Throughout the nervous system, the Cl- -outward transporter NKCC1 and the Cl- -inward transporter KCC2 play an important role in setting the intracellular chloride concentration. Both transporters are co-expressed in many neurons. This requires tight and reciprocal regulation, due to their opposite direction of Cl- transport. Here, we investigated the role of cholesterol-rich membrane microdomains, so-called membrane rafts, for NKCC1 and KCC2 function. Membrane rafts were isolated from mature rat brainstems at postnatal days (P) 25–30 via solubilization in Brij 58 followed by sucrose-density fl otation experiments. This analysis revealed that NKCC1 was codistributed with the membrane raft marker fl otillin-1 in lower density fractions. In contrast, two populations were observed for KCC2. One was solubilized, whereas the other population was detergent insoluble. Studies in HEK293 cells demonstrated that disruption of membrane rafts by combined treatment of methyl-b-cyclodextrin and sphingomyelinase impaired NKCC1 mediated 86Rb+ uptake, whereas it increased KCC2 dependent 86Rb+ uptake. From these data we conclude that NKCC1 requires association with membrane rafts for transport activity, whereas KCC2 is inhibited by membrane rafts. In agreement with this conclusion, transport inactive KCC2 in the neonatal brainstem (P0–2) is exclusively present in membrane rafts. Taken together, our analysis identifi es a cellular mechanism that might be involved in reciprocal regulation of NKCC1 and KCC2.
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