The European Eel NCCβ Gene Encodes a Thiazide-resistant Na-Cl Cotransporter

Abstract

The thiazide-sensitive Na-Cl cotransporter (NCC) is the major pathway for salt reabsorption in the mammalian distal convoluted tubule. NCC plays a key role in the regulation of blood pressure. Its inhibition with thiazides constitutes the primary baseline therapy for arterial hypertension. However, the thiazide-binding site in NCC is unknown. Mammals have only one gene encoding for NCC. The eel, however, contains a duplicate gene. NCCα is an ortholog of mammalian NCC and is expressed in the kidney. NCCβ is present in the apical membrane of the rectum. Here we cloned and functionally characterized NCCβ from the European eel. The cRNA encodes a 1043-amino acid membrane protein that, when expressed in Xenopus oocytes, functions as an Na-Cl cotransporter with two major characteristics, making it different from other known NCCs. First, eel NCCβ is resistant to thiazides. Single-point mutagenesis supports that the absence of thiazide inhibition is, at least in part, due to the substitution of a conserved serine for a cysteine at position 379. Second, NCCβ is not activated by low-chloride hypotonic stress, although the unique Ste20-related proline alanine-rich kinase (SPAK) binding site in the amino-terminal domain is conserved. Thus, NCCβ exhibits significant functional differences from NCCs that could be helpful in defining several aspects of the structure-function relationship of this important cotransporter.

Tissue and salinity specific Na+/Cl− cotransporter (NCC) orthologues involved in the adaptive osmoregulation of sea lamprey (Petromyzon marinus)

“…This is further supported by our inability to identify a gene encoding NKCC2 in the published genomes of sea lamprey, western brook lamprey, Pacific lamprey, or Arctic lamprey. We cannot rule out the possibility that a sea lamprey NKCC2 could be insensitive to bumetanide 67 , 68 and that nkcc2 is present but has yet to be detected in the available lamprey genomes. Note that no significant effects in I sc were obtained with either chlorothiazide or hydrochlorothiazide, which were initially employed to target a putative NCC.…”

Structure-function relationships in the sodium chloride cotransporter

“…(D) Amino acid sequence alignments analysis of the ion binding sites in human CCCs. Copied from Nan et al (2022) , supplemented with details from studies done by Pacheco-Alvarez et al (2006) , Moreno et al (2004) , Moreno et al (2006) , Moreno et al (2016) , Moreno et al (2022) , Monroy et al (2000) , Castañeda-Bueno et al (2010) , Tutakhel et al (2018) . Created by BioRender.com .…”

“…NCC is specifically inhibited by thiazide-type diuretics, with a higher affinity in the rNCC orthologue than in the flNCC ( Figure 1C ). Throughout the last few years, the kinetic properties of sNCC, eNCC and jNCC have also been studied, as shown in Figures 1A, C a lower affinity for transported ions is found in these aquatic species ( Moreno et al, 2016 ; Moreno et al, 2019 ; Moreno et al, 2022 ). As we previously described, different NCC species demonstrate different K m values of the ions transported.…”

The Post-Translational Modification Networking in WNK-Centric Hypertension Regulation and Electrolyte Homeostasis

“…NCC contributes to renal electrolyte transport by reabsorbing one sodium ion along with a chloride ion, and is ablated by thiazide diuretics. However, the specific amino acid residues that thiazide binds to inhibit NCC remain unknown [ 207 , 208 ].…”