Coordinate modulation of Na-K-2Cl cotransport and K-Cl cotransport by cell volume and chloride

Abstract

Na-K-2Cl cotransporter (NKCC) and K-Cl cotransporter (KCC) play key roles in cell volume regulation and epithelial Cl(-) transport. Reductions in either cell volume or cytosolic Cl(-) concentration ([Cl(-)](i)) stimulate a corrective uptake of KCl and water via NKCC, whereas cell swelling triggers KCl loss via KCC. The dependence of these transporters on volume and [Cl(-)](i) was evaluated in model duck red blood cells. Replacement of [Cl(-)](i) with methanesulfonate elevated the volume set point at which NKCC activates and KCC inactivates. The set point was insensitive to cytosolic ionic strength. Reducing [Cl(-)](i) at a constant driving force for inward NKCC and outward KCC caused the cells to adopt the new set point volume. Phosphopeptide maps of NKCC indicated that activation by cell shrinkage or low [Cl(-)](i) is associated with phosphorylation of a similar constellation of Ser/Thr sites. Like shrinkage, reduction of [Cl(-)](i) accelerated NKCC phosphorylation after abrupt inhibition of the deactivating phosphatase with calyculin A in vivo, whereas [Cl(-)] had no specific effect on dephosphorylation in vitro. Our results indicate that NKCC and KCC are reciprocally regulated by a negative feedback system dually modulated by cell volume and [Cl(-)]. The major effect of Cl(-) on NKCC is exerted through the volume-sensitive kinase that phosphorylates the transport protein.

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*♦

“…The prevailing notion holds that phosphorylation activates NKCCs and inactivates KCCs (31,58,65,66). This elegant concept of reciprocal regulation of Cl Ϫ -inward and Cl Ϫ -outward transporters was originally based on the observations that cell swelling, NEM, staurosporine, and serine-threonine phosphatase 1 inhibited NKCC1 (67)(68)(69) and activated KCCs (33,58,67). This view was subsequently substantiated by the reciprocal effect of WNKs on KCCs and NKCC/NCC transport activities (31,70) (39) and concomitantly caused depolarizing action of GABA in neurons (71).…”

WNK3 modulates transport of Cl - in and out of cells: Implications for control of cell volume and neuronal excitability

“…5B), suggesting that it is the (or one of the) long-sought Cl Ϫ -responsive kinases that regulate cell volume in response to osmotic stress and͞or changes in [Cl Ϫ ] i (2,6,52). Hypertonicity increases the activity of NKCC1 and inhibits KCCs by stimulating the phosphorylation of both proteins, resulting in increased [Cl Ϫ ] i .…”

Potassium-Chloride Cotransporter Mediates Cell Cycle Progression and Proliferation of Human Corneal Epithelial Cells

“…46,47 It has been proposed that protein phosphatases (PPs) inhibit the volume-sensitive kinase responsible for the coordinated phosphorylation status of these transporters. 48,49 Coordinated activation of these transporters may explain how stimulation of oppositely directed ion transport mechanisms leads to a similar outcome, i.e., cell proliferation. Further evidence in support of PPs involvement in eliciting coordinated control of KCC and NKCC activation, and, subsequently, stimulation of cell proliferation, is provided by studies describing the role of PPs (i.e., PP2A, and MAP kinase phosphatase-1) in mediating crosstalk between the p44/42MAPK and p38MAPK pathways induced by either growth factors or hypotonicity.…”