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Research paper thumbnail of A single residue in transmembrane domain 11 defines the different affinity for thiazides between the mammalian and flounder NaCl transporters

AJP: Renal Physiology, 2010

Little is known about the residues that control the binding and affinity of thiazide-type diureti... more 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 22Na+ 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, th...

Research paper thumbnail of A single residue in transmembrane domain 11 defines the different affinity for thiazides between the mammalian and flounder NaCl transporters

AJP: Renal Physiology, 2010

Little is known about the residues that control the binding and affinity of thiazide-type diureti... more 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 22Na+ 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, th...

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