Cloning and Characterization of KCC3 and KCC4, New Members of the Cation-Chloride Cotransporter Gene Family

doi:10.1074/jbc.274.23.16355

Cloning and Characterization of KCC3 and KCC4, New Members of the Cation-Chloride Cotransporter Gene Family*

From the Departments of ^‡Medicine, ^**Pharmacology, and ^§§Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee 37232 and the ^¶Molecular Physiology Unit, Instituto Nacional de la Nutrición Salvador Zubirán and Instituto de Investigaciones Biomédicas UNAM, Mexico City, Mexico

Abstract

The K⁺-Cl⁻cotransporters (KCCs) belong to the gene family of electroneutral cation-chloride cotransporters, which also includes two bumetanide-sensitive Na⁺-K⁺-2Cl⁻cotransporters and a thiazide-sensitive Na⁺-Cl⁻ cotransporter. We have cloned cDNAs encoding mouse KCC3, human KCC3, and human KCC4, three new members of this gene family. The KCC3 and KCC4 cDNAs predict proteins of 1083 and 1150 amino acids, respectively. The KCC3 and KCC4 proteins are 65–71% identical to the previously characterized transporters KCC1 and KCC2, with which they share a predicted membrane topology. The four KCC proteins differ at amino acid residues within key transmembrane domains and in the distribution of putative phosphorylation sites within the amino- and carboxyl-terminal cytoplasmic domains. The expression of mouse KCC3 in Xenopus laevis oocytes reveals the expected functional characteristics of a K⁺Cl⁻ cotransporter: Cl⁻-dependent uptake of⁸⁶Rb⁺ which is strongly activated by cell swelling and weakly sensitive to furosemide. A direct functional comparison of mouse KCC3 to rabbit KCC1 indicates that KCC3 has a much greater volume sensitivity. The human KCC3 and KCC4 genes are located on chromosomes 5p15 and 15q14, respectively. Although widely expressed, KCC3 transcripts are the most abundant in heart and kidney, and KCC4 is expressed in muscle, brain, lung, heart, and kidney. The unexpected molecular heterogeneity of K⁺-Cl⁻ cotransport has implications for the physiology and pathophysiology of a number of tissues.

Footnotes

↵* This work was supported by National Institutes of Health Grants K-11 DK02103 (to D. B. M.) and R-29 HL-49251 (to E. D.) and by grants from the Mexican Council of Science and Technology (CONACYT, M3840) and the Howard Hughes Medical Institute (75197-553601) (to G. G).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) AF087436 (mKCC3), AF105365 (hKCC3), and AF105366 (hKCC4).
↵§ To whom correspondence should be addressed: Div. of Nephrology, Vanderbilt University Medical Ctr., MCN S-3223, Nashville, TN 37232. Tel.: 615-343-2853; Fax: 615-343-7156; E-mail:david.mount{at}mcmail.vanderbilt.edu.
↵‖ Supported by a scholarship from Dirección Genaral del Personal Académico of the National University of Mexico.
↵‡ Established investigator of the American Heart Association.
↵2 D. B. Mount and E. Delpire, unpublished data.
Abbreviations:

KCC

K⁺-Cl⁻ cotransporter

BSC or NKCC

bumetanide-sensitive Na⁺-K⁺-2Cl⁻cotransporter

TSC or NCC

thiazide-sensitive Na⁺-Cl⁻ cotransporter

h

human (e.g. hKCC)

m

mouse (e.g. mKCC)

⁸⁶Rb⁺

rubidium

EST

expressed sequence tag

bp

base pair(s)

kb

kilobase (or kilobase pairs)

IMAGE

integrated molecular analysis of genomes and their expression

TIGR

The Institute for Genome Research

UTR

untranslated region

RT-PCR

reverse transcriptase polymerase chain reaction

RACE

rapid amplification of conserved ends

VNTR

variable number of tandem repeats

TM

transmembrane segment

PKC

protein kinase C
- Received February 25, 1999.
- Revision received March 29, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.