Implications of the N-terminal heterogeneity for the neuronal K-Cl cotransporter KCC2 function

Markkanen, Marika; Ludwig, Anastasia; Khirug, Stanislav; Pryazhnikov, Evgeny; Soni, Shetal; Khiroug, Leonard; Delpire, Eric; Rivera, Claudio; Airaksinen, Matti S.; Uvarov, Pavel

doi:10.1016/j.brainres.2017.08.034

Cited by 26 publications

(17 citation statements)

References 78 publications

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“…One potent mechanism to rapidly and reversibly regulate the intrinsic transport rate and cell-surface abundance of KCC2 is phosphorylation and dephosphorylation of serine, threonine, and tyrosine residues (23). Recently, some progress in our understanding of the regulatory impact of specific phosphosites (27,34,35,39,41,43) and underlying kinases and phosphates (35)(36)(37)(43)(44)(45)(46) has been made.…”

Section: Discussionmentioning

confidence: 99%

Phosphoregulation of the intracellular termini of K+-Cl− cotransporter 2 (KCC2) enables flexible control of its activity

Cordshagen

Busch

Winklhofer

et al. 2018

Journal of Biological Chemistry

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The pivotal role of K-Cl cotransporter 2 (KCC2) in inhibitory neurotransmission and severe human diseases fosters interest in understanding posttranslational regulatory mechanisms such as (de)phosphorylation. Here, the regulatory role of the five phosphosites Ser, Thr, Ser, Thr, and Thr was investigated by the use of alanine and aspartate mutants. Tl-based flux analyses in HEK-293 cells demonstrated increased transport activity for S932D (mimicking phosphorylation) and T1008A (mimicking dephosphorylation), albeit to a different extent. Increased activity was due to changes in intrinsic activity, as it was not caused by increased cell-surface abundance. Substitutions of Ser, Thr, or Thr had no effect. Additionally, we show that the indirect actions of the known KCC2 activators staurosporine and -ethylmaleimide (NEM) involved multiple phosphosites. S31D, T34A, S932A/D, T999A, or T1008A/D abrogated staurosporine mediated stimulation, and S31A, T34D, or S932D abolished NEM-mediated stimulation. This demonstrates for the first time differential effects of staurosporine and NEM on KCC2. In addition, the staurosporine-mediated effects involved both KCC2 phosphorylation and dephosphorylation with Ser and Thr being target sites. In summary, our data reveal a complex phosphoregulation of KCC2 that provides the transporter with a toolbox for graded activity and integration of different signaling pathways.

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Section: Discussionmentioning

confidence: 99%

Phosphoregulation of the intracellular termini of K+-Cl− cotransporter 2 (KCC2) enables flexible control of its activity

Cordshagen

Busch

Winklhofer

et al. 2018

Journal of Biological Chemistry

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“…ZT-1a inhibits NKCC1 but stimulates KCC3 activity. 86 Rb + flux assays have been utilized extensively as a reliable measurement of cation chloride cotransporter activity [51][52][53][54] . Therefore, the ability of ZT-1a to decrease inhibitory phosphorylation of KCC3 at Thr991/Thr1048 prompted us to assess ZT-1a's effect on KCC3 activity by measuring hypotonicity-stimulated 86 Rb + uptake in isotonic or hypotonic low Cl − conditions (Fig.…”

Section: Zt-1a Reduces Spak-dependent CCC Phosphorylation In Cellsmentioning

confidence: 99%

Modulation of brain cation-Cl− cotransport via the SPAK kinase inhibitor ZT-1a

et al. 2020

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The SLC12A cation-Cl − cotransporters (CCC), including NKCC1 and the KCCs, are important determinants of brain ionic homeostasis. SPAK kinase (STK39) is the CCC master regulator, which stimulates NKCC1 ionic influx and inhibits KCC-mediated efflux via phosphorylation at conserved, shared motifs. Upregulation of SPAK-dependent CCC phosphorylation has been implicated in several neurological diseases. Using a scaffold-hybrid strategy, we develop a novel potent and selective SPAK inhibitor, 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano) methyl)-2-methylphenyl)-2-hydroxybenzamide ("ZT-1a"). ZT-1a inhibits NKCC1 and stimulates KCCs by decreasing their SPAK-dependent phosphorylation. Intracerebroventricular delivery of ZT-1a decreases inflammation-induced CCC phosphorylation in the choroid plexus and reduces cerebrospinal fluid (CSF) hypersecretion in a model of post-hemorrhagic hydrocephalus. Systemically administered ZT-1a reduces ischemia-induced CCC phosphorylation, attenuates cerebral edema, protects against brain damage, and improves outcomes in a model of stroke. These results suggest ZT-1a or related compounds may be effective CCC modulators with therapeutic potential for brain disorders associated with impaired ionic homeostasis.

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“…In vitro and in vivo analyses revealed that members of the with-no-lysine kinase (WNKs) family in combination with their downstream targets STE20/SPS1-related proline/alanine rich kinase (SPAK) and oxidative stress response kinase (OSR1) are the most prominent kinases that regulate KCC2 and NKCC1 activity in a reciprocal way. SPAK/OSR1, activated by WNK1, phosphorylate Thr 6 and Thr 1007 of KCC2 [48,59,[61][62][63][64]. WNKs also interact with a yet unknown kinase to phosphorylate Thr 906 of KCC2 [48,63].…”

Section: Introductionmentioning

confidence: 99%

Staurosporine and NEM mainly impair WNK-SPAK/OSR1 mediated phosphorylation of KCC2 and NKCC1

et al. 2020

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The pivotal role of KCC2 and NKCC1 in development and maintenance of fast inhibitory neurotransmission and their implication in severe human diseases arouse interest in posttranscriptional regulatory mechanisms such as (de)phosphorylation. Staurosporine (broad kinase inhibitor) and N-ethylmalemide (NEM) that modulate kinase and phosphatase activities enhance KCC2 and decrease NKCC1 activity. Here, we investigated the regulatory mechanism for this reciprocal regulation by mass spectrometry and immunoblot analyses using phospho-specific antibodies. Our analyses revealed that application of staurosporine or NEM dephosphorylates Thr 1007 of KCC2, and Thr 203 , Thr 207 and Thr 212 of NKCC1. Dephosphorylation of Thr 1007 of KCC2, and Thr 207 and Thr 212 of NKCC1 were previously demonstrated to activate KCC2 and to inactivate NKCC1. In addition, application of the two agents resulted in dephosphorylation of the T-loop and S-loop phosphorylation sites Thr 233 and Ser 373 of SPAK, a critical kinase in the WNK-SPAK/OSR1 signaling module mediating phosphorylation of KCC2 and NKCC1. Taken together, these results suggest that reciprocal regulation of KCC2 and NKCC1 via staurosporine and NEM is based on WNK-SPAK/OSR1 signaling. The key regulatory phospho-site Ser 940 of KCC2 is not critically involved in the enhanced activation of KCC2 upon staurosporine and NEM treatment, as both agents have opposite effects on its phosphorylation status. Finally, NEM acts in a tissue-specific manner on Ser 940 , as shown by comparative analysis in HEK293 cells and immature cultured hippocampal neurons. In summary, our analyses identified phospho-sites that are responsive to staurosporine or NEM application. This provides important information towards a better understanding of the cooperative interactions of different phospho-sites.

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Implications of the N-terminal heterogeneity for the neuronal K-Cl cotransporter KCC2 function

Cited by 26 publications

References 78 publications

Phosphoregulation of the intracellular termini of K+-Cl− cotransporter 2 (KCC2) enables flexible control of its activity

Phosphoregulation of the intracellular termini of K+-Cl− cotransporter 2 (KCC2) enables flexible control of its activity

Modulation of brain cation-Cl− cotransport via the SPAK kinase inhibitor ZT-1a

Staurosporine and NEM mainly impair WNK-SPAK/OSR1 mediated phosphorylation of KCC2 and NKCC1

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