Differences in the large extracellular loop between the K(+)-Cl(-) cotransporters KCC2 and KCC4.

K(+)Cl(-) cotransporters (KCCs) play fundamental physiological roles in processes such as inhibitory neurotransmission and cell volume regulation. Mammalian genomes encode four distinct KCC paralogs, which share basic transport characteristics but differ significantly in ion affinity, pharmacology, and relative sensitivity to cell volume. Studies to identify divergence in functional characteristics have thus far focused on the cytoplasmic termini. Here, we investigated sequence requirements of the large extracellular loop (LEL) for function in KCC2 and KCC4. Mutation of all four evolutionarily conserved cysteines abolished KCC2 transport activity. This behavior differs from that of its closest relative, KCC4, which is insensitive to this mutation. Chimeras supported the differences in the LEL of the two cotransporters, because swapping wild-type LEL resulted in functional KCC2 but rendered KCC4 inactive. Insertion of the quadruple cysteine substitution mutant of the KCC4 loop, which was functional in the parental isoform, abolished transport activity in KCC2. Dose-response curves of wild-type and chimeric KCCs revealed that the LEL contributes to the different sensitivity to loop diuretics; a KCC2 chimera containing the KCC4 LEL displayed an IC(50) of 396.5 mum for furosemide, which was closer to KCC4 (548.8 mum) than to KCC2 (184.4 mum). Cell surface labeling and immunocytochemistry indicated that mutations do not affect trafficking to the plasma membrane. Taken together, our results show a dramatic and unexpected difference in the sequence requirements of the LEL between the closely related KCC2 and KCC4. Furthermore, they demonstrate that evolutionarily highly conserved amino acids can have different functions within KCC members.

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.

CIP1 is an activator of the K+-Cl- cotransporter KCC2.

In most neurons, efficient setting of the intracellular Cl(-)-concentration requires the coordinated regulation of the Cl(-)-inward transporter NKCC1 and the Cl(-)-outward transporter KCC2. Previously, the cation-chloride cotransporter interacting protein 1 (CIP1) was shown to inactivate NKCC1. Here, we investigated its role for KCC2 activity. After co-expression of CIP1 and KCC2 in HEK-293 cells, a physical and functional interaction was observed. CIP1 co-purified with KCC2 in pull-down experiments and significantly increased KCC2 transport activity, as determined by 86Rb+ flux measurements. RT-PCR analysis demonstrated a ubiquitous expression during postnatal development of the rat. Real-time PCR revealed a two-fold down-regulation of CIP1 during maturation of the rat brain. Taken together, our data identify CIP1 as a potent activator of KCC2. Furthermore, the results support previous data of heteromer formation among members of the cation-chloride cotransporter gene family.

Hypothyroidism impairs chloride homeostasis and onset of inhibitory neurotransmission in developing auditory brainstem and hippocampal neurons.

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.