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 |
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Authors: | Maren Weber Anna-Maria Hartmann Timo Beyer Anne Ripperger Hans Gerd Nothwang |
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Affiliation: | 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 ;the ¶Research Center for Neurosensory Sciences, Carl von Ossietzky University Oldenburg, 26111 Oldenburg, Germany |
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Abstract: | 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, Ser25, Ser26, Ser937, Ser1022, Ser1025, and Ser1026. 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 (KCC2WT). Tl+ flux measurements demonstrated unchanged transport activity for Ser25, Ser26, Ser1022, Ser1025, and Ser1026 mutants. In contrast, KCC2S937D, mimicking phosphorylation, resulted in a significant up-regulation of transport activity. Aspartate substitution of Thr934, a neighboring putative phosphorylation site, resulted in a comparable increase in KCC2 transport activity. Both KCC2T934D and KCC2S937D mutants were inhibited by the kinase inhibitor staurosporine and by N-ethylmaleimide, whereas KCC2WT, KCC2T934A, and KCC2S937A 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 KCC2T934D and KCC2S937D compared with KCC2WT. 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. |
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Keywords: | Cell Culture Chloride Transport Evolution Neurophysiology Phosphorylation Post-translational Modification Protein Conformation Cation Chloride Cotransporter Mutation |
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