A single residue substitution confers thiazide sensitivity to the European eel thiazide‐resistant NaCl cotransporter, NCCb isoform

The thiazide sensitive Na‐Cl cotransporter (NCC) is the major pathway for salt reabsorption in the distal convoluted tubule of the mammalian kidney and is the receptor for the thiazide diuretics, which constitute one of the baseline therapies for arterial hypertension worldwide. Yet, the binding site for thiazides at the NCC is still unknown. In the eel the NCC gene is duplicated. The NCCα is an orthologue for mammalian NCC and is expressed at the eel's kidney, while the duplicated gene, the NCCβ is expressed along the intestine and is not present in mammals. We recently reported that the NCCβ of the European eel (eNCCβ) is a thiazide‐ resistant NaCl cotransporter (Moreno et al, JBC 2016). Here, we used “clustal omega” software to analyze all the NCC sequences known to this moment and compared them to the eNCCβ, searching for residues that are conserved in all NCCs, except in the eNCCβ. Using site‐directed mutagenesis we substituted specific amino acid residues in the sequence of eNCCβ, for those that are highly preserved in all other NCC sequences. The mutant cDNAs were sequenced to avoid unwanted mutations. Assessment of the functional expression was performed in Xenopus laevis oocytes, microinjected with cRNA from the wild‐type and mutated eNCCβ, by measuring the tracer 22Na+ uptake in the absence or presence of extracellular chloride or of hydrochlorothiazide. Sixteen different amino acid residues of the eNCCβ, localized at the first seven transmembrane regions (TM), were individually substituted for the corresponding residue in rat NCC. The wild type eNCCβ cRNA injection induced after two days of incubation the expression of Na‐influx pathway that was chloride dependent, but not affected by thiazide diuretics, up to 10‐3 M concentration. In 15 out of 16 mutant clones, NaCl transport activity was present, but no thiazide sensitivity was observed. However, the substitution of a phenylalanine for a tyrosine at the position 174 (eNCCβ‐F174Y), which is localized at the extracellular loop that binds TM1 to TM2, conferred over 90% of inhibition with hydrochlorothiazide, with an IC50 of about 10‐7 M. In addition, the tracer 22Na+ uptake induced by eNCCβ‐F174Y was inhibited by five different thiazide diuretic tested, but not by furosemide. A substitution of a phenylalanine for tyrosine residue at position 174 of the eel NCCβ isoform confers sensibility to thiazide diuretics in the eel NCCβ cotransporter. Support or Funding Information Supported by Conacyt grant numbers 222918 and 188712 to EM and GG, respectively This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .