The Na+:Cl- cotransporter is activated and phosphorylated at the amino-terminal domain upon intracellular chloride depletion.
The renal Na(+):Cl(-) cotransporter rNCC is mutated in human disease, is the therapeutic target of thiazide-type diuretics, and is clearly involved in arterial blood pressure regulation. rNCC belongs to an electroneutral cation-coupled chloride cotransporter family (SLC12A) that has two major branches with inverse physiological functions and regulation: sodium-driven cotransporters (NCC and NKCC1/2) that mediate cellular Cl(-) influx are activated by phosphorylation, whereas potassium-driven cotransporters (KCCs) that mediate cellular Cl(-) efflux are activated by dephosphorylation. A cluster of three threonine residues at the amino-terminal domain has been implicated in the regulation of NKCC1/2 by intracellular chloride, cell volume, vasopressin, and WNK/STE-20 kinases. Nothing is known, however, about rNCC regulatory mechanisms. By using rNCC heterologous expression in Xenopus laevis oocytes, here we show that two independent intracellular chloride-depleting strategies increased rNCC activity by 3-fold. The effect of both strategies was synergistic and dose-dependent. Confocal microscopy of enhanced green fluorescent protein-tagged rNCC showed no changes in rNCC cell surface expression, whereas immunoblot analysis, using the R5-anti-NKCC1-phosphoantibody, revealed increased phosphorylation of rNCC amino-terminal domain threonine residues Thr(53) and Thr(58). Elimination of these threonines together with serine residue Ser(71) completely prevented rNCC response to intracellular chloride depletion. We conclude that rNCC is activated by a mechanism that involves amino-terminal domain phosphorylation.
Pacheco-Alvarez
Diana
D
Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónomade México, Tlalpan 14000, Mexico City, Mexico.
Cristóbal
Pedro San
PS
Meade
Patricia
P
Moreno
Erika
E
Vazquez
Norma
N
Muñoz
Eva
E
Díaz
Abigail
A
Juárez
María Eugenia
ME
Giménez
Ignacio
I
Gamba
Gerardo
G
D000595:Amino Acid Sequence; D000818:Animals; D002712:Chlorides / Q000737:chemistry / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D049992:Sodium Chloride Symporters / Q000378:metabolism*; D013912:Threonine / Q000737:chemistry / Q000378:metabolism; D014982:Xenopus laevis
D002712:Chlorides; D049992:Sodium Chloride Symporters; D013912:Threonine