Moreno Erika, Cristóbal Pedro San, Rivera Manuel, Vázquez Norma, Bobadilla Norma A, Gamba Gerardo
Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México Tlalpan 14000, Mexico City, Mexico; Instituto de Ciencias de la Salud, Universidad Autónoma del Estado de Hidalgo, Pachuca, Hidalgo 42160, México.
Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México Tlalpan 14000, Mexico City, Mexico.
J Biol Chem. 2006 Jun 23;281(25):17266-17275. doi: 10.1074/jbc.M602614200. Epub 2006 Apr 19.
The thiazide-sensitive Na+-Cl- cotransporter (NCC) is the major pathway for salt reabsorption in the distal convoluted tubule, serves as a receptor for thiazide-type diuretics, and is involved in inherited diseases associated with abnormal blood pressure. Little is known regarding the structure-function relationship in this cotransporter. Previous studies from our group reveal that mammalian NCC exhibits higher affinity for ions and thiazides than teleost NCC and suggest a role for glycosylation upon thiazide affinity. Here we have constructed a series of chimeric and mutant cDNAs between rat and flounder NCC to define the role of glycosylation status, the amino-terminal domain, the carboxyl-terminal domain, the extracellular glycosylated loop, and the transmembrane segments upon affinity for Na+, Cl-, and metolazone. Xenopus laevis oocytes were used as the heterologous expression system. We observed that elimination of glycosylation sites in flounder NCC did not affect the affinity of the cotransporter for metolazone. Also, swapping the amino-terminal domain, the carboxyl-terminal domain, the glycosylation sites, or the entire extracellular glycosylation loop between rat and flounder NCC had no effect upon ions or metolazone affinity. In contrast, interchanging transmembrane regions between rat and flounder NCC revealed that affinity-modifying residues for chloride are located within the transmembrane 1-7 region and for thiazides are located within the transmembrane 8-12 region, whereas both segments seem to be implicated in defining sodium affinity. These observations strongly suggest that binding sites for chloride and thiazide in NCC are different.
噻嗪类敏感的Na⁺-Cl⁻共转运体（NCC）是远曲小管中盐重吸收的主要途径，是噻嗪类利尿剂的受体，并参与与血压异常相关的遗传性疾病。关于该共转运体的结构-功能关系知之甚少。我们小组先前的研究表明，哺乳动物NCC对离子和噻嗪类药物的亲和力高于硬骨鱼NCC，并提示糖基化对噻嗪类药物亲和力有作用。在此，我们构建了一系列大鼠和比目鱼NCC之间的嵌合和突变cDNA，以确定糖基化状态、氨基末端结构域、羧基末端结构域、细胞外糖基化环和跨膜区段对Na⁺、Cl⁻和美托拉宗亲和力的作用。非洲爪蟾卵母细胞用作异源表达系统。我们观察到，去除比目鱼NCC中的糖基化位点并不影响共转运体对美托拉宗的亲和力。此外，交换大鼠和比目鱼NCC之间的氨基末端结构域、羧基末端结构域、糖基化位点或整个细胞外糖基化环对离子或美托拉宗亲和力没有影响。相反，交换大鼠和比目鱼NCC之间的跨膜区域显示，氯离子亲和力修饰残基位于跨膜1-7区域，噻嗪类药物亲和力修饰残基位于跨膜8-12区域，而这两个区段似乎都与确定钠亲和力有关。这些观察结果强烈表明，NCC中氯离子和噻嗪类药物的结合位点不同。
