Activity-dependent cleavage of the K-Cl cotransporter KCC2 mediated by calcium-activated protease calpain.
The K-Cl cotransporter KCC2 plays a crucial role in neuronal chloride regulation. In mature central neurons, KCC2 is responsible for the low intracellular Cl(-) concentration ([Cl(-)](i)) that forms the basis for hyperpolarizing GABA(A) receptor-mediated responses. Fast changes in KCC2 function and expression have been observed under various physiological and pathophysiological conditions. Here, we show that the application of protein synthesis inhibitors cycloheximide and emetine to acute rat hippocampal slices have no effect on total KCC2 protein level and K-Cl cotransporter function. Furthermore, blocking constitutive lysosomal degradation with leupeptin did not induce significant changes in KCC2 protein levels. These findings indicate a low basal turnover rate of the total KCC2 protein pool. In the presence of the glutamate receptor agonist NMDA, the total KCC2 protein level decreased to about 30% within 4 h, and this effect was blocked by calpeptin and MDL-28170, inhibitors of the calcium-activated protease calpain. Interictal-like activity induced by incubation of hippocampal slices in an Mg(2+)-free solution led to a fast reduction in KCC2-mediated Cl(-) transport efficacy in CA1 pyramidal neurons, which was paralleled by a decrease in both total and plasmalemmal KCC2 protein. These effects were blocked by the calpain inhibitor MDL-28170. Taken together, these findings show that calpain activation leads to cleavage of KCC2, thereby modulating GABAergic signaling.
Department of Biosciences and Neuroscience Center, University of Helsinki, FI-00014 Helsinki, Finland.
Martin
M
Puskarjov
Faraz
F
Ahmad
Kai
K
Kaila
Peter
P
Blaesse
D000200:Action Potentials / Q000187:drug effects; D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism; D061207:Calcium Ionophores / Q000494:pharmacology; D002154:Calpain / Q000378:metabolism* / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004640:Emetine / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D006624:Hippocampus / Q000166:cytology; D066298:In Vitro Techniques; D015759:Ionomycin / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D016202:N-Methylaspartate / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D017966:Pyramidal Cells / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D018709:Statistics, Nonparametric; D027981:Symporters / Q000378:metabolism*; D014633:Valine / Q000031:analogs & derivatives / Q000494:pharmacology; D000096922:K Cl- Cotransporters
D002118:Calcium; D061207:Calcium Ionophores; D002154:Calpain; D003513:Cycloheximide; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D016291:Dizocilpine Maleate; D004640:Emetine; D018691:Excitatory Amino Acid Antagonists; D015759:Ionomycin; D007976:Leupeptins; D008274:Magnesium; D016202:N-Methylaspartate; D011500:Protein Synthesis Inhibitors; D027981:Symporters; D014633:Valine; D000096922:K Cl- Cotransporters; C056639:2-amino-5-phosphopentanoic acid; C032854:leupeptin; C058076:calpain inhibitor III