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

Sensen Zhang,Haiteng Deng,Wei Zhuo,Xianbin Meng,Wenwen Zeng,Maojun Yang,Laixing Zhang,Perrine Friedel,Yang Liu,Yuebin Zhang,Jun Zhou,Biff Forbush,Guohui Li,Kasturi Roy,Tianya Liu,Suma Somasekharan

doi:10.1038/s42003-021-01750-w

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.

Highlights

NKCC and KCC transporters mediate coupled transport of Na++K++Cl− and K++Cl− across the plasma membrane, regulating cell Cl− concentration and cell volume and playing critical roles in transepithelial salt and water transport and in neuronal excitability
Changes in Cl− regulation are likely to have an important role in the molecular etiology of a number of psychiatric and neurological diseases— since some of the deleterious effects on neuronal development and function have been connected with elevated [Cl−]i and/or cell volume and increased strength of GABA signaling, a number of recent efforts have focused on inhibition of NKCC1, inhibition of WNK-SPAK/OSR1, or on finding agonists to increase KCC2 transport activity and membrane trafficking[19,26]
Based on biochemical stability and structural homogeneity, we identified hNKCC1a and mKCC2a as the top two candidates suited for high-resolution studies (Supplementary Fig. 1)—these candidate proteins were purified from mammalian cells through Strep-tag II affinity chromatography and reconstituted in digitonin buffer for cryoelectron microscopy (cryo-EM) study

Summary

Introduction

NKCC and KCC transporters mediate coupled transport of Na++K++Cl− and K++Cl− across the plasma membrane, regulating cell Cl− concentration and cell volume and playing critical roles in transepithelial salt and water transport and in neuronal excitability. We present the cryoelectron 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. Changes in Cl− regulation are likely to have an important role in the molecular etiology of a number of psychiatric and neurological diseases— since some of the deleterious effects on neuronal development and function have been connected with elevated [Cl−]i and/or cell volume and increased strength of GABA signaling, a number of recent efforts have focused on inhibition of NKCC1, inhibition of WNK-SPAK/OSR1, or on finding agonists to increase KCC2 transport activity and membrane trafficking[19,26]. Four CCC structures, including the DrNKCC140, human NKCC1(K289N_G351R)[41], human KCC139, and mouse KCC442 have been reported at 2.9–3.7 Å resolution, and these have revealed putative ion-binding sites and key residues likely involved in transport conformational change

Methods

Results

Conclusion