Ion and diuretic specificity of chimeric proteins between apical Na(+)-K(+)-2Cl(-) and Na(+)-Cl(-) cotransporters.
The mammalian kidney bumetanide-sensitive Na(+)-K(+)-2Cl(-) and thiazide-sensitive Na(+)-Cl(-) cotransporters are the major pathways for salt reabsorption in the thick ascending limb of Henle's loop and distal convoluted tubule, respectively. These cotransporters serve as receptors for the loop- and thiazide-type diuretics, and inactivating mutations of corresponding genes are associated with development of Bartter's syndrome type I and Gitleman's disease, respectively. Structural requirements for ion translocation and diuretic binding specificity are unknown. As an initial approach for analyzing structural determinants conferring ion or diuretic preferences in these cotransporters, we exploited functional differences and structural similarities between Na(+)-K(+)-2Cl(-) and Na(+)-Cl(-) cotransporters to design and study chimeric proteins in which the NH(2)-terminal and/or COOH-terminal domains were switched between each other. Thus six chimeric proteins were produced. Using the heterologous expression system of Xenopus laevis oocytes, we observed that four chimeras exhibited functional activity. Our results revealed that, in the Na(+)-K(+)-2Cl(-) cotransporter, ion translocation and diuretic binding specificity are determined by the central hydrophobic domain. Thus NH(2)-terminal and COOH-terminal domains do not play a role in defining these properties. A similar conclusion can be suggested for the Na(+)-Cl(-) cotransporter.
Tovar-Palacio
Claudia
C
Molecular Physiology Unit, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Tlalpan 14000, Mexico City, Mexico.
Bobadilla
Norma A
NA
Cortés
Paulina
P
Plata
Consuelo
C
de los Heros
Paola
P
Vázquez
Norma
N
Gamba
Gerardo
G
D000818:Animals; D001581:Benzothiadiazines; D002034:Bumetanide / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016764:Cell Polarity / Q000502:physiology; D003001:Cloning, Molecular; D004232:Diuretics / Q000494:pharmacology; D005260:Female; D008138:Loop of Henle / Q000378:metabolism*; D016296:Mutagenesis; D009865:Oocytes; D017434:Protein Structure, Tertiary; D051381:Rats; D011955:Receptors, Drug / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D049993:Sodium Chloride Symporter Inhibitors / Q000494:pharmacology; D049992:Sodium Chloride Symporters; D028021:Sodium-Potassium-Chloride Symporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D064466:Solute Carrier Family 12, Member 1; D064486:Solute Carrier Family 12, Member 3; D027981:Symporters; D014982:Xenopus laevis
D001581:Benzothiadiazines; D002352:Carrier Proteins; D004232:Diuretics; D011955:Receptors, Drug; D011993:Recombinant Fusion Proteins; C578716:Slc12a1 protein, rat; C578876:Slc12a3 protein, rat; D049993:Sodium Chloride Symporter Inhibitors; D049992:Sodium Chloride Symporters; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; D064486:Solute Carrier Family 12, Member 3; D027981:Symporters; C064415:thiazide receptor; D002034:Bumetanide