Molecular Cloning of a Sixth Member of the K+-dependent Na+/Ca2+ Exchanger Gene Family, NCKX6

Xinjiang Cai,Jonathan Lytton

doi:10.1074/jbc.m310908200

Abstract

Bioinformatic and molecular cloning tools were used to identify and isolate cDNA clones from mouse and human tissues that encode the sixth member of the K(+)-dependent Na+/Ca2+ exchanger family, NCKX6. The mouse NCKX6 protein is 585 amino acids long and shares about 62% sequence similarity with previously identified exchangers in the alpha-repeat regions but has little primary sequence similarity outside these regions. NCKX6 transcripts of 4 kb are abundantly expressed in all tissues examined and are thus more broadly distributed than previously described NC(K)X family members. Two alternatively spliced products of this novel gene were identified that encode proteins of different length. The short isoform differs from the full-length isoform at the C-terminal hydrophobic domain as a result of a shift in the reading frame caused by the deletion of two exons. Both NCKX6 isoforms were expressed in HEK-293 cells. Functional analysis by digital imaging of fura-2 loaded transfected HEK-293 cells demonstrated that the short isoform exhibited K(+)-dependent Na+/Ca2+ exchange activity whereas the full-length isoform did not. The latter was retained within the endoplasmic reticulum, whereas the short isoform was present at the plasma membrane in transfected cells. Immunofluorescence studies examining NCKX6 expression in native tissue using an NCKX6-specific antibody showed intense labeling of the cardiac sarcolemmal membrane. The discovery of NCKX6 therefore reveals a novel member of the Na+/Ca2+ exchanger superfamily whose ubiquitous expression in all tissues suggests an important role for K(+)-dependent Na+/Ca2+ exchange in maintaining cellular Ca2+ homeostasis in diverse tissues and cell types.

Highlights

Calcium signaling plays an important role in mediating numerous cellular processes in virtually all types of animal cells [1]
We have identified a novel Naϩ/Ca2ϩ exchanger molecule, NCKX6, which is ubiquitously expressed in various tissues examined and shares a moderate degree of sequence similarity in the ␣-repeat regions with previously cloned Naϩ/Ca2ϩ exchangers (NCX) and NCKX exchangers
Protein possesses a topology in the N-terminal domain very similar to known NCX and NCKX members: a signal peptide, M0, a glycosylated extracellular loop, and a cluster of five transmembrane segments (TMS) followed by a large intracellular loop

Summary

EXPERIMENTAL PROCEDURES

All molecular procedures were performed according to standard protocols [27, 28] or the directions of reagent manufacturers, unless noted otherwise. The longer clone, encoding the full-length NCKX6 protein, was identical to the novel mouse homolog previously identified in GenBankTM (accession number AF261233). While this manuscript was being prepared, the sequence data for the short clone was deposited in GenBankTM with accession number BC043689 by the Mammalian Gene Collection group at the National Institutes of Health. University of Calgary), which is an expression construct that encodes a fusion protein between EGFP and the C-terminal 113 amino acids of a recently identified Golgi protein Golgin-67 [35] This construct was co-transfected into HEK-293 cells with the FLAGtagged mouse full-length NCKX6 clone. Visualization and processing of images were performed as described above

RESULTS

DISCUSSION

Xinjiang Cai and Jonathan Lytton