The structural basis of function and regulation of neuronal cotransporters NKCC1 and KCC2

Abstract

NKCC and KCC transporters mediate coupled transport of Na++K++Cl− and K++Cl− across the plasma membrane, thus regulating cell Cl− concentration and cell volume and playing critical roles in transepithelial salt and water transport and in neuronal excitability. The function of these transporters has been intensively studied, but a mechanistic understanding has awaited structural studies of the transporters. Here, we present the cryo-electron microscopy (cryo-EM) structures of the two neuronal cation-chloride cotransporters human NKCC1 (SLC12A2) and mouse KCC2 (SLC12A5), along with computational analysis and functional characterization. These structures highlight essential residues in ion transport and allow us to propose mechanisms by which phosphorylation regulates transport activity.

Cation-coupled chloride cotransporters: chemical insights and disease implications

“…The first high-resolution structures of zebrafish [Danio rerio (Dr)] NKCC1 [20] and human (h)KCC1 [21] were reported in 2019. In 2020-2021, six independent studies reported new structures of hNKCC1, mKCC2, and hKCC2-4 transporters, alone or in complex with inhibitors (Table 1 and Box 2) [20][21][22][23][24][25][26][27][28]. These structural data confirmed the predicted LeuT structural fold with a large ordered and glycosylated loop in the EC domain and two cytosolic domains (NT and CT) with numerous phosphorylation sites.…”

“…An NT domain peptide was also resolved in the mKCC2, hKCC2a-b, hKCC3b, and hKCC4a structures, where it sterically closes the cytosolic vestibule and blocks transport activity [24][25][26]. Deletion or mutation of the NT domains in KCC2b and KCC3b enhances transport activity [24][25][26], which supports the role of the NT domain as an autoinhibitory element. This closed (autoinhibitory) state is well characterized in the KCC2a and KCC4a structures, where an NT domain peptide is…”

“…Ion translocation occurs via the alternating access model, which involves switching between an inward-open (IO) ↔ outward-open (OO) conformational state that exposes the ion-binding sites to either side of the membrane (Box 1) [18,19]. New structural insights from recent cryo-EM structures Five CCC members have been resolved with cryo-EM (i.e., NKCC1 and KCC1-4 [20][21][22][23][24][25][26]), confirming the dimeric organization of CCCs, with the exception of mouse (m)KCC4 resolved as a monomer [27]. The first high-resolution structures of zebrafish [Danio rerio (Dr)] NKCC1 [20] and human (h)KCC1 [21] were reported in 2019.…”

“…This is central to understanding the mechanism for ion permeation in CCCs. In particular, some of the solved IO conformation structures of the CCC dimers (i.e., DrNKCC1, hKCC1, mKCC2, hKCC2a-b, hKCC3a-b, hKCC4a [20,[23][24][25][26]) showed the CT dimeric domain in the full-length molecular assembly for the first time. These structures also revealed that the CT domain of one protomer is located under the TM domain of the other protomer, creating a domain-swap configuration that stabilizes the dimerization.…”

All-atom Simulations Uncover the Molecular Terms of NKCC1 Transport Mechanism

“…16,18,19 Specifically, NKCC1 presents a homodimeric transmembrane domain, and N-terminal and C-terminal domains that contribute to dimer assembly and possibly also modulate ions trafficking. 20,21 A recent cryo-EM structure of dimeric zebrafish NKCC1 20 grasped the transporter in its inward-facing state (Figure 1), also tracing two Cland one K + ions in their binding sites. The position of the Na + binding site was, instead, inferred based on structural homology with other transporters.…”