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J. Biol. Chem., Vol. 282, Issue 42, 30570-30576, October 19, 2007

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

Pavel Uvarov¹², Anastasia Ludwig¹, Marika Markkanen, Priit Pruunsild^¶, Kai Kaila^||, Eric Delpire^**, Tônis Timmusk^¶, Claudio Rivera, and Matti S. Airaksinen³

From the Neuroscience Center, Viikinkaari 4, Institute of Biotechnology, Viikinkaari 9, and ^||Department of Biological and Environmental Sciences, Viikinkaari 1, University of Helsinki, 00014 Helsinki, Finland, the ^¶Department of Gene Technology, Tallinn University of Technology, Akadeemia tee 15, Tallinn 19086, Estonia, and the ^**Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee 37232

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.

Received for publication, June 21, 2007 , and in revised form, August 20, 2007.

^* This work was supported by grants from the Academy of Finland, the University of Helsinki, and the Sigrid Jusélius Foundation (to M. S. A. and C. R.) and by Wellcome Trust, the Estonian Science Foundation, and the Estonian Ministry of Education and Research (to T. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) EF641113.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3 and supplemental Table S1.

¹ Both authors contributed equally to this work.

² To whom correspondence may be addressed. E-mail: pavel.uvarov{at}helsinki.fi. ³ To whom correspondence may be addressed. E-mail: matti.airaksinen{at}helsinki.fi.

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