Functional differences between flounder and rat thiazide-sensitive Na-Cl cotransporter.
The purpose of the present study was to determine the major functional, pharmacological, and regulatory properties of the flounder thiazide-sensitive Na-Cl cotransporter (flTSC) to make a direct comparison with our recent characterization of the rat TSC (rTSC; Monroy A, Plata C, Hebert SC, and Gamba G. Am J Physiol Renal Physiol 279: F161-F169, 2000). When expressed in Xenopus laevis oocytes, flTSC exhibits lower affinity for Na(+) than for Cl(-), with apparent Michaelis-Menten constant (K(m)) values of 58.2 +/- 7.1 and 22.1 +/- 4.2 mM, respectively. These K(m) values are significantly higher than those observed in rTSC. The Na(+) and Cl(-) affinities decreased when the concentration of the counterion was lowered, suggesting that the binding of one ion increases the affinity of the transporter for the other. The effect of several thiazides on flTSC function was biphasic. Low concentrations of thiazides (10(-9) to 10(-7) M) resulted in activation of the cotransporter, whereas higher concentrations (10(-6) to 10(-4) M) were inhibitory. In rTSC, this biphasic effect was observed only with chlorthalidone. The affinity for thiazides in flTSC was lower than in rTSC, but the affinity in flTSC was not affected by the Na(+) or the Cl(-) concentration in the uptake medium. In addition to thiazides, flTSC and rTSC were inhibited by Hg(2+), with an apparent higher affinity for rTSC. Finally, flTSC function was decreased by activation of protein kinase C with phorbol esters and by hypertonicity. In summary, we have found significant regulatory, kinetic, and pharmacological differences between flTSC and rTSC orthologues.
Vázquez
Norma
N
Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán and Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Tlalpan 14000, Mexico City, Mexico.
Monroy
Adriana
A
Dorantes
Elisa
E
Muñoz-Clares
Rosario A
RA
Gamba
Gerardo
G
D000818:Animals; D001581:Benzothiadiazines; D002752:Chlorthalidone / Q000494:pharmacology; D004232:Diuretics; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005432:Flounder / Q000378:metabolism*; D006982:Hypertonic Solutions; D066298:In Vitro Techniques; D007700:Kinetics; D008628:Mercury / Q000633:toxicity; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D049993:Sodium Chloride Symporter Inhibitors / Q000494:pharmacology*; D028021:Sodium-Potassium-Chloride Symporters / Q000187:drug effects / Q000378:metabolism*; D013045:Species Specificity; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
D001581:Benzothiadiazines; D004232:Diuretics; D006982:Hypertonic Solutions; D049993:Sodium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D011493:Protein Kinase C; D008628:Mercury; D002752:Chlorthalidone