Molecular Motions Involved in Na-K-Cl Cotransporter-mediated Ion Transport and Transporter Activation Revealed by Internal Cross-linking between Transmembrane Domains 10 and 11/12

Abstract

Background: Na-K-Cl cotransporters (NKCCs) are responsible for volume and chloride homeostasis and chloride transport and are targets of loop diuretic drugs. Results: Cross-links between transmembrane domains (TMs) 10 and 11/12 of NKCC1 are identified as inhibitory and stimulatory. Conclusion: Activation of NKCC1 involves movement of TM12 relative to TM10. Significance: This identifies movement of TM12 as a key step in the molecular mechanism of NKCC activation.

Structure of the human cation–chloride cotransporter NKCC1 determined by single-particle electron cryo-microscopy

“…Ion transport pathways. From a bird's eye view, NKCC1 shares many structural hallmarks in its substrates (i.e., ions) transport pathway with other APC transporters, such as LeuT that mediates Na + -coupled leucine uptake in Aquifex aeolicus 41 , in line with previous modeling and mutagenesis studies 24 . LeuT arguably represents the best understood APC transporter with respect to its transport mechanisms due in no small part to insights gained by a series of LeuT structures captured in outward-open, occluded, or inward-open states 42,43 .…”

“…This pseudo-symmetric topology of two inverted repeats is the linchpin of the alternating-access mechanism whereby transporters isomerize among outward-open, occluded, and inward-open states to translocate substrates across membranes 23 . Hydropathy plot analyses and structure modeling based on related APC transporter structures predict that all CCCs share a common structural architecture with twelve TM helices harboring a 5 + 5 inverted repeat fold flanked by cytoplasmic N-and C-terminal domains 1,24,25 . CCCs are homo-or hetero-dimers and their Cterminal domains contribute to dimer assembly [26][27][28][29][30] .…”

Potentiating KCC2 activity is sufficient to limit the onset and severity of seizures

“…These mutations must be altering the functional capacity of KCC2 through direct alteration of its kinetic properties. This may be through modulation of the net velocity of Cl − transport in a manner similar to phosphorylation-initiated cross-linking of NKCC1 transmembrane domains that ultimately lock it in active/inactive states (42). On the other hand, these KCC2 mutations could increase the affinity of KCC2 for Cl − .…”

A Novel Regulatory Locus of Phosphorylation in the C Terminus of the Potassium Chloride Cotransporter KCC2 That Interferes with N-Ethylmaleimide or Staurosporine-mediated Activation*♦

“…These findings also imply that drugs aimed to modify KCC2 phosphorylation for therapeutic benefit (36,73) have to be investigated under different conditions as their effect might depend on the conformational state of KCC2 and therefore be highly context-specific. Functional cross-talk between different regions has also been suggested for NKCC1, where phosphorylation of the N terminus causes movement of the C terminus, which in turn entails altered interactions between different transmembrane domains (74).…”