Brain-type creatine kinase activates neuron-specific K+-Cl- co-transporter KCC2.
GABA, a major inhibitory neurotransmitter in the adult CNS, is excitatory at early developmental stages as a result of the elevated intracellular Cl- concentration ([Cl-]i). This functional switch is primarily attributable to the K+-Cl- co-transporter KCC2, the expression of which is developmentally regulated in neurons. Previously, we reported that KCC2 interacts with brain-type creatine kinase (CKB). To elucidate the functional significance of this interaction, HEK293 cells were transfected with KCC2 and glycine receptor alpha2 subunit, and gramicidin-perforated patch-clamp recordings were performed to measure the glycine reversal potential (Egly), giving an estimate of [Cl-]i. KCC2-expressing cells displayed the expected changes in Egly following alterations in the extracellular K+ concentration ([K+]o) or administration of an inhibitor of KCCs, suggesting that the KCC2 function was being properly assessed. When added into KCC2-expressing cells, dominant-negative CKB induced a depolarizing shift in Egly and reduced the hyperpolarizing shift in Egly seen in response to a lowering of [K+]o compared with wild-type CKB. Moreover, 2,4-dinitrofluorobenzene (DNFB), an inhibitor of CKs, shifted Egly in the depolarizing direction. In primary cortical neurons expressing CKB, the GABA reversal potential was also shifted in the depolarizing direction by DNFB. Our findings suggest that, in the cellular micro-environment, CKB activates the KCC2 function.
Inoue
Koichi
K
Department of Physiology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan. inok@hama-med.ac.jp
Yamada
Junko
J
Ueno
Shinya
S
Fukuda
Atsuo
A
D000818:Animals; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D052276:Creatine Kinase, BB Form / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D004139:Dinitrofluorobenzene / Q000494:pharmacology; D004594:Electrophysiology; D005799:Genes, Dominant; D006096:Gramicidin / Q000494:pharmacology; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D009474:Neurons / Q000378:metabolism* / Q000502:physiology; D009994:Osmolar Concentration; D018408:Patch-Clamp Techniques; D051381:Rats; D018009:Receptors, Glycine / Q000235:genetics / Q000378:metabolism; D027981:Symporters / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000378:metabolism; D000096922:K Cl- Cotransporters
D002712:Chlorides; D052276:Creatine Kinase, BB Form; D004139:Dinitrofluorobenzene; D006096:Gramicidin; D018009:Receptors, Glycine; D027981:Symporters; D005680:gamma-Aminobutyric Acid; D000096922:K Cl- Cotransporters; C501174:Glra2 protein, rat