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-β-cyclodextrin (MβCD) 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 MβCD 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 MβCD 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.