Molecular Characterization of a Putative K-Cl Cotransporter in Rat Brain

Abstract

Using a combination of data base searching, polymerase chain reaction, and library screening, we have identified a putative K-Cl cotransporter isoform (KCC2) in rat brain that is specifically localized in neurons. A cDNA of 5566 bases was obtained from overlapping clones and encoded a protein of 1116 amino acids with a deduced molecular mass of 123.6 kDa. Over its full length, the amino acid sequence of KCC2 is 67% identical to the widely distributed K-Cl cotransporter isoform (KCC1) identified in rat brain and rabbit kidney (Gillen, C., Brill, S., Payne, J.A., and Forbush, B., III (1996) J. Biol. Chem. 271, 16237-16244) but only ϳ25% identical to other members of the cation-chloride cotransporter gene family, including "loop" diuretic-sensitive Na-K-Cl cotransport and thiazide-sensitive Na-Cl cotransport. Based on analysis of the primary structure as well as homology with other cation-chloride cotransporters, we predict 12 transmembrane segments bounded by N-and C-terminal cytoplasmic regions. Four sites for N-linked glycosylation are predicted on an extracellular intermembrane loop between putative transmembrane segments 5 and 6. Northern blot analysis using a KCC2-specific cDNA probe revealed a very highly expressed ϳ5.6-kilobase transcript only in brain. Reverse transcriptase-polymerase chain reaction revealed that KCC1 was present in rat primary astrocytes and rat C6 glioma cells but that KCC2 was completely absent from these cells, suggesting KCC2 was not of glial cell origin. In situ hybridization studies demonstrated that the KCC2 transcript was expressed at high levels in neurons throughout the central nervous system, including CA1-CA4 pyramidal neurons of the hippocampus, granular cells and Purkinje neurons of the cerebellum, and many groups of neurons throughout the brainstem.

A Furosemide-Sensitive K+–Cl−Cotransporter Counteracts Intracellular Cl−Accumulation and Depletion in Cultured Rat Midbrain Neurons

“…Depolarizing GABA A responses, however, can be caused by bicarbonate efflux in combination with C l Ϫ influx or combined Cl Ϫ and HCO 3 Ϫ efflux (Grover et al, 1993;Kaila, 1994;Thompson, 1994;Staley et al, 1995;Perkins and Wong, 1996;Kaila et al, 1997). To be able to predict the direction of C l Ϫ currents flowing during GABA A receptor-mediated inhibition it is essential to understand the regulation of C l Ϫ homeostasis that provides the transmembrane gradient.A recently cloned K ϩ -Cl Ϫ cotransporter gene (KCC2) represents a perfect candidate for the regulation of neuronal Cl Ϫ homeostasis (Payne et al, 1996). In contrast to the ubiquitous presence of the K ϩ -Cl Ϫ cotransporter KCC1, expression of the K ϩ -Cl Ϫ cotransporter KCC2 is detected in C NS only and seems to be neuron specific.…”

“…A recently cloned K ϩ -Cl Ϫ cotransporter gene (KCC2) represents a perfect candidate for the regulation of neuronal Cl Ϫ homeostasis (Payne et al, 1996). In contrast to the ubiquitous presence of the K ϩ -Cl Ϫ cotransporter KCC1, expression of the K ϩ -Cl Ϫ cotransporter KCC2 is detected in C NS only and seems to be neuron specific.…”

“…In vivo additional factors determine gradients for IPSCs through GABA A or glycine channels, complicating the analysis of their regulation. Despite this fact, a furosemide-sensitive K ϩ -Cl Ϫ cotransporter is likely to operate in central neurons because the KCC2 mRNA is expressed in hippocampal neurons (Payne et al, 1996). The permeability for HCO 3 Ϫ shifts E GABA by 10 -15 mV (depending on pH i ) in a positive direction (Alvarez-Leefmans, 1990;Kaila, 1994).…”

Mice Lacking the Basolateral Na-K-2Cl Cotransporter Have Impaired Epithelial Chloride Secretion and Are Profoundly Deaf

“…1 the basolateral or secretory isoform of the Na-K2Cl cotransporter (1,2), is a member of the cation-coupled chloride transporter family (3,4), which includes the apical Na-K-2Cl cotransporter of the renal thick ascending limb (5,6), the NaCl cotransporter of the renal distal convoluted tubule (6), and four KCl cotransporters (7)(8)(9). NKCC1 mediates the coupled electroneutral transport of 1 Na ϩ , 1 K ϩ , and 2 Cl Ϫ ions across the plasma membrane (4), driven by the inwardly directed Na ϩ and Cl Ϫ gradients that occur under physiological conditions.…”

Pallidal control of substantia nigra dopaminergic neuron firing pattern and its relation to extracellular neostriatal dopamine levels

“…As a ligand-gated chloride channel, GABA A receptor function is dependent on cellular chloride regulatory mechanisms (Kaila, 1994). In most mature CNS neurons, a neuron-specific K + Cl À cotransporter (KCC2) is responsible for extruding intracellular chloride, thereby generating an inwardly directed chloride gradient which makes increases in chloride conductance hyperpolarizing (Payne et al, 1996(Payne et al, , 2003DeFazio et al, 2000). Light and electron microscopic immunocytochemical labeling revealed the expression of KCC2 by GABAergic pars reticulata neurons (Gulacsi et al, 2003) (Fig.…”