Molecular and evolutionary insights into the structural organization of cation chloride cotransporters

Abstract

Cation chloride cotransporters (CCC) play an essential role for neuronal chloride homeostasis. K+-Cl− cotransporter (KCC2), is the principal Cl−-extruder, whereas Na+-K+-Cl− cotransporter (NKCC1), is the major Cl−-uptake mechanism in many neurons. As a consequence, the action of the inhibitory neurotransmitters gamma-aminobutyric acid (GABA) and glycine strongly depend on the activity of these two transporters. Knowledge of the mechanisms involved in ion transport and regulation is thus of great importance to better understand normal and disturbed brain function. Although no overall 3-dimensional crystal structures are yet available, recent molecular and phylogenetic studies and modeling have provided new and exciting insights into structure-function relationships of CCC. Here, we will summarize our current knowledge of the gross structural organization of the proteins, their functional domains, ion binding and translocation sites, and the established role of individual amino acids (aa). A major focus will be laid on the delineation of shared and distinct organizational principles between KCC2 and NKCC1. Exploiting the richness of recently generated genome data across the tree of life, we will also explore the molecular evolution of these features.

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

“…We presume it is highly flexible in KCC4. The C-terminal domain, however, is well conserved, has documented roles in regulation, expression, and trafficking 1, 2,13,14,16 , and mediates homodimerization of NKCC1 and the Archaean CCC (MaCCC) 30, 31 .…”

“…Comparison of residues in KCC4 that correspond to those in the NKCC1 homodimerization interface 30 suggest KCC4 could similarly self-interact. An intriguing hypothesis is that monomeric and dimeric KCC4 are functionally distinct and modulated differently; monomers may function independently of flexibly attached CTDs, while dimerization, which involves close juxtaposition of CTDs and transmembrane regions in NKCC1, could enable regulation of transporter activity through CTD posttranslational modifications 14, 22, 23, 38 . Consistent with this notion, a monomeric to dimeric transition in KCC2 has been correlated with an increase in transporter activity 22 and can be regulated by phosphorylation of the CTD 38 , while proteolytic cleavage of the KCC2 CTD correlates with a decrease in activity 23 .…”

“…First, the scaffold is followed by a C-terminal domain (CTD) important for regulating expression, trafficking, and activity including through phosphorylation or dephosphorylation of CTD sites in response to cell swelling 16–19 . Second, CCCs contain a “long extracellular loop” with predicted disulfide bonds and glycosylation sites that differs in position and structure between CCCs; it is formed by the region between TM5-TM6 in KCCs and between TM7-TM8 in NKCCs 14, 20 .…”

Role of NKCC1 and KCC2 in Epilepsy: From Expression to Function

“…NKCC1 and KCC2 are comprised of 12 membrane-spanning segments, 6 extracellular loops, and intracellular N-and C-terminals. They differ in the position of regulatory sequences, phosphorylation sites, and long extracellular loops (25,31,32). In immature neurons, an age-specific upregulation of NKCC1 and a relative deficiency in KCC2 loads more Cl − into the cell, resulting in a net Cl − outflow and subsequent depolarization when GABA activates GABAa Rs.…”

Chloride co‐transporters as possible therapeutic targets for stroke

“…Homo-and hetero-oligomers have been described for almost all CCCs (e.g. associations of KCC1 and KCC3, KCC2 and KCC4, and NKCC1 and KCC4, have been found); however, there is, to date, no conclusive data on how the different oligomerization patterns affect protein function (Simard et al 2007;Hartmann and Nothwang 2014).…”