Hyperpolarizing GABAergic transmission requires the KCC2 C-terminal ISO domain.
KCC2 is the neuron-specific member of the of K(+)-Cl(-) cotransporter gene family. It is also the only member of its family that is active under physiologically normal conditions, in the absence of osmotic stress. By extruding Cl(-) from the neuron under isotonic conditions, this transporter maintains a low concentration of neuronal Cl(-), which is essential for fast inhibitory synaptic transmission by GABA and glycine in the mature nervous system. The other members of this K(+)-Cl(-) cotransporter gene family are exclusively swelling-activated. Here we demonstrate that a 15 aa region near the end of the C terminus, unique to KCC2 (termed the ISO domain), is required for KCC2 to cotransport K(+) and Cl(-) out of the neuron under isotonic conditions. We made this discovery by overexpressing chimeric KCC2-KCC4 cDNA constructs in cultured hippocampal neurons prepared from Sprague Dawley rat embryos and assaying neuronal Cl(-) through gramicidin perforated patch-clamp recordings. We found that when neurons had been transfected with a chimeric KCC2 that lacked the unique ISO domain, hyperpolarizing responses to GABA were abolished. This finding indicates that the ISO domain is required for neuronal Cl(-) regulation. Furthermore, we discovered that when KCC2 lacks the ISO domain, it still retains swelling-activated transport, which demonstrates that there are exclusive molecular determinants of isotonic and swelling-induced K(+)-Cl(-) cotransport in neurons.
Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada M5S 3G5.
Brooke A
BA
Acton
Vivek
V
Mahadevan
Adrianna
A
Mercado
Pavel
P
Uvarov
Yanli
Y
Ding
Jessica
J
Pressey
Matti S
MS
Airaksinen
David B
DB
Mount
Melanie A
MA
Woodin
D000818:Animals; D001693:Biological Transport, Active / Q000502:physiology; D048429:Cell Size; D002712:Chlorides / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D006624:Hippocampus / Q000166:cytology; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D017136:Ion Transport; D018613:Microscopy, Confocal; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D027981:Symporters / Q000502:physiology*; D009435:Synaptic Transmission / Q000502:physiology*; D014981:Xenopus; D005680:gamma-Aminobutyric Acid / Q000502:physiology*; D000096922:K Cl- Cotransporters
D002712:Chlorides; D004247:DNA; D011188:Potassium; D027981:Symporters; D005680:gamma-Aminobutyric Acid; D000096922:K Cl- Cotransporters