D004594:Electrophysiology; D006031:Glycosylation; D015151:Immunoblotting; D007700:Kinetics; D008138:Loop of Henle / Q000502:physiology*; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D009865:Oocytes; D020033:Protein Isoforms; D051381:Rats; D028021:Sodium-Potassium-Chloride Symporters / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D064466:Solute Carrier Family 12, Member 1; D014982:Xenopus laevis
D018118:Chloride Channels; D020033:Protein Isoforms; C578716:Slc12a1 protein, rat; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1