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 neuron-specific cation chloride cotransporter KCC2 plays a crucial role in hyperpolarizing synaptic inhibition. Transporter dysfunction is associated with various neurological disorders, raising interest in regulatory mechanisms. Phosphorylation has been identified as a key regulatory process. Here, we retrieved experimentally observed phosphorylation sites of KCC2 from public databases and report on the systematic analysis of six phosphorylated serines, Ser(25), Ser(26), Ser(937), Ser(1022), Ser(1025), and Ser(1026). Alanine or aspartate substitutions of these residues were analyzed in HEK-293 cells. All mutants were expressed in a pattern similar to wild-type KCC2 (KCC2(WT)). Tl(+) flux measurements demonstrated unchanged transport activity for Ser(25), Ser(26), Ser(1022), Ser(1025), and Ser(1026) mutants. In contrast, KCC2(S937D), mimicking phosphorylation, resulted in a significant up-regulation of transport activity. Aspartate substitution of Thr(934), a neighboring putative phosphorylation site, resulted in a comparable increase in KCC2 transport activity. Both KCC2(T934D) and KCC2(S937D) mutants were inhibited by the kinase inhibitor staurosporine and by N-ethylmaleimide, whereas KCC2(WT), KCC2(T934A), and KCC2(S937A) were activated. The inverse staurosporine effect on aspartate versus alanine substitutions reveals a cross-talk between different phosphorylation sites of KCC2. Immunoblot and cell surface labeling experiments detected no alterations in total abundance or surface expression of KCC2(T934D) and KCC2(S937D) compared with KCC2(WT). These data reveal kinetic regulation of transport activity by these residues. In summary, our data identify a novel key regulatory phosphorylation site of KCC2 and a functional interaction between different conformation-changing post-translational modifications. The action of pharmacological agents aimed to modulate KCC2 activity for therapeutic benefit might therefore be highly context-specific.
From the Neurogenetics Group, Center of Excellence Hearing4All, School of Medicine and Health Sciences.
Maren
M
Weber
From the Neurogenetics Group, Center of Excellence Hearing4All, School of Medicine and Health Sciences, Systematics and Evolutionary Biology Group, Institute for Biology and Environmental Sciences, and.
Anna-Maria
AM
Hartmann
From the Neurogenetics Group, Center of Excellence Hearing4All, School of Medicine and Health Sciences.
Timo
T
Beyer
From the Neurogenetics Group, Center of Excellence Hearing4All, School of Medicine and Health Sciences.
Anne
A
Ripperger
From the Neurogenetics Group, Center of Excellence Hearing4All, School of Medicine and Health Sciences, the Research Center for Neurosensory Sciences, Carl von Ossietzky University Oldenburg, 26111 Oldenburg, Germany hans.g.nothwang@uni-oldenburg.de.
Hans Gerd
HG
Nothwang
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D057225:Data Mining; D030562:Databases, Protein; D005033:Ethylmaleimide / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010802:Phylogeny; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D019311:Staurosporine / Q000494:pharmacology*; D027981:Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000096922:K Cl- Cotransporters
D005033:Ethylmaleimide; D010750:Phosphoproteins; D019311:Staurosporine; D027981:Symporters; D000096922:K Cl- Cotransporters
Cation Chloride Cotransporter; Cell Culture; Chloride Transport; Evolution; Mutation; Neurophysiology; Phosphorylation; Post-translational Modification; Protein Conformation