Functional differences between flounder and rat thiazide-sensitive Na-Cl cotransporter

Abstract

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.

A Single Nucleotide Polymorphism Alters the Activity of the Renal Na+:Cl- Cotransporter and Reveals a Role for Transmembrane Segment 4 in Chloride and Thiazide Affinity

“…This expression system has been shown to be an excellent tool for a robust and reproducible expression of TSC in our hands (2,12,16,24,25,32) and also in other laboratories (13,27,33,34). In contrast, TSC expression in transfected eukaryotic cells has not been successful in many laboratories, including our own.…”

“…Supporting this hypothesis, we have shown that in TSC the higher affinity for chloride is accompanied by a higher affinity for thiazide diuretics. On one hand, the affinity for thiazides is shifted to the left when dose-response curves are performed in low extracellular Cl Ϫ concentrations (16,32), and on the other hand, the prevention of glycosylation in rat TSC increases the affinity for both extracellular Cl Ϫ and thiazides (24). In the present study, we observed that G264A substitution produces a dramatic increase in Cl Ϫ affinity, together with an increase in the affinity for the thiazide-type diuretic metolazone.…”

Functional Expression of the Human Thiazide-Sensitive NaCl Cotransporter in Madin-Darby Canine Kidney Cells

“…This shows that PKC does not act directly or indirectly on hNCC activity and/or cell surface expression in these cells. In contrast, PMA was found to markedly decrease Na ϩ uptake in Xenopus laevis oocytes expressing either flounder (20) or rat (21) NCC. This suggests that PKC affects NCC activity and/or trafficking differentially in polarized MDCK cells and oocytes.…”

“…Of all species studied thus far, only hNCC contains a potential phosphorylation site for PKA (36). This explains the lack of effect of cAMP on thiazide-sensitive Na ϩ uptake in Xenopus laevis oocytes expressing flounder NCC (20) and suggests that this site is important in cAMP-dependent activation of hNCCmediated Na ϩ transport. At present, it is unclear whether and which hormones or paracrine factors act through cAMP to regulate hNCC in DCT.…”

“…By use of this expression system, it was found that neither cyclic adenosine monophosphate (cAMP) nor cyclic guanosine monophosphate (cGMP) affected Na ϩ uptake mediated by flounder NCC (11). On the other hand, the phorbol ester phorbol 12-myriatate 13-acetate (PMA), which is a potent activator of protein kinase C (PKC), markedly decreased Na ϩ uptake by oocytes expressing either flounder (20) or rat (21) NCC. It was not investigated, however, whether PKC decreased the activity or the plasma membrane expression of NCC.…”