Abstract
K(+)-dependent Na(+)/Ca(2+) exchangers belong to the solute carrier 24 (SLC24A1-5) gene family of membrane transporters. Five different gene products (NCKX1-5) have been identified in humans, which play key roles in biological processes including vision, olfaction, and skin pigmentation. NCKXs are bi-directional membrane transporters that transport 1 Ca(2+)+K(+) ions in exchange for 4 Na(+) ions. Recent studies have linked mutations in the SLC24A4 (NCKX4) and SLC24A5 (NCKX5) genes to amylogenesis imperfecta (AI) and non-syndromic oculocutaneous albinism (OCA6), respectively. Here, we introduced mutations found in patients with AI and OCA6 into human SLC24A4 (NCKX4) cDNA leading to single residue substitutions in the mutant NCKX4 proteins. We measured NCKX-mediated Ca(2+) transport activity of WT and mutant NCKX4 proteins expressed in HEK293 cells. Three mutant NCKX4 cDNAs represent mutations found in the SCL24A4 gene and three represent mutations found in the SCL24A5 gene involving residues conserved between NCKX4 and NCKX5. Five mutant proteins had no observable NCKX activity, whereas one mutation resulted in a 78% reduction in transport activity. Total protein expression and trafficking to the plasma membrane (the latter with one exception) were not affected in the HEK293 cell expression system. We also analyzed two mutations in a Drosophila NCKX gene that have been reported to result in an increased susceptibility for seizures, and found that both resulted in mutant proteins with significantly reduced but observable NCKX activity. The data presented here support the genetic analyses that mutations in SLC24A4 and SLC24A5 are responsible for the phenotypic defects observed in human patients.
Highlights
DMEM and cells were split to 500,000 cells in 10-cm plates and left to grow overnight
It is believed that all NCKXs share a common topology with 10 TMSs divided into two conserved clusters of five TMSs connected by a divergent cytoplasmic loop (Fig. 2A)
NCKX4 transfected into HEK293 cells yields strong functional protein activity allowing for high resolution activity measurements unlike the Nckx-x and NCKX5 clones
Summary
All chemicals were purchased from Sigma and all tissue culture supplies were purchased from ThermoFisher Scientific. PcDNA Preparation of the Mutant NCKX4 Proteins—The NCKX4 cDNA used in our study (accession number AF520705) was modified by introducing the Myc tag (EQKLISEEDL) into the extracellular loop (after amino acid 56 and includes Asp of the WT sequence) that precedes TMS1 to allow for easy detection of the expressed NCKX4 protein by Western blotting. The seven mutations in the NCKX4 protein summarized in Table 1 were prepared using the site-directed mutagenesis PCR technique; briefly, primers that included specific point mutations for the seven amino acid substitutions (G141S, A142T, A146E, A146V, S213R, L436R, and S499C) were prepared (Table 2) and used for the PCR. The PCR product was subcloned into pcDNA3.1 flanked by the ApaI and BamHI restriction sites. All products were sequenced to verify that the correct mutation was introduced (University of Calgary Sequencing Facility)
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