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

doi:10.1074/jbc.271.27.16245

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

A NEURONAL-SPECIFIC ISOFORM*

From the Departments of Human Physiology and
^§ Biological Chemistry, University of California School of Medicine, Davis, California 95616

^‡ To whom correspondence should be addressed. Tel.: 916-752-1359; Fax: 916-752-5423.

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 ∼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.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The 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 GenBank™/EMBL Data Bank with accession number(s) U55816[GenBank].
↵¹ The abbreviations used are:

CCC

cation-chloride cotransporter

KCC

K-Cl cotransporter

NKCC

Na-K-Cl cotransporter

NCC and TSC

thiazide-sensitive Na-Cl cotransporter

IPSP

inhibitory postsynaptic potential

GABA

γ-aminobutyric acid

EST

expressed sequenced tag

TM

transmembrane segment

E_Cl

equilibrium potential for Cl⁻

[X]_i

intracellular concentration

PCR

polymerase chain reaction

RT-PCR

reverse transcriptase PCR

bp

base pair(s)

nt

nucleotide(s).
- Received April 3, 1996.