N-Glycosylation at two sites critically alters thiazide binding and activity of the rat thiazide-sensitive Na(+):Cl(-) cotransporter.
The rat thiazide-sensitive Na-Cl cotransporter (rNCC) is expressed in the renal distal convoluted tubule and is the site of action of an important class of antihypertensive agents, the thiazide diuretics. The amino acid sequence contains two potential N-linked glycosylation consensus sites, N404 and N424. Either enzymatic deglycosylation or tunicamycin reduced the cotransporter to its core molecular weight (113 kD). Glycosylation site single mutants expressed in oocytes ran as thick bands at 115 kD, consistent with the high-mannose glycoprotein. The double mutant produced the single thin 113-kD band seen in the deglycosylated cotransporter. Functional expression of cotransporters in Xenopus laevis oocytes revealed that the mutants displayed drastically decreased thiazide-sensitive (22)Na(+) uptake compared with wild-type NCC. Analysis of enhanced green fluorescence protein (EGFP)-tagged cotransporters demonstrated that this decrease in function is predominantly secondary to decreased surface expression. The elimination of glycosylation in the double mutant increased thiazide sensitivity by more than two orders of magnitude and also increased Cl(-) affinity. Thus, we have demonstrated that rNCC is N-glycosylated in vivo at two sites, that glycosylation is essential for efficient function and surface expression of the cotransporter, and that the elimination of glycosylation allows much greater access of thiazide diuretics to their binding site.
Hoover
Robert S
RS
Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico.
Poch
Esteban
E
Monroy
Adriana
A
Vázquez
Norma
N
Nishio
Toshiyuki
T
Gamba
Gerardo
G
Hebert
Steven C
SC
D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002712:Chlorides / Q000493:pharmacokinetics; D005260:Female; D006031:Glycosylation; D008297:Male; D009154:Mutation; D051381:Rats; D017207:Rats, Sprague-Dawley; D011955:Receptors, Drug / Q000235:genetics / Q000378:metabolism*; D012964:Sodium / Q000493:pharmacokinetics; D049992:Sodium Chloride Symporters; D064486:Solute Carrier Family 12, Member 3; D027981:Symporters; D013829:Thiadiazines / Q000378:metabolism*; D014982:Xenopus laevis
D002352:Carrier Proteins; D002712:Chlorides; D011955:Receptors, Drug; C578876:Slc12a3 protein, rat; D049992:Sodium Chloride Symporters; D064486:Solute Carrier Family 12, Member 3; D027981:Symporters; D013829:Thiadiazines; C064415:thiazide receptor; D012964:Sodium