Molecular basis for regulation of human potassium chloride cotransporters

Abstract

The Solute Carrier Family 12 (SLC12) encodes electroneutral cation-chloride cotransporters (CCCs) that are fundamental in cell volume regulation and chloride homeostasis. Dysfunction of CCCs engenders abnormality in renal function and neuro-system development. Here we presented structure of the full length human potassium-chloride co-transporter 2 (KCC2) and 3 (KCC3), the KCC3 mutants in phosphorylation mimic (P-mimic) and dephosphorylation mimic (DP-mimic) status, and KCC3 in complex with [(DihydroIndenyl)Oxy] Alkanoic acid (DIOA), a specific inhibitor of KCCs, at resolution of 2.7 Å -3.6 Å. A small N-terminal loop is bound at the intracellular vestibule of the transport path, arresting the transporter in an autoinhibition state. The C-terminal domain (CTD) of KCCs is structure-solved for the first time, revealing two conserved phosphorylation harboring segments (PHSs), which exhibit different conformation between P-mimic and DP-mimic mutants, explaining the inhibitory effect of phosphorylation. DIOA is located in between the two transmembrane domains, tightly bound to the loop between TM10 and TM11, locking the transporter in inward-facing conformation. Together, our study makes important steps in understanding the sophisticated regulation mechanisms of KCCs, with prospect for the specific drug development.

Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs

“…KCC4 is observed in an inward-open conformation that exposes the inside of the transporter to the cytoplasm through a wide and electropositive tunnel that may serve to concentrate the less abundant intracellular substrate Cl -. This conformation is similar to that observed in NKCC1 and KCC1 and presumably represents the lowest energy state for both CCCs in symmetrical salt concentrations in the absence of a transmembrane electrical gradient 30,61 . We identify K + and Clions around central discontinuities in TM1 and TM6.…”

“…However, comparison to the NKCC1 homodimerization interface 30 suggest KCC4 could similarly self-interact and mutational data suggests this interaction increases KCC4 activity. A recent report of KCC2 and KCC3 structures appears to show a homodimeric interaction through the CTDs very similar to that observed in NKCC1 61 . If monomeric and dimeric KCC4 are functionally distinct, regulated oligomerization in cells would be an opportunity to modulate transport activity.…”

Structural changes in the extracellular loop 2 of the murine KCC2 potassium chloride cotransporter modulate ion transport

“…As in all LeuT-fold transporters, TMs 1-5 and TMs 6-10 are symmetry related and inversely orientated (5+5 TM inverted repeat structure) (38-42). Recent 3D reconstruction from single particle cryo-electron microscopy (cryo-EM) provided insight into the highly conserved ion binding sites in KCCs, and NKCC1 (35,36,(43)(44)(45)(46)(47)(48) suggesting a similar mechanism of Clcoupling and K +transport (43,49,50). Although the K + and Claffinities vary among CCCs (4,49), the coordination residues of these ion binding sites are highly conserved.…”

“…The termini are important for trafficking, isotonic activity, and regulation via post-translational modifications ( 6 , 31 , 32 , 33 , 34 , 35 , 36 ). The N-terminal domains of KCCs have an autoinhibitory function locking the transporter in the inward-facing state to prevent intracellular solvent access to the ion-binding sites formed by residues in TM1, 3, 6, and 8 ( 35 , 36 , 37 ). As in all LeuT-fold transporters, TMs 1 to 5 and TMs 6 to 10 are symmetry related and inversely orientated (5 + 5 TM inverted repeat structure) ( 38 , 39 , 40 , 41 , 42 ).…”

“…Recent three-dimensional reconstruction from single-particle cryo-EM provided insight into the highly conserved ion-binding sites in KCCs and NKCC1 ( 35 , 36 , 37 , 43 , 44 , 45 , 46 , 47 ), suggesting a similar mechanism of Cl − coupling and K + transport ( 43 , 48 , 49 ). Although the K + and Cl − affinities vary among CCCs ( 4 , 48 ), the coordination residues of these ion-binding sites are highly conserved.…”