title: A single residue in transmembrane domain 11 defines the different affinity for thiazides between the mammalian and flounder NaCl transporters.
abstract: 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.
authors: Molecular Physiology Unit, Vasco de Quiroga No. 15, Tlalpan 14000, Mexico City, Mexico.
authors: María
authors: M
authors: Castañeda-Bueno
authors: Norma
authors: N
authors: Vázquez
authors: Ismael
authors: I
authors: Bustos-Jaimes
authors: Damian
authors: D
authors: Hernández
authors: Erika
authors: E
authors: Rodríguez-Lobato
authors: Diana
authors: D
authors: Pacheco-Alvarez
authors: Raquel
authors: R
authors: Cariño-Cortés
authors: Erika
authors: E
authors: Moreno
authors: Norma A
authors: NA
authors: Bobadilla
authors: Gerardo
authors: G
authors: Gamba
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D015153:Blotting, Western; D004232:Diuretics / Q000378:metabolism*; D005432:Flounder / Q000378:metabolism*; D006801:Humans; D008788:Metolazone / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000502:physiology; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D011817:Rabbits; D051381:Rats; D049992:Sodium Chloride Symporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013045:Species Specificity; D049971:Thiazides / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D004232:Diuretics; D049992:Sodium Chloride Symporters; D049971:Thiazides; D008788:Metolazone