Opposite effect of membrane raft perturbation on transport activity of KCC2 and NKCC1.
In the majority of neurons, the intracellular Cl(-) concentration is set by the activity of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) and the K(+)-Cl(-) cotransporter (KCC2). Here, we investigated the cotransporters' functional dependence on membrane rafts. In the mature rat brain, NKCC1 was mainly insoluble in Brij 58 and co-distributed with the membrane raft marker flotillin-1 in sucrose density flotation experiments. In contrast, KCC2 was found in the insoluble fraction as well as in the soluble fraction, where it co-distributed with the non-raft marker transferrin receptor. Both KCC2 populations displayed a mature glycosylation pattern. Disrupting membrane rafts with methyl-beta-cyclodextrin (MbetaCD) increased the solubility of KCC2, yet had no effect on NKCC1. In human embryonic kidney-293 cells, KCC2 was strongly activated by a combined treatment with MbetaCD and sphingomyelinase, while NKCC1 was inhibited. These data indicate that membrane rafts render KCC2 inactive and NKCC1 active. In agreement with this, inactive KCC2 of the perinatal rat brainstem largely partitioned into membrane rafts. In addition, the exposure of the transporters to MbetaCD and sphingomyelinase showed that the two transporters differentially interact with the membrane rafts. Taken together, membrane raft association appears to represent a mechanism for co-ordinated regulation of chloride transporter function.
Hartmann
Anna-Maria
AM
Department of Neurogenetics, Institute for Biology and Environmental Sciences, Carl von Ossietzky University, Oldenburg, Germany.
Blaesse
Peter
P
Kranz
Thorsten
T
Wenz
Meike
M
Schindler
Jens
J
Kaila
Kai
K
Friauf
Eckhard
E
Nothwang
Hans Gerd
HG
D000818:Animals; D000831:Animals, Newborn; D001933:Brain Stem / Q000166:cytology / Q000254:growth & development / Q000378:metabolism; D002412:Cations / Q000378:metabolism; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D006031:Glycosylation; D006706:Homeostasis / Q000502:physiology; D006801:Humans; D007668:Kidney / Q000166:cytology; D021962:Membrane Microdomains / Q000378:metabolism*; D009433:Neural Inhibition / Q000502:physiology; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D012995:Solubility; D064506:Solute Carrier Family 12, Member 2; D027981:Symporters / Q000378:metabolism*; D009435:Synaptic Transmission / Q000502:physiology*; D000096922:K Cl- Cotransporters
D002412:Cations; D002712:Chlorides; D002784:Cholesterol; D003902:Detergents; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D027981:Symporters; D000096922:K Cl- Cotransporters; C578877:SLC12A2 protein, human; C578879:Slc12a2 protein, rat