Potassium-dependent Sodium-calcium Exchanger Research Articles

Chemical Imaging of Retinal Pigment Epithelium in Frozen Sections of Zebrafish Larvae Using ToF-SIMS.

Variants of the SLC24A5 gene, which encodes a putative potassium-dependent sodium-calcium exchanger (NCKX5) that most likely resides in the melanosome or its precursor, affect pigmentation in both humans and zebrafish (Danio rerio). This finding suggests that genetic variations influencing human skin pigmentation alter melanosome biogenesis via ionic changes. Gaining an understanding of how changes in the ionic environment of organelles impact melanosome morphogenesis and pigmentation will require a spatially resolved way to characterize the chemical environment of melanosomes in pigmented tissue such as retinal pigment epithelium (RPE). The imaging mass spectrometry technique most suited for this type of cell and tissue analysis is time-of-flight secondary ion mass spectrometry (ToF-SIMS) because it is able to detect many biochemical species with high sensitivity and with submicron spatial resolution. Here, we describe chemical imaging of the RPE in frozen-hydrated sections of larval zebrafish using cryo-ToF-SIMS. To facilitate the data interpretation, positive and negative polarity ToF-SIMS image data were transformed into a single hyperspectral data set and analyzed using principal component analysis. The combination of a novel protocol and the use of multivariate data analysis allowed us to discover new marker ions that are attributable to leucodopachrome, a metabolite specific to the biosynthesis of eumelanin. The described methodology may be adapted for the investigation of other classes of molecules in frozen tissues from zebrafish and other organisms.

Expression of potassium-dependent sodium-calcium exchanger in the murine lens

Loss of intracellular calcium homeostasis may contribute to the opacification of lens tissue during cortical cataract formation. In healthy lenses, the concentration of intracellular calcium is maintained at levels far below electrochemical equilibrium but the identity of the calcium extrusion mechanism in lens fiber cells has remained elusive. Previous studies focused on the role of plasma membrane calcium ATPases and sodium-calcium exchangers. Here, we examined the expression of mRNA transcripts encoding potassium-dependent sodium-calcium exchangers (Nckx's, encoded by the Slc24 gene family) in the mouse lens. The most abundant of the five Slc24 family members was Slc24a4 (Nckx4). Notably, Slc24a4 was the only family member with increased expression in fiber cells. Using an antibody raised against recombinant mouse Nckx4, we showed that the protein is expressed strongly in the outer cortical fibers, consistent with results of in situ hybridization experiments and earlier mass spectrometry analysis. To test the role of Nckx4 directly, we generated mice in which Slc24a4 was deleted conditionally in lens tissue. In conditional knockout animals, the level of Nckx4 protein was reduced to background levels without a discernible effect on lens growth or transparency. Thus, despite its relative abundance in the lens, Nckx4 does not appear to have an indispensable role in the maintenance of lens clarity.

The genome draft of coconut (Cocos nucifera).

Coconut palm (Cocos nucifera,2n = 32), a member of genus Cocos and family Arecaceae (Palmaceae), is an important tropical fruit and oil crop. Currently, coconut palm is cultivated in 93 countries, including Central and South America, East and West Africa, Southeast Asia and the Pacific Islands, with a total growth area of more than 12 million hectares [1]. Coconut palm is generally classified into 2 main categories: “Tall” (flowering 8–10 years after planting) and “Dwarf” (flowering 4–6 years after planting), based on morphological characteristics and breeding habits. This Palmae species has a long growth period before reproductive years, which hinders conventional breeding progress. In spite of initial successes, improvements made by conventional breeding have been very slow. In the present study, we obtained de novo sequences of the Cocos nucifera genome: a major genomic resource that could be used to facilitate molecular breeding in Cocos nucifera and accelerate the breeding process in this important crop. A total of 419.67 gigabases (Gb) of raw reads were generated by the Illumina HiSeq 2000 platform using a series of paired-end and mate-pair libraries, covering the predicted Cocos nucifera genome length (2.42 Gb, variety “Hainan Tall”) to an estimated ×173.32 read depth. A total scaffold length of 2.20 Gb was generated (N50 = 418 Kb), representing 90.91% of the genome. The coconut genome was predicted to harbor 28 039 protein-coding genes, which is less than in Phoenix dactylifera (PDK30: 28 889), Phoenix dactylifera (DPV01: 41 660), and Elaeis guineensis (EG5: 34 802). BUSCO evaluation demonstrated that the obtained scaffold sequences covered 90.8% of the coconut genome and that the genome annotation was 74.1% complete. Genome annotation results revealed that 72.75% of the coconut genome consisted of transposable elements, of which long-terminal repeat retrotransposons elements (LTRs) accounted for the largest proportion (92.23%). Comparative analysis of the antiporter gene family and ion channel gene families between C. nucifera and Arabidopsis thaliana indicated that significant gene expansion may have occurred in the coconut involving Na+/H+ antiporter, carnitine/acylcarnitine translocase, potassium-dependent sodium-calcium exchanger, and potassium channel genes. Despite its agronomic importance, C. nucifera is still under-studied. In this report, we present a draft genome of C. nucifera and provide genomic information that will facilitate future functional genomics and molecular-assisted breeding in this crop species.

A SLC24A2 Gene Variant Uncovered in Pancreatic Ductal Adenocarcinoma by Whole Exome Sequencing.

Pancreatic ductal adenocarcinoma (PDAC) presents as an aggressive malignancy caused by environmental and genetic factors. In order to identify causal genes for PDAC, we performed whole exome sequencing (WES) to detect gene mutations in seven pairs of PDAC tissue and adjacent non-tumor tissue samples. Finally, we found a new nonsynonymous single nucleotide variant (nsSNV) in solute carrier 24 family member 2 (SLC24A2) gene resulting in the substitution of native glutamic acid (E) into aspartic acid (D) at position of 287 amino acid (E287D) in SLC24A2 protein, and confirmed this variant by Sanger gene sequencing. SLC24A2 is a potassium-dependent sodium-calcium exchanger and can transport metal ion across cell membrane. Multiple in silico variants' effects analyses methods including SIFT, PolyPhen, PROVEAN, and PANTHER demonstrated this variant had probably damaging effects, which was consistent with the results obtained from Mutation Taster software analysis with a probability of 0.99999997 to be "disease causing." The three dimension (3D) structure analysis results suggested this variant had little effects on the solubility and hydrophobicity of the protein; but it could decrease the protein stability by increasing the total protein structure energy (-8874.33 kJ/mol for the mutant and -8963.54 kJ/mol for the native) and by causing the mutant protein decreasing three stabilizing residues. Less stability of the mutant 287D protein than the native E287 protein was also supported by I-Mutant and Western-blotting analysis results. Overall, a new mutation in SLC24A2 gene was identified to decrease the stability of SLC24A2, which may have potential clinical usages.

A critical role for the potassium-dependent sodium-calcium exchanger NCKX2 in protection against focal ischemic brain damage.

The superfamily of cation/Ca2+ plasma-membrane exchangers contains two branches, the K+-independent Na+-Ca2+ exchangers (NCXs) and the K+-dependent Na+-Ca2+ exchangers (NCKXs), widely expressed in mammals. NCKX2 is the major neuronally expressed isoform among NCKX members. Despite its importance in maintaining Na+, Ca2+, and K+ homeostasis in the CNS, the role of NCKX2 during cerebral ischemia, a condition characterized by an alteration of ionic concentrations, has not yet been investigated. The present study examines NCKX2 role in the development of ischemic brain damage in permanent middle cerebral artery occlusion (pMCAO) and transient middle cerebral artery occlusion. Furthermore, to evaluate the effect of nckx2 ablation on neuronal survival, nckx2-/- primary cortical neurons were subjected to oxygen glucose deprivation plus reoxygenation. NCKX2 mRNA and protein expression was evaluated in the ischemic core and surrounding ipsilesional areas, at different time points after pMCAO in rats. In ischemic core and in periinfarctual area, NCKX2 mRNA and protein expression were downregulated. In addition, NCKX2 knock-down by antisense oligodeoxynucleotide and NCKX2 knock-out by genetic disruption dramatically increased infarct volume. Accordingly, nckx2-/- primary cortical neurons displayed a higher vulnerability and a greater [Ca2+]i increase under hypoxic conditions, compared with nckx2+/+ neurons. In addition, NCKX currents both in the forward and reverse mode of operation were significantly reduced in nckx2-/- neurons compared with nckx2+/+ cells. Overall, these results indicate that NCKX2 is involved in brain ischemia, and it may represent a new potential target to be investigated in the study of the molecular mechanisms involved in cerebral ischemia.

SLC24A5 Encodes a trans-Golgi Network Protein with Potassium-dependent Sodium-Calcium Exchange Activity That Regulates Human Epidermal Melanogenesis

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.

Molecular Cloning of a Fourth Member of the Potassium-dependent Sodium-Calcium Exchanger Gene Family, NCKX4

We report here the identification and characterization of a fourth member of the potassium-dependent sodium-calcium exchanger gene family, NCKX4 (gene SLC24A4), which mapped to the chromosomal region 14q32. Human NCKX4 encoded a protein of 605 amino acids that displayed a high level of sequence identity to previously described family members, rod NCKX1 (gene SLC24A1), cone/neuronal NCKX2 (gene SLC24A2), and ubiquitous NCKX3 (gene SLC24A3), in the hydrophobic regions surrounding the alpha-repeat sequences thought to form the ion-binding pocket used for transport. The protein product of the NCKX4 gene shared the highest level of amino acid identity, as well as an almost identical arrangement of exon boundaries, with NCKX3, indicating that these two genes have arisen from a recent duplication event. NCKX4 transcripts were abundantly expressed in all brain regions, aorta, lung, and thymus, as well as at a lower level in many other tissues. The NCKX4 protein demonstrated potassium-dependent sodium calcium exchanger activity when assayed in transfected HEK293 cells using digital imaging of fura-2 fluorescence. The discovery of NCKX4, as far as can be ascertained from the current version of the human genome sequence, completes the mammalian potassium-dependent sodium-calcium exchanger gene family.

Potassium-dependent sodium-calcium exchange through the eye of the fly.

In this review, we describe the characterization of a Drosophila sodium/calcium-potassium exchanger, Nckx30C. Sodium/calcium (-potassium) exchangers (NCX and NCKX) are required for the rapid removal of calcium in excitable cells. The deduced protein topology for NCKX30C is similar to that of mammalian NCKX, with 5 hydrophobic domains in the amino terminus separated from 6 at the carboxy-terminal end by a large intracellular loop. NCKX30C functions as a potassium-dependent sodium-calcium exchanger and is expressed in adult neurons and during ventral nerve cord development in the embryo. Nckx30C is expressed in a dorsal/ventral pattern in the eye-antennal disc, suggesting that large fluxes of calcium may be occurring during imaginal disc development in the larvae. NCKX30C may play a critical role in modulating calcium during development as well as in the removal of calcium and maintenance of calcium homeostasis in adults.

Molecular Cloning of a Third Member of the Potassium-dependent Sodium-Calcium Exchanger Gene Family,NCKX3

We describe here the identification and characterization of a novel member of the family of K(+)-dependent Na(+)/Ca(2+) exchangers, NCKX3 (gene SLC24A3). Human NCKX3 encodes a protein of 644 amino acids that displayed a high level of sequence identity to the other family members, rod NCKX1 and cone/neuronal NCKX2, in the hydrophobic regions surrounding the "alpha -repeat" sequences thought to form the ion-binding pocket for transport. Outside of these regions NCKX3 showed no significant identity to other known proteins. As anticipated from this sequence similarity, NCKX3 displayed K(+)-dependent Na(+)/Ca(2+) exchanger activity when assayed in heterologous expression systems, using digital imaging of fura-2 fluorescence, electrophysiology, or radioactive (45)Ca(2+) uptake. The N-terminal region of NCKX3, although not essential for expression, increased functional activity at least 10-fold and may represent a cleavable signal sequence. NCKX3 transcripts were most abundant in brain, with highest levels found in selected thalamic nuclei, in hippocampal CA1 neurons, and in layer IV of the cerebral cortex. Many other tissues also expressed NCKX3 at lower levels, especially aorta, uterus, and intestine, which are rich in smooth muscle. The discovery of NCKX3 thus expands the K(+)-dependent Na(+)/Ca(2+) exchanger family and suggests this class of transporter has a more widespread role in cellular Ca(2+) handling than previously appreciated.

Molecular Cloning and Functional Expression of the Potassium-Dependent Sodium–Calcium Exchanger from Human and Chicken Retinal Cone Photoreceptors

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.

Molecular Cloning of a Novel Potassium-dependent Sodium-Calcium Exchanger from Rat Brain

We have isolated a novel cDNA clone from rat cerebral cortex encoding a protein of 670 amino acids (NCKX2) that has significant similarity to the 1199-amino acid-long Na/Ca-K exchanger of bovine rod outer segment (NCKX1). NCKX2 transcripts are 10.5 kilobase pairs in length and are expressed abundantly in neurons throughout the brain and with much lower abundance in selected other tissues. The predicted topology of the rat NCKX2 protein is very similar to that of bovine NCKX1, beginning with a solitary transmembrane segment (M0), which is removed as a "signal peptide" in bovine NCKX1, an extracellular loop, a cluster of five transmembrane spanning segments (M1 to M5), a long cytoplasmic loop, and a final hydrophobic cluster (M6 to M11). Within the hydrophobic clusters, rat NCKX2 shares 80% identity and 91% similarity with bovine NCKX1. The two larger hydrophilic loops are much shorter in NCKX2 than in NCKX1, accounting largely for the difference in length between the two proteins, and are dissimilar in sequence except for a 32-amino acid stretch with 69% identity in the cytosolic loop. NCKX2 was epitope-tagged in the extracellular domain and was shown to be expressed at the surface of transfected HEK cells. Analysis of NCKX2 function by fluorescent imaging of fura-2-loaded transfected cells demonstrated that NCKX2 is a potassium-dependent sodium/calcium exchanger.