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

Robert S. Hoover, Esteban Poch^¶, Adriana Monroy^*, Norma Vázquez^*, Toshiyuki Nishio, Gerardo Gamba^* and Steven C. Hebert

*Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán and Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico; Department of Cellular and Molecular Physiology, Yale University Medical School, New Haven, Connecticut; Department of Pediatrics, Tohoku University School of Medicine, Sendai, Japan; and ^¶ Servei de Nefrologia, Hospital Clinic, Universitat de Barcelona, Barcelona, Spain.

Correspondence to Dr. Steven C. Hebert, Professor and Chairman, Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street P.O. Box 208026, New Haven, CT 06520-8026. Phone: 203-785-6696; Fax: 203-785-7678;

ABSTRACT. 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 ²²Na⁺ 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. E-mail: steven.hebert@yale.edu

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