Serine 575 accounts for difference in thiazide affinity between mammalian and flounder NCC

Thiazide diuretics are recommended as the first line pharmacological therapy for the treatment of arterial hypertension. Little is known, however, about residues defining the binding and/or affinity for thiazide type diuretics into its protein target, the renal Na‐Cl cotransporter (NCC). We have previously shown that affinity for thiazides is higher in rat (rNCC) than in flounder (flNCC). A chimeric analysis approach between flNCC and rNCC showed that transmembrane regions (TM) 8–12 contain information defining the difference in metolazone affinity (Moreno et al. JBC 2006). In the present study, alignment analysis revealed that there are only 6 non‐conservative variations between flNCC and mammalian NCC in TM 8–12, located as follows: two in TM9, three in TM11, and one in TM12. We used site directed mutagenesis to generate rNCC containing flNCC residues and thiazide affinity was defined using Xenopus laevis oocytes in which wild type or mutant NCC activity was assessed by measuring 22 Na + uptake in the presence of increasing concentrations of metolazone. We observed that mutations in TM11 conferred rNCC an flNCC‐like affinity, which could be explained solely by the substitution of a single residue, the S575C. Supporting this observation, the substitution C576S conferred flNCC an rNCC‐like affinity. Interestingly, S575C mutation also rendered rNCC more active. Mutation of S575 in rNCC for other residues such as alanine, aspartate, and lysine did not alter metolazone affinity, suggesting that reduced affinity in flNCC could be due to the formation of a disulfide bond. We conclude that difference in metolazone affinity between rat and flounder NCC can be explained by a single residue.