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.

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Thyroid hormone (TH) deficiency during perinatal life causes a multitude of functional and morphological deficits in the brain. In rats and mice, TH dependency of neural maturation is particularly evident during the first 1-2 weeks of postnatal development. During the same period, synaptic transmission via the inhibitory transmitters glycine and GABA changes from excitatory depolarizing effects to inhibitory hyperpolarizing ones in most neurons [depolarizing-hyperpolarizing (D/H) shift]. The D/H shift is caused by the activation of the K(+)-Cl(-) co-transporter KCC2 which extrudes Cl(-) from the cytosol, thus generating an inward-directed electrochemical Cl(-) gradient. Here we analyzed whether the D/H shift and, consequently, the onset of inhibitory neurotransmission are influenced by TH. Gramicidin perforated-patch recordings from auditory brainstem neurons of experimentally hypothyroid rats revealed depolarizing glycine effects until postnatal day (P)11, i.e. almost 1 week longer than in control rats, in which the D/H shift occurred at approximately P5-6. Likewise, until P12-13 the equilibrium potential E(Gly) in hypothyroids was more positive than the membrane resting potential. Normal E(Gly) could be restored upon TH substitution in P11-12 hypothyroids. These data demonstrate a disturbed Cl(-) homeostasis following TH deficiency and point to a delayed onset of synaptic inhibition. Interestingly, immunohistochemistry demonstrated an unchanged KCC2 distribution in hypothyroids, implying that TH deficiency did not affect KCC2 gene expression but may have impaired the functional status of KCC2. Hippocampal neurons of hypothyroid P16-17 rats also demonstrated an impaired Cl(-) homeostasis, indicating that TH may have promoted the D/H shift and maturation of synaptic inhibition throughout the brain.

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