Cryo-EM structure of the human sodium-chloride cotransporter NCC

Abstract

The sodium-chloride cotransporter NCC mediates the coupled import of sodium and chloride across the plasma membrane, playing vital roles in kidney extracellular fluid volume and blood pressure control. Here, we present the full-length structure of human NCC, with 2.9 Å for the transmembrane domain and 3.8 Å for the carboxyl-terminal domain. NCC adopts an inward-open conformation and a domain-swap dimeric assembly. Conserved ion binding sites among the cation-chloride cotransporters and the Na2 site are observed in our structure. A unique His residue in the substrate pocket in NCC potentially interacts with Na1 and Cl1 and might also mediate the coordination of Na2 through a Ser residue. Putative observed water molecules are indicated to participate in the coordination of ions and TM coupling. Together with transport activity assays, our structure provides the first glimpse of NCC and defines ion binding sites, promoting drug development for hypertension targeting on 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 .…”

“…As we previously described, different NCC species demonstrate different K m values of the ions transported. Currently, it is known that NCC’s ion binding sites are highly conserved in other members of the SLC12 family ( Nan et al, 2022 ); however, it is difficult to comprehend the conformational changes that occur in NCCs of different species due to the differing percentages of identity in their amino acid sequences. At this moment, the role of residues near the ion binding sites remains unclear; nevertheless, they are certainly responsible for determining the different affinities for ion transport.…”

“…By searching the human genome database (HGMD, professional 2018.3), over 500 different mutations along NCC have been linked to Gitelman’s disease. Nan et al (2022) recently mapped out the numerous Gitelman syndrome-related mutations onto their NCC cryo-EM (single-particle Cryogenic Electron Microscopy) structure and classified them in five groups according to their function or location. In Figure 2C , the mutations appear as various colored spheres: located extracellularly (yellow); in the ion translocation pathway (pink); other TM domains (cyan); TM-C-terminal interface (red); and in the N-terminal domain (green).…”

“…Recently, a high resolution cryo-EM structure of NCC was achieved ( Nan et al, 2022 ). The map of the TM regions permitted the construction of precise atomic models of this region and EL4 as well.…”

“…The Cl − 1 binding site is coordinated by the main-chain amide group of glycine-152 (G152), valine-153 (V153), and isoleucine-154 (I154 of TM1) and the hydroxyl group of serine-350 (S350), that also weakly coordinates this ion. The Na + 1 binding site is accommodated by the main-chain carbonyl groups of asparagine-149 (N149), isoleucine-150 (I150), proline-349 (P349), and threonine-352 (T352) ( Figure 2B ), Nan et al (2022) . Notably, mutations in I150M, V153M, I154F y P349L, can cause Gitelman syndrome, demonstrating the important role of these residues in NCC transport activity ( Figure 2C ).…”