Molecular characterization of a putative K-Cl cotransporter in rat brain

A neuronal-specific isoform

John A Payne, Tamara J. Stevenson, Lucy F. Donaldson

Research output: Contribution to journalArticle

407 Citations (Scopus)

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 kids. 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 ~25% 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 ~5.6-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.

Original languageEnglish (US)
Pages (from-to)16245-16252
Number of pages8
JournalJournal of Biological Chemistry
Volume271
Issue number27
DOIs
StatePublished - 1996

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Neurons
Rats
Brain
Protein Isoforms
Polymerase chain reaction
Cations
Chlorides
Complementary DNA
Sodium Potassium Chloride Symporter Inhibitors
Thiazides
Pyramidal Cells
Purkinje Cells
Glycosylation
Amino Acids
Reverse Transcriptase Polymerase Chain Reaction
Glioma
Neuroglia
Astrocytes
Northern Blotting
Cerebellum

ASJC Scopus subject areas

  • Biochemistry

Cite this

Molecular characterization of a putative K-Cl cotransporter in rat brain : A neuronal-specific isoform. / Payne, John A; Stevenson, Tamara J.; Donaldson, Lucy F.

In: Journal of Biological Chemistry, Vol. 271, No. 27, 1996, p. 16245-16252.

Research output: Contribution to journalArticle

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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 kids. 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 ~25{\%} 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 ~5.6-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.",
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