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N. J. Mangini, B. G. Kennedy, J. C. Valencia, V. A. Canfield, K. C. Cheng; Expression and Localization in RPE of a Novel Cation Exchanger (NCKX5/SLC24A5) That Plays a Role in Melanogenesis. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2528.
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NCKX5 (SLC24A5), a newly-described K-dependent, Na/Ca exchanger, localizes to intracellular organelles, including melansomes. A SNP in NCKX5 makes the largest known contribution to skin color differences between humans of African and European ancestry (Science, 310, 2005). Pigmentation is a risk factor for age-related macular degeneration (AMD) and the gene encoding NCKX5 is located on chromosome 15q21, a region that also has a susceptibility locus associated with AMD (Am. J. Hum Genet. 74, 2004). This work examines NCKX5 expression and localization in native and cultured human RPE as a first step to defining the relevance of this transporter to RPE melanization and any relation to AMD susceptibility.
NCKX5 gene expression was assessed by RT-PCR analysis of total RNA from freshly-isolated RPE (2 donors); primary RPE cell cultures (3 different donors) and two established RPE cell lines (D407 and ARPE19). Polyclonal antibodies were made in chickens and rabbits against synthetic peptides corresponding to NCKX5-specific sequences. Protein expression was examined by Western blotting. NCKX5 localization in native RPE tissue and cultured hRPE was examined by fluorescence and confocal immunohistochemistry.
The NCKX5-identity of PCR DNAs and two full-length cDNAs from a Caucasian donor was confirmed by sequencing. Both cDNAs were thr111 alleles; one cDNA (ACC# DQ665307) was a splice variant missing exon 2. Western blotting with chicken and rabbit NCKX5 antibodies detected a major band at ~ 59 kD (presumed to be the full size protein) in all hRPE lysates. In cultured hRPE punctuate immunocytochemical staining was detected in the cytoplasm. In native tissue, staining was also punctuate and more clearly localized to organelles, presumably including melanosomes.
The present work shows that HRPE express the NCKX5/SLC25A5 gene encoding a putative K-dependent Na/Ca exchanger. Our findings suggest that alternative splicing of the transcript occurs. Immunohistochemical studies with NCKX5-specific antibodies document that the protein localizes to intracellular organelles, likely including melanosomes, in RPE.
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