A Novel N-terminal Isoform of the Neuron-specific K-Cl Cotransporter KCC2

Abstract

The neuronal K-Cl cotransporter KCC2 maintains the low intracellular chloride concentration required for the hyperpolarizing actions of inhibitory neurotransmitters ␥-aminobutyric acid and glycine in the central nervous system. This study shows that the mammalian KCC2 gene (alias Slc12a5) generates two neuron-specific isoforms by using alternative promoters and first exons. The novel KCC2a isoform differs from the only previously known KCC2 isoform (now termed KCC2b) by 40 unique N-terminal amino acid residues, including a putative Ste20-related proline alanine-rich kinase-binding site. Ribonuclease protection and quantitative PCR assays indicated that KCC2a contributes 20 -50% of total KCC2 mRNA expression in the neonatal mouse brain stem and spinal cord. In contrast to the marked increase in KCC2b mRNA levels in the cortex during postnatal development, the overall expression of KCC2a remains relatively constant and makes up only 5-10% of total KCC2 mRNA in the mature cortex. A rubidium uptake assay in human embryonic kidney 293 cells showed that the KCC2a isoform mediates furosemide-sensitive ion transport activity comparable with that of KCC2b. Mice that lack both KCC2 isoforms die at birth due to severe motor defects, including disrupted respiratory rhythm, whereas mice with a targeted disruption of the first exon of KCC2b survive for up to 2 weeks but eventually die due to spontaneous seizures. We show that these mice lack KCC2b but retain KCC2a mRNA. Thus, distinct populations of neurons show a differential dependence on the expression of the two isoforms: KCC2a expression in the absence of KCC2b is presumably sufficient to support vital neuronal functions in the brain stem and spinal cord but not in the cortex.

A Single Seizure Episode Leads to Rapid Functional Activation of KCC2 in the Neonatal Rat Hippocampus

“…The available data indicate that the latter isoform is required for the developmental E GABA shift, but little is known about the functional significance of KCC2a (Uvarov et al, 2007(Uvarov et al, , 2009). Nevertheless, the parallel activitydependent upregulation of the two isoforms is interesting because of their capability of heterodimerization (Uvarov et al, 2009 Ϫ extrusion mechanism.…”

“…2 E-G). Two splice variants of KCC2 (KCC2a and b) have recently been described (Uvarov et al, 2007). These isoforms are coexpressed in most brain regions and seem to be able to form heterodimers (Uvarov et al, 2009).…”

Early Growth Response 4 Mediates BDNF Induction of Potassium Chloride Cotransporter 2 Transcription

“…The shift in polarity of GABA A responses occurs in rodent hippocampus during the first 2 postnatal weeks (Farrant and Kaila, 2007). We have shown that the KCC2 gene generates two isoforms, KCC2a and KCC2b, by an alternative promoter and first exon usage (Uvarov et al, 2007). During development, expression levels of KCC2a mRNA in hippocampus are relatively constant, whereas KCC2b mRNA increases significantly.…”

“…During development, expression levels of KCC2a mRNA in hippocampus are relatively constant, whereas KCC2b mRNA increases significantly. This indicates that the developmental increase in the expression of KCC2 mRNA and protein is mainly due to the KCC2b isoform (Uvarov et al, 2007(Uvarov et al, , 2009.…”

Similar effects of all WNK3 variants on SLC12 cotransporters

“…KCC2a (26) was obtained by cutting and pasting KCC2 exon 1a amplified from the human fetal brain cDNA library in a process similar to that described above. KCC2a construction was verified by DNA sequencing.…”