Phosphoregulation of K⁺‐Cl⁻ cotransporter 4 during changes in intracellular Cl⁻ and cell volume

Abstract

It has long been stated that the K(+)-Cl(-) cotransporters (KCCs) are activated during cell swelling through dephosphorylation of their cytoplasmic domains by a protein phosphatase (PP) but that other enzymes are involved by targeting this PP or the KCCs directly. To date, however, the role of signaling intermediates in KCC regulation has been deduced from indirect evidence rather than in vitro phosphorylation studies, and examined after simulation of ion transport through cell swelling or N-ethylmaleimide treatment. In this study, the oocyte expression system was used to examine the effects of changes in cell volume (C(VOL)) and intracellular [Cl(-)] ([Cl(-)](i)) on the activity and phosphorylation levels (P(LEV)) of KCC4, and determine whether these effects are mediated by PP1 or phorbol myristate acetate (PMA)-sensitive effectors. We found that (1) low [Cl(-)](i) or low C(VOL) leads to decreased activity but increased P(LEV), (2) high C(VOL) leads to increased activity but no decrease in P(LEV) and (3) calyculin A (Cal A) or PMA treatment leads to decreased activity but no increase in P(LEV). Thus, we have shown for the first time that one of the KCCs can be regulated through direct phosphorylation, that changes in [Cl(-)](i) or C(VOL) modify the activity of signaling enzymes at carrier sites, and that the effectors directly involved do not include a Cal A-sensitive PP in contrast to the widely held view. J. Cell. Physiol. 219: 787-796, 2009. (c) 2009 Wiley-Liss, Inc.

Hyperpolarizing GABAergic Transmission Requires the KCC2 C-Terminal ISO Domain

“…4A), compared to neurons transfected with KCC2 (E Cl = −97.56 ± 4.49 mV, n = 09p > 0.001). However, it is known that K + -Cl − cotransporter activity (including KCC4) is increased at higher levels of intracellular Cl − (Gillen and Forbush, 1999, Bergeron et al, 2006, Bergeron et al, 2009), thus we asked whether increased levels of neuronal Cl − were required to reveal whether the addition of the ISO domain to KCC4 could confer this cotransporter with constitutive isotonic activity. We performed this experiment by loading neurons with 50 mM intracellular Cl − , and found that the ISO domain could in fact confer KCC4 with constitutive isotonic activity.…”

“…Increased intracellular Cl − was likely required to demonstrate the sufficiency of the ISO domain for conferring KCC4 with isotonic transport because K + -Cl − cotransporter activity (including KCC4) is increased at higher [Cl − ] i (Gillen and Forbush, 1999, Bergeron et al, 2006, Bergeron et al, 2009). This finding demonstrating the sufficiency of the ISO domain is supported by results obtained in Xenopus oocytes where the same chimeric construct (KCC4-ISO) was sufficient to mediate constitutive activity of the transport.…”

WNK3 is a Putative Chloride-sensing Kinase

“…Phosphorylation of NKCC1 [53], NKCC2 [54], and NCC [55] is associated with activation of the cotransporters by either cell shrinkage or intracellular chloride depletion. In contrast, phosphorylation of KCCs is associated with inhibition of the cotransporters [56]. In this regard, several conserved threonine/serine residues in the amino terminus of NKCC1/2 and NCC have been shown to be phosphorylated in response to activating stimuli [54,[57][58][59][60][61].…”

Frog Oocytes to Unveil the Structure and Supramolecular Organization of Human Transport Proteins

“…In contrast, strong signals were obtained for the His-tagged protein markers (lane: M; positive control). (D) and (E) Monomer (below 150 kDa marker) and dimer (∼250 kDa) bands of non- and glycosylated, recombinant mouse KCC4 [12], [13]. (F) and (G) Monomeric (∼75 kDa [16]), dimeric (∼150 kDa) and oligomeric glycosylated, recombinant human EAAC1.…”

“…The function of wt-KCC4 and multi-KCC4 expressing oocytes was determined as previously described [12], [13]. Briefly, oocytes were incubated for 1 h in a low Cl − /hypotonic solution (∼5 mM Cl − and 125 mOsm) to activate KCC4, incubated for another 45 min in a physiological solution (86 mM Cl − and ∼200 mOsm) supplemented with 5 mM Rb + (used as a tracer of K + ) and 10 mM ouabain, and bathed several times in a wash solution supplemented with 10 mM ouabain, 250 mM bumetanide and 250 mM furosemide.…”