The European and Japanese eel NaCl cotransporters exhibit chloride currents and are resistant to thiazide type diuretics

The thiazide-sensitive Na+-Cl- cotransporter (NCC) is the major pathway for salt reabsorption in the mammalian distal convoluted tubule, and the inhibition of its function with thiazides is widely used for the treatment of arterial hypertension. In mammals and teleosts, NCC is present as one ortholog that is mainly expressed in the kidney. One exception, however, is the eel, which has two genes encoding NCC. The eNCC alpha is located in the kidney and eNCC beta, which is present in the apical membrane of the rectum. Interestingly, the European eNCCb functions as a Na+-Cl- cotransporter that is nevertheless resistant to thiazides and is not activated by low-chloride hypotonic stress. However, in the Japanese eel rectal sac, a thiazide-sensitive NaCl transport mechanism has been described. The protein sequences between eNCC beta and jNCC beta are 98% identical. Here, by site-directed mutagenesis, we transformed eNCC beta into jNCC beta. Our data showed that jNCC beta, similar to eNCC beta, is resistant to thiazides. In addition, both NCC beta proteins have high transport capacity with respect to their renal NCC orthologs and, in contrast to known NCCs, exhibit electrogenic properties that are reduced when residue I172 is substituted by A, G, or M. This is considered a key residue for the chloride ion-binding sites of NKCC and KCC. We conclude that NCCb proteins are not sensitive to thiazides and have electrogenic properties dependent on Cl-, and site I172 is important for the function of NCC beta.