Hoover Robert S, Poch Esteban, Monroy Adriana, Vázquez Norma, Nishio Toshiyuki, Gamba Gerardo, Hebert Steven C
Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico.
J Am Soc Nephrol. 2003 Feb;14(2):271-82. doi: 10.1097/01.asn.0000043903.93452.d0.
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.
大鼠噻嗪类敏感性钠氯共转运体（rNCC）在肾远曲小管中表达，是一类重要的抗高血压药物——噻嗪类利尿剂的作用位点。其氨基酸序列包含两个潜在的N-连接糖基化共有位点，即N404和N424。酶促去糖基化或衣霉素处理均可使共转运体的分子量降至其核心分子量（113 kD）。在卵母细胞中表达的糖基化位点单突变体在115 kD处呈现为厚条带，与高甘露糖糖蛋白一致。双突变体产生了去糖基化共转运体中所见的单一细的113-kD条带。非洲爪蟾卵母细胞中共转运体的功能表达显示，与野生型NCC相比，突变体的噻嗪类敏感性（22）Na⁺摄取显著降低。对增强型绿色荧光蛋白（EGFP）标记的共转运体的分析表明，功能的这种降低主要是由于表面表达减少所致。双突变体中糖基化的消除使噻嗪类敏感性提高了两个以上数量级，同时也增加了对Cl⁻的亲和力。因此，我们证明了rNCC在体内有两个位点发生N-糖基化，糖基化对于共转运体的有效功能和表面表达至关重要，并且糖基化的消除使噻嗪类利尿剂能够更容易地接近其结合位点。
