Molecular Physiology Unit, Vasco de Quiroga No. 15, Tlalpan 14000, Mexico City, Mexico.
Am J Physiol Renal Physiol. 2010 Nov;299(5):F1111-9. doi: 10.1152/ajprenal.00412.2010. Epub 2010 Aug 18.
Little is known about the residues that control the binding and affinity of thiazide-type diuretics for their protein target, the renal Na(+)-Cl(-) cotransporter (NCC). Previous studies from our group have shown that affinity for thiazides is higher in rat (rNCC) than in flounder (flNCC) and that the transmembrane region (TM) 8-12 contains the residues that produce this difference. Here, an alignment analysis of TM 8-12 revealed that there are only six nonconservative variations between flNCC and mammalian NCC. Two are located in TM9, three in TM11, and one in TM12. We used site-directed mutagenesis to generate rNCC containing flNCC residues, and thiazide affinity was assessed using Xenopus laevis oocytes. Wild-type or mutant NCC activity was measured using (22)Na(+) uptake in the presence of increasing concentrations of metolazone. Mutations in TM11 conferred rNCC an flNCC-like affinity, which was caused mostly by the substitution of a single residue, S575C. Supporting this observation, the substitution C576S conferred to flNCC an rNCC-like affinity. Interestingly, the S575C mutation also rendered rNCC more active. Substitution of S575 in rNCC for other residues, such as alanine, aspartate, and lysine, did not alter metolazone affinity, suggesting that reduced affinity in flNCC is due specifically to the presence of a cysteine. We conclude that the difference in metolazone affinity between rat and flounder NCC is caused mainly by a single residue and that this position in the protein is important for determining its functional properties.
关于噻嗪类利尿剂与蛋白靶标——肾脏钠-氯共转运体（NCC）结合和亲和力的控制残基知之甚少。我们之前的研究表明，与 flNCC 相比，噻嗪类药物在大鼠（rNCC）中的亲和力更高，并且 TM8-12 区含有产生这种差异的残基。在此，对 TM8-12 的序列比对分析表明，flNCC 和哺乳动物 NCC 之间只有 6 个非保守性差异。其中 2 个位于 TM9，3 个位于 TM11，1 个位于 TM12。我们使用定点突变技术生成了含有 flNCC 残基的 rNCC，并使用 Xenopus laevis 卵母细胞评估了噻嗪类药物的亲和力。野生型或突变型 NCC 的活性通过在存在不同浓度的美托拉宗的情况下测量（22）Na+摄取来评估。TM11 中的突变使 rNCC 具有类似于 flNCC 的亲和力，这主要是由于单个残基 S575C 的取代所致。这一观察结果得到了支持，因为 C576S 的取代赋予了 flNCC 类似于 rNCC 的亲和力。有趣的是，S575C 突变也使 rNCC 更活跃。在 rNCC 中，将 S575 替换为其他残基，如丙氨酸、天冬氨酸和赖氨酸，不会改变美托拉宗的亲和力，这表明 flNCC 中亲和力的降低是由于半胱氨酸的存在所致。我们得出结论，大鼠和比目鱼 NCC 中美托拉宗亲和力的差异主要是由单个残基引起的，该位置对于确定其功能特性很重要。
