A novel N-terminal isoform of the neuron-specific K-Cl cotransporter KCC2.
The neuronal K-Cl cotransporter KCC2 maintains the low intracellular chloride concentration required for the hyperpolarizing actions of inhibitory neurotransmitters gamma-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.
Uvarov
Pavel
P
Neuroscience Center, Viikinkaari 4, University of Helsinki, Finland. pavel.uvarov@helsinki.fi
Ludwig
Anastasia
A
Markkanen
Marika
M
Pruunsild
Priit
P
Kaila
Kai
K
Delpire
Eric
E
Timmusk
Tônis
T
Rivera
Claudio
C
Airaksinen
Matti S
MS
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001933:Brain Stem / Q000378:metabolism* / Q000473:pathology; D002540:Cerebral Cortex / Q000378:metabolism* / Q000473:pathology; D005665:Furosemide / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D017136:Ion Transport / Q000187:drug effects / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics; D012119:Respiration / Q000235:genetics; D012413:Rubidium / Q000494:pharmacology; D012640:Seizures / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D049994:Sodium Potassium Chloride Symporter Inhibitors / Q000494:pharmacology; D013116:Spinal Cord / Q000378:metabolism* / Q000473:pathology; D027981:Symporters / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D000096922:K Cl- Cotransporters
D005665:Furosemide; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D012413:Rubidium; D049994:Sodium Potassium Chloride Symporter Inhibitors; D027981:Symporters; D000096922:K Cl- Cotransporters