A non-synonymous single nucleotide polymorphism in the human SLC24A5 gene is associated with natural human skin color variation. Multiple sequence alignments predict that this gene encodes a member of the potassium-dependent sodium-calcium exchanger family denoted NCKX5. In cultured human epidermal melanocytes we show using affinity-purified antisera that native human NCKX5 runs as a triplet of approximately 43 kDa on SDS-PAGE and is partially localized to the trans-Golgi network. Removal of the NCKX5 protein through small interfering RNA-mediated knockdown disrupts melanogenesis in human and murine melanocytes, causing a significant reduction in melanin pigment production. Using a heterologous expression system, we confirm for the first time that NCKX5 possesses the predicted exchanger activity. Site-directed mutagenesis of NCKX5 and NCKX2 in this system reveals that the non-synonymous single nucleotide polymorphism in SLC24A5 alters a residue that is important for NCKX5 and NCKX2 activity. We suggest that NCKX5 directly regulates human epidermal melanogenesis and natural skin color through its intracellular potassium-dependent exchanger activity.
Membrane microdomains (lipid rafts) are enriched in selected signaling molecules and may compartmentalize receptor-mediated signals. Here, we report that in primary human B lymphocytes and in Ramos B cells B cell receptor (BCR) stimulation induces rapid and transient redistribution of a subset of engaged BCRs to lipid rafts and phosphorylation of raft-associated tyrosine kinase substrates. Cholesterol sequestration disrupted the lipid rafts, preventing BCR redistribution, but did not inhibit tyrosine kinase activation or phosphorylation of mitogen-activated protein kinase/extracellular regulated kinase. However, raft disruption enhanced the release of calcium from intracellular stores, suggesting that rafts may sequester early signaling events that down-regulate calcium flux. Consistent with this, BCR stimulation induced rapid and transient translocation of the Src homology 2 domain-containing inositol phosphatase, SHIP, into lipid rafts.
Light causes a rapid lowering of cytosolic free calcium in the outer segments of both retinal rod and cone photoreceptors. This light-induced lowering of calcium is caused by extrusion via a Na-Ca exchanger located in the rod and cone outer segment plasma membrane and plays a key role in the process of light adaptation. The Na-Ca exchanger in retinal rod outer segment was shown earlier to be a novel Na-Ca+K exchanger (NCKX), and its cDNA was obtained by molecular cloning from several mammalian species. On the other hand, the proper identity of the retinal cone Na-Ca exchanger, in terms of both functional characteristics (e.g., requirement for and transport of potassium) and molecular identity, has not yet been elucidated. Here, we report the molecular cloning, intraretinal localization by in situ hybridization, and initial functional characterization of the chicken and human cone-specific Na-Ca exchangers. In addition we report the chicken rod-specific NCKX. We identified NCKX transcripts in both human and chicken cones and observed strong potassium-dependent Na-Ca exchange activity after heterologous expression of human and chicken cone NCKX cDNAs in cultured insect cells. In situ hybridization in chicken retina showed abundant rod NCKX transcripts only in rod photoreceptors, whereas abundant cone NCKX transcripts were found in most, if not all, cone photoreceptors and also in a subpopulation of retinal ganglion cells. A detailed comparison with the previously described retinal rod and brain NCKX cDNAs is presented.
SUMMARY1. Intact rod outer segments (r.o.s.) isolated from bovine retinas were used to measure net Ca2+ fluxes using the optical Ca2+ indicator Arsenazo III. Ca2+ fluxes were observed, which could change the internal Ca2+ content of isolated r.o.s. by as much as 0 5 mM s-5.2. The Ca2+ content of isolated intact r.o.s. was strongly dependent on the Na/Ca ratio in the isolation medium, and could be made less than 01 mol Ca2+ mold1 rhodopsin (zero Ca2+ in isolation medium) or up to 7 mol Ca2+ mold1 rhodopsin (zero Na+ in isolation medium).3. Ca2+ efflux from r.o.s. rich in Ca2+ was observed only when Na+ was added to the external medium (as opposed to any other alkali cation); in Ca2+-depleted r.o.s. Ca2+ uptake required the presence of internal Na+ and was inhibited selectively by external Na+. These results suggest that Na-Ca exchange across the plasma membrane operated freely in both directions and controlled the internal Ca2+ concentration in r.o.s.4. Na+-stimulated Ca2+ efflux depended on the external Na+ concentration in a sigmoidal way. This suggests that the simultaneous binding of two Na ions is rate limiting for transport.5. In Ca2+-depleted r.o.s. and in the absence of external Na+, 1 mol Ca2+ mold1 rhodopsin (or 3 mM-total Ca2+) could be taken up within 1 min by intact r.o.s. at a free external Ca2+ concentration of about 1 /tM. 6. Only part of the internal Ca2+ was available for Na-Ca exchange. The external Na+ and K+ concentration as well as the temperature were factors controlling the accessibility of internal Ca2+ to participate in Na-Ca exchange.7. Ca2+ fluxes in r.o.s. with a permeabilized plasma membrane but intact disk membranes were very similar to those observed in intact r.o.s.; Na-Ca exchange could operate in both directions across the disk membrane.8. In addition to Na-Ca exchange, leaky r.o.s. also showed a guanosine 3', 5'-cyclic monophosphate (cyclic GMP)-induced Ca2+ release that was about I of the rate of Present address:
The retinal rod Na/Ca-K exchanger (NCKX) is a unique calcium extrusion protein utilizing both inward sodium gradient and outward potassium gradient. Three mammalian rod NCKX cDNAs have been cloned to date, but quantitative analysis of NCKX function in heterologous systems has proven difficult. Here, we describe a simple system for quantitative analysis of NCKX function; stable transformation of cultured insect cells with the novel pEA1/153A vector containing NCKX cDNAs was combined with measurements of potassium-dependent 45 Ca uptake in sodium-loaded cells. We carried out structure-function studies on NCKX with the following results: 1) two-thirds of the full-length sequence of bovine NCKX could be deleted without affecting potassiumdependent calcium transport and without affecting key properties of the potassium binding site; 2) the affinity of NCKX for potassium was about 10-fold greater in choline medium when compared with lithium medium; this shift was observed in rod outer segments or in cells expressing full-length rod NCKX, the above deletion mutant, or a distantly related NCKX paralog cloned from Caenorhabditis elegans. We conclude that the potassium binding site is highly conserved among members of the NCKX family and is formed by residues located within the two sets of transmembrane spanning segments in the NCKX sequence.Calcium extrusion across the plasma membrane of cells is vital to all cells, in view of the ubiquitous role of calcium as second messenger and since sustained elevated calcium levels rapidly lead to cell death (1). Calcium extrusion against a large electrochemical calcium gradient is mediated by two classes of plasma membrane proteins, an ATP-driven calcium pump and Na/Ca exchangers. Two groups of plasma membrane Na/Ca exchangers can be distinguished: those that neither require nor transport potassium (the NCX family) and those that require and, in the case of the rod photoreceptor NCKX1, have been demonstrated to transport potassium (the NCKX family) (for recent reviews, see Refs. 2 and 3). To date, three NCKX1 cDNAs have been cloned from mammalian rod photoreceptors (4 -6) and one NCKX2 cDNA from rat brain (7). Furthermore, several potential NCKX paralogs present in lower organisms have been identified on the basis of analysis of sequences obtained from genomic sequencing projects (2, 8). Studies on functional properties of the "in situ" Na/Ca-K exchanger have been limited thus far to NCKX1 found in the plasma membrane of the outer segments of retinal rod photoreceptors (reviewed in Refs. 9 -11). Sequence comparison of the three mammalian NCKX1 orthologs cloned to date reveals a remarkably low sequence identity (ϳ65%) in contrast to sequence identities of Ͼ90% observed for other sodium-coupled transporters. We examined functional activity of heterologously expressed dolphin, bovine, and human NCKX1 in several cell systems and only observed consistent and robust functional expression with the dolphin NCKX1 cDNA (6). Comparing the mammalian rod NCKX1 sequences with the sequence fro...
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