N-Glycosylation at Two Sites Critically Alters Thiazide Binding and Activity of the Rat Thiazide-sensitive Na+:Cl- Cotransporter

R. S. Hoover
2003 Journal of the American Society of Nephrology  
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
more » ... kD, consistent with the highmannose 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 ϩ uptake compared with wild-type NCC. Analysis of enhanced green fluorescence protein (EGF-P)-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 Ϫ 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. Sodium chloride cotransport sensitive to thiazides is the dominant mechanism mediating Na ϩ and Cl Ϫ reabsorption in the early distal convoluted tubule of the mammalian kidney, as has been demonstrated by in vivo microperfusion studies (1). The thiazide-sensitive Na-Cl cotransporter (NCC) has been cloned by our group from the winter flounder urinary bladder (2) and from the rat kidney (3). More recently the cotransporter was cloned from human (4) and mouse (5) kidney. NCC belongs to the electroneutral cotransporter family of proteins that also includes the cloned bumetanide-sensitive Na-K-2Cl cotransporters (3,6,7) and the more recently cloned K ϩ -Cl Ϫ cotrans-porters (8 -11). The members of this family share a great degree of similarity in amino acid sequence and proposed topology. Functional expression of NCC in Xenopus laevis oocytes yielded 22 Na ϩ uptake that was Cl Ϫ -dependent and thiazide-sensitive (2,3,12), and a polyclonal antibody raised against the N-terminus of the rNCC protein immunolocalized the cotransporter to the apical membrane of the distal convoluted tubule (13). In addition, several mutations in the coding sequence of the human NCC gene have been genetically linked to the Gitelman syndrome variant of familial hypokalemic metabolic alkalosis, demonstrating the relevance of this cotransporter to human pathophysiolgy (4). Moreover, this cotransporter is the site of action of one of the most commonly used classes of anti-hypertensive agents, the thiazide diuretics. On the basis of hydropathy analysis, the basic topology of the Na-Cl and Na-K-2Cl cotransporters consists of a central hydrophobic core region containing 12 putative transmembrane-spanning segments between long hydrophilic, putatively intracellular amino and carboxyl termini. Some experimental evidence exists that this is the correct topology for the Na-K-2Cl cotransporter (14). The predicted molecular weight of the core NCC protein is 110 kD (3). However, the higher molec-
doi:10.1097/01.asn.0000043903.93452.d0 pmid:12538726 fatcat:mslxjnzp4nfubcx7wgkmgqpm4a