We describe here the identification and characterization of a novel member of the family of K+-dependent Na+/Ca2+ 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 "α -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+/Ca2+exchanger activity when assayed in heterologous expression systems, using digital imaging of fura-2 fluorescence, electrophysiology, or radioactive 45Ca2+ 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+/Ca2+ exchanger family and suggests this class of transporter has a more widespread role in cellular Ca2+ handling than previously appreciated. We describe here the identification and characterization of a novel member of the family of K+-dependent Na+/Ca2+ 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 "α -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+/Ca2+exchanger activity when assayed in heterologous expression systems, using digital imaging of fura-2 fluorescence, electrophysiology, or radioactive 45Ca2+ 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+/Ca2+ exchanger family and suggests this class of transporter has a more widespread role in cellular Ca2+ handling than previously appreciated. Na+/Ca2+ exchanger expressed sequence tags kilobase pair 3-(N-morpholino)propane-sulfonic acid Na+/Ca2+ + K+ exchanger polymerase chain reaction reverse transcription-coupled polymerase chain reaction Plasma membrane Na+/Ca2+ exchangers are an important component of intracellular Ca2+ homeostasis and have been extensively studied in various cell systems (1Blaustein M.P. Lederer W.J. Physiol. Rev. 1999; 79: 763-854Crossref PubMed Scopus (1443) Google Scholar). Na+/Ca2+ exchangers are encoded by a protein superfamily present in organisms ranging from bacteria to man (2Philipson K.D. Nicoll D.A. Annu. Rev. Physiol. 2000; 62: 111-133Crossref PubMed Scopus (442) Google Scholar). All the members of this family share sequence similarity in two hydrophobic and internally homologous domains, commonly referred to as α-repeats (3Schwarz E.M. Benzer S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10249-10254Crossref PubMed Scopus (180) Google Scholar). Two groups within the Na+/Ca2+ exchanger superfamily have been characterized so far in considerable detail and consist of structurally and functionally distinct proteins. Na+/Ca2+ exchangers (NCX)1 are thought to catalyze the extrusion of one intracellular Ca2+ ion in exchange for three extracellular Na+ ions (Ref. 1Blaustein M.P. Lederer W.J. Physiol. Rev. 1999; 79: 763-854Crossref PubMed Scopus (1443) Google Scholar but see Ref. 4Fujioka Y. Komeda M. Matsuoka S. J. Physiol. ( Lond. ). 2000; 523: 339-351Crossref PubMed Scopus (83) Google Scholar). Na+/Ca2+ + K+ exchangers (NCKX), on the other hand, are thought to transport one intracellular Ca2+ and one K+ ion in exchange for four extracellular Na+ ions (5Schnetkamp P.P.M. Cell Calcium. 1995; 18: 322-330Crossref PubMed Scopus (47) Google Scholar).The NCX family of exchangers is best exemplified by the mammalian cardiac Na+/Ca2+ exchanger, NCX1, first cloned from canine heart (6Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-565Crossref PubMed Scopus (627) Google Scholar), which plays a crucial role in the relaxation process of heart muscle by extruding the Ca2+ that enters at the beginning of systole. NCX1 is also expressed in a variety of other tissues (7Kofuji P. Hadley R.W. Kieval R.S. Lederer W.J. Schulze D.H. Am. J. Physiol. 1992; 263: C1241-C1249Crossref PubMed Google Scholar, 8Lee S.-L., Yu, A.S.L. Lytton J. J. Biol. Chem. 1994; 269: 14849-14852Abstract Full Text PDF PubMed Google Scholar, 9Quednau B.D. Nicoll D.A. Philipson K.D. Am. J. Physiol. 1997; 272: C1250-C1261Crossref PubMed Google Scholar) suggesting an important role in the physiological processes of different cell types. The tissue-specific expression pattern of NCX1 has been demonstrated to be under the control of a multipartite promoter (10Barnes K.V. Cheng G. Dawson M.M. Menick D.R. J. Biol. Chem. 1997; 272: 11510-11517Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 11Nicholas S.B. Yang W. Lee S.L. Zhu H. Philipson K.D. Lytton J. Am. J. Physiol. 1998; 274: H217-H232PubMed Google Scholar, 12Scheller T. Kraev A. Skinner S. Carafoli E. J. Biol. Chem. 1998; 273: 7643-7649Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Moreover, a complex pattern of alternative splicing in the large intracellular loop of NCX1 generates isoforms of the protein, which are also expressed in a highly tissue-specific way (7Kofuji P. Hadley R.W. Kieval R.S. Lederer W.J. Schulze D.H. Am. J. Physiol. 1992; 263: C1241-C1249Crossref PubMed Google Scholar, 8Lee S.-L., Yu, A.S.L. Lytton J. J. Biol. Chem. 1994; 269: 14849-14852Abstract Full Text PDF PubMed Google Scholar, 9Quednau B.D. Nicoll D.A. Philipson K.D. Am. J. Physiol. 1997; 272: C1250-C1261Crossref PubMed Google Scholar). Functional studies have revealed complex regulatory mechanisms of the NCX1 protein, some of which differ between alternatively spliced isoforms (2Philipson K.D. Nicoll D.A. Annu. Rev. Physiol. 2000; 62: 111-133Crossref PubMed Scopus (442) Google Scholar, 13Dyck C. Omelchenko A. Elias C.L. Quednau B.D. Philipson K.D. Hnatowich M. Hryshko L.V. J. Gen. Physiol. 1999; 114: 701-711Crossref PubMed Scopus (84) Google Scholar). The NCX protein family contains two other members, products of genes NCX2 andNCX3, whose expression is restricted largely to brain and skeletal muscle (9Quednau B.D. Nicoll D.A. Philipson K.D. Am. J. Physiol. 1997; 272: C1250-C1261Crossref PubMed Google Scholar, 14Li Z. Matsuoka S. Hryshko L.V. Nicoll D.A. Bersohn M.M. Burke E.P. Lifton R.P. Philipson K.D. J. Biol. Chem. 1994; 269: 17434-17439Abstract Full Text PDF PubMed Google Scholar, 15Nicoll D.A. Quednau B.D. Qui Z. Xia Y.-R. Lusis A.J. Philipson K.D. J. Biol. Chem. 1996; 271: 24914-24921Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). All three NCX proteins share a high degree of sequence identity, especially within the transmembrane spanning domains, and are believed to share the same overall topology, modeled to have two clusters of hydrophobic membrane-spanning helices separated by a large hydrophilic, intracellular loop. The hydrophobic domains are thought to pack together in the membrane, forming the ion translocation pathway (16Qiu Z. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2001; 276: 194-199Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Indeed, the three NCX proteins show very similar functional properties when assayed in heterologous expression systems (17Linck B. Qiu Z. He Z. Tong Q. Hilgemann D.W. Philipson K.D. Am. J. Physiol. 1998; 274: C415-C423Crossref PubMed Google Scholar, 18Iwamoto T. Shigekawa M. Am. J. Physiol. 1998; 275: C423-C430Crossref PubMed Google Scholar).The second major group in the Na+/Ca2+exchanger superfamily is exemplified by the Na+/Ca2++K+ exchanger from retinal rod outer segments, NCKX1. NCKX1 was first cloned from bovine retina (19Reiländer H. Achilles A. Friedel U. Maul G. Lottspeich F. Cook N.J. EMBO J. 1992; 11: 1689-1695Crossref PubMed Scopus (159) Google Scholar) and, more recently, from other species including dolphin, rat, buffalo, and man (20Poon S. Leach S. Li X.F. Tucker J.E. Schnetkamp P.P.M. Lytton J. Am. J. Physiol. 2000; 278: C651-C660Crossref Google Scholar, 21Cooper C.B. Winkfein R.J. Szerencsei R.T. Schnetkamp P.P.M. Biochemistry. 1999; 38: 6276-6283Crossref PubMed Scopus (32) Google Scholar, 22Tucker J.E. Winkfein R.J. Cooper C.B. Schnetkamp P.P.M. Investig. Ophthalmol. Vis. Sci. 1998; 39: 435-440PubMed Google Scholar). This protein plays a critical role in the visual transduction process of the mammalian retina (5Schnetkamp P.P.M. Cell Calcium. 1995; 18: 322-330Crossref PubMed Scopus (47) Google Scholar). In darkness, the cyclic nucleotide-gated ion channels of the outer segment are largely open, and both Na+ and Ca2+ ions flow in. Ca2+ homeostasis must still be maintained under these conditions of membrane depolarization and reduced sodium gradient, and NCKX1 is the principal means by which Ca2+ is extruded from rod outer segments. Such a function could not be achieved by a molecule operating with the 3:1 Na+:Ca2+ stoichiometry of NCX1, and indeed it has been demonstrated that NCKX1 couples the entry of four Na+ ions in exchange for the exit of one Ca2+ and one K+ ion (23Cervetto L. Lagnado L. Perry R.J. Robinson D.W. McNaughton P.A. Nature. 1989; 337: 740-743Crossref PubMed Scopus (284) Google Scholar, 24Schnetkamp P.P.M. Basu D.K. Szerencsei R.T. Am. J. Physiol. 1989; 257: C153-C157Crossref PubMed Google Scholar).Whereas NCX1 proteins from mammalian species are over 90% identical in their amino acid sequences, NCKX1 orthologs display relatively low sequence identities of around 60%, largely due to differences in the two large hydrophilic loops, an extracellular one near the N terminus and a cytoplasmic one near the center of the molecule. NCKX1 has been modeled to have a similar arrangement of transmembrane-spanning segments as NCX1, although actual amino acid sequence similarity is very limited and restricted only to the two α-repeats, as mentioned above. The cloning of NCKX1 from rat eye (20Poon S. Leach S. Li X.F. Tucker J.E. Schnetkamp P.P.M. Lytton J. Am. J. Physiol. 2000; 278: C651-C660Crossref Google Scholar) also revealed the presence of alternatively spliced isoforms in this species, which differ by the arrangement of four exons at the N terminus of the large intracellular loop. Interestingly, the equivalent region of bovine NCKX1, but apparently not the alternatively spliced region in rat NCKX1, is responsible for producing a functionally silent protein when expressed in heterologous systems (20Poon S. Leach S. Li X.F. Tucker J.E. Schnetkamp P.P.M. Lytton J. Am. J. Physiol. 2000; 278: C651-C660Crossref Google Scholar, 21Cooper C.B. Winkfein R.J. Szerencsei R.T. Schnetkamp P.P.M. Biochemistry. 1999; 38: 6276-6283Crossref PubMed Scopus (32) Google Scholar).Functional measurements have pointed toward the presence of K+-dependent Na+/Ca2+exchangers in tissues other than eye (25Dahan D. Spanier R. Rahamimoff H. J. Biol. Chem. 1991; 266: 2067-2075Abstract Full Text PDF PubMed Google Scholar, 26Kimura M. Aviv A. Reeves J.P. J. Biol. Chem. 1993; 268: 6874-6877Abstract Full Text PDF PubMed Google Scholar). Molecular evidence has recently confirmed expression of NCKX1 in cells of hematopoietic origin (27Kimura M. Jeanclos E.M. Donnelly R.J. Lytton J. Reeves J.P. Am. J. Physiol. 1999; 277: H911-H917PubMed Google Scholar) and identified a second K+-dependent Na+/Ca2+ exchanger (NCKX2) in brain neurons and cone photoreceptors (28Prinsen C.F. Szerencsei R.T. Schnetkamp P.P.M. J. Neurosci. 2000; 20: 1424-1434Crossref PubMed Google Scholar, 29Tsoi M. Rhee K.-H. Bungard D. Li X.-F. Lee S.-L. Auer R.N. Lytton J. J. Biol. Chem. 1998; 273: 4115-4162Abstract Full Text Full Text PDF Scopus (103) Google Scholar). Moreover, sequence analysis of the genomes of model organisms, such as Drosophila andCaenorhabditis elegans, has revealed several new hypothetical proteins with similarity to the NCKX family (2Philipson K.D. Nicoll D.A. Annu. Rev. Physiol. 2000; 62: 111-133Crossref PubMed Scopus (442) Google Scholar, 3Schwarz E.M. Benzer S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10249-10254Crossref PubMed Scopus (180) Google Scholar,29Tsoi M. Rhee K.-H. Bungard D. Li X.-F. Lee S.-L. Auer R.N. Lytton J. J. Biol. Chem. 1998; 273: 4115-4162Abstract Full Text Full Text PDF Scopus (103) Google Scholar). 2A. Kraev, unpublished observations.2A. Kraev, unpublished observations. Two of these have recently been characterized functionally (30Szerencsei R.T. Tucker J.E. Cooper C.B. Winkfein R.J. Farrell P.J. Iatrou K. Schnetkamp P.P.M. J. Biol. Chem. 2000; 275: 669-676Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 31Haug-Collet K. Pearson B. Webel R. Szerencsei R.T. Winkfein R.J. Schnetkamp P.P.M. Colley N.J. J. Cell Biol. 1999; 147: 659-670Crossref PubMed Scopus (56) Google Scholar). In addition, analysis of NCKX mRNA expression using probes from regions of sequence conservation under conditions of reduced stringency has revealed evidence for further, as yet uncharacterized, mammalian members of the NCKX family (20Poon S. Leach S. Li X.F. Tucker J.E. Schnetkamp P.P.M. Lytton J. Am. J. Physiol. 2000; 278: C651-C660Crossref Google Scholar). 3Analysis of the completed sequence of the human genome (53Lander E.S. Linton L.M. Birren B. Nusbaum C. Zody M.C. Baldwin J. Devon K. Dewar K. Doyle M. Fitzhugh W. Funke R. Gage D. Harris K. Heaford A. Howland J. et al.Nature. 2001; 409: 860-921Crossref PubMed Scopus (17490) Google Scholar, 54Venter J.C. Adams M.D. Myers E.W. Li P.W. Mural R.J. Sutton G.G. Smith H.O. Yandell M. Evans C.A. Holt R.A. Gocayne J.D. Amanatides P. Ballew R.M. Huson D.H. Russo J. et al.Science. 2001; 291: 1304-1351Crossref PubMed Scopus (10466) Google Scholar) reveals another putative NCKX family member on chromosome 14. This gene was given the designation NCKX4(SLC24A4), although it has not yet been characterized functionally.3Analysis of the completed sequence of the human genome (53Lander E.S. Linton L.M. Birren B. Nusbaum C. Zody M.C. Baldwin J. Devon K. Dewar K. Doyle M. Fitzhugh W. Funke R. Gage D. Harris K. Heaford A. Howland J. et al.Nature. 2001; 409: 860-921Crossref PubMed Scopus (17490) Google Scholar, 54Venter J.C. Adams M.D. Myers E.W. Li P.W. Mural R.J. Sutton G.G. Smith H.O. Yandell M. Evans C.A. Holt R.A. Gocayne J.D. Amanatides P. Ballew R.M. Huson D.H. Russo J. et al.Science. 2001; 291: 1304-1351Crossref PubMed Scopus (10466) Google Scholar) reveals another putative NCKX family member on chromosome 14. This gene was given the designation NCKX4(SLC24A4), although it has not yet been characterized functionally. These findings lend support to the idea that the NCKX gene family predates the evolution of vertebrate vision and that the encoded proteins are an essential component of intracellular Ca2+ homeostasis in many different cells and tissues. A more generalized role for NCKX proteins in current schemes of cellular Ca2+ homeostasis has yet to be considered, largely because there has been no systematic analysis of their expression in tissues other than retina (28Prinsen C.F. Szerencsei R.T. Schnetkamp P.P.M. J. Neurosci. 2000; 20: 1424-1434Crossref PubMed Google Scholar).In this study, we have identified, starting from genome project EST data in three mammalian species, a novel member of the K+-dependent Na+/Ca2+exchanger gene family, NCKX3. We demonstrate that transcripts from this gene are prominent in brain and other tissues and that, when expressed in HEK293 cells, NCKX3 indeed encodes a K+-dependent Na+/Ca2+exchanger.DISCUSSIONIn this study we have described the cloning and characterization of a cDNA encoding a novel, third, member of the family of K+-dependent Na+/Ca2+exchangers, NCKX3. Like the other two members, rod NCKX1 and cone/neuronal NCKX2, NCKX3 demonstrated K+-dependent Na+/Ca2+exchange activity when measured with the fluorescent Ca2+dye fura2, with ion currents, or with 45Ca2+uptake. Although we did not formally determine whether K+was actually transported together with Na+ and Ca2+, there was an absolute requirement for K+in all of the functional assays. That NCKX3 can be measured electrically, with charge moving in the same direction as Na+, also places limits on the ionic stoichiometry of transport. Combined with the high degree of amino acid sequence similarity to NCKX1, which has been clearly established to transport 4 Na+ in exchange for 1 K+ and 1 Ca2+(23Cervetto L. Lagnado L. Perry R.J. Robinson D.W. McNaughton P.A. Nature. 1989; 337: 740-743Crossref PubMed Scopus (284) Google Scholar, 24Schnetkamp P.P.M. Basu D.K. Szerencsei R.T. Am. J. Physiol. 1989; 257: C153-C157Crossref PubMed Google Scholar), it seems likely that NCKX3 has a similar stoichiometry.Previous studies with the cardiac Na+/Ca2+exchanger, NCX1, had implicated the so-called α-repeat regions of the molecule in forming the binding pocket for ion translocation (45Nicoll D.A. Ottolia M. Lu L. Lu Y. Philipson K.D. J. Biol. Chem. 1999; 274: 910-917Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 46Nicoll D.A. Hryshko L.V. Matsuoka S. Frank J.S. Philipson K.D. J. Biol. Chem. 1996; 271: 13385-13391Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Functional studies on a deletion mutant of bovine NCKX1 and a C. elegans paralog (cNCKX) have suggested similar regions also specify the transport sites needed for K+-dependent Na+/Ca2+exchangers (30Szerencsei R.T. Tucker J.E. Cooper C.B. Winkfein R.J. Farrell P.J. Iatrou K. Schnetkamp P.P.M. J. Biol. Chem. 2000; 275: 669-676Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). A comparison of the sequences between NCKX3 and these molecules (Fig. 2) shows the high sequence similarity anticipated within the α-repeats. NCKX3 is, however, significantly more divergent in these regions than the NCKX1/NCKX2/cNCKX trio. Intriguingly, these positions of divergence often correspond to identities or similarities with NCX1. Nevertheless, NCKX3 is clearly K+-dependent, in contrast to NCX1, and thus this new sequence information places further limits on the amino acids likely to contribute to the ionic specificity of the K+-dependent class of Na+/Ca2+ exchangers.Hydropathy analysis as well as our in vitro translation experiments support a proposed transmembrane topology for NCKX3 that is largely similar to those proposed previously for other family members (Fig. 1). The NCKX3 protein begins with a functionally dispensable region that may encode a putative "signal" peptide, in a manner analogous to that reported for NCX1 (41Sahin-Toth M. Nicoll D.A. Frank J.S. Philipson K.D. Friedlander M. Biochem. Biophys. Res. Commun. 1995; 212: 968-974Crossref PubMed Scopus (25) Google Scholar, 42Loo T.W. Ho C. Clarke D.M. J. Biol. Chem. 1995; 270: 19345-19350Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 43Furman I. Cook O. Kasir J. Low W. Rahamimoff H. J. Biol. Chem. 1995; 270: 19120-19127Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The presence of this region in the expressed protein significantly improved the level of functional expression of NCKX3 (Fig. 9). Thus, whereas all cDNA constructs demonstrated K+-dependent Na+/Ca2+ exchange activity when assayed using fura-2 fluorescent digital imaging, only the HuNCKX3-L clone expressed activity above background in either electrophysiological or45Ca2+ uptake assays. Since in vitrotranscription and translation experiments (Fig. 5) did not reveal a significant difference in the efficiency of protein expression, the longer NCKX3 protein species containing the N-terminal hydrophobic M0 sequence may be targeted or delivered to the plasma membrane more efficiently than the protein lacking this sequence.The processed NCKX3 protein consists of a short, glycosylated, extracellular loop at the N terminus followed by a cluster of five hydrophobic, putative transmembrane, segments, a long hydrophilic loop, and finally, a second cluster of hydrophobic regions. The long central loop, which is presumed to be cytoplasmic based on a comparison to NCKX1 and NCX1, contains consensus sequences for several protein kinases. Recent studies using cysteine-scanning mutagenesis of NCX1 have revealed that the C-terminal hydrophobic region of that molecule is composed of four helical transmembrane segments and a pore-like re-entrant loop structure that extends into the membrane (45Nicoll D.A. Ottolia M. Lu L. Lu Y. Philipson K.D. J. Biol. Chem. 1999; 274: 910-917Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 47Iwamoto T. Uehara A. Imanaga I. Shigekawa M. J. Biol. Chem. 2000; 275: 38571-38580Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 48Iwamoto T. Nakamura T.Y. Pan Y. Uehara A. Imanaga I. Shigekawa M. FEBS Lett. 1999; 446: 264-268Crossref PubMed Scopus (90) Google Scholar). Hydropathy analysis of NCKX3 suggests a somewhat different topology, with five transmembrane spans separating the central cytoplasmic loop from the protein C terminus, which would reside on the extracellular side of the membrane. The validity of such a model, and the notion that the K+-dependent Na+/Ca2+ exchangers (NCKX) may have a topology different from those of the K+-independent (NCX) family, will need to be tested.The exon boundaries for human NCKX3 are indicated in Fig. 1, and their location in genomic sequence on chromosome 20 is shown in Fig. 3. The following two features of the NCKX3 gene are striking: (i) the distance over which the exons, especially the first three, are spaced; (ii) the arrangement of exons in comparison to the genomic structure of other Na+/Ca2+ exchanger genes. The NCX1 (SLC8A1), NCX3(SLC8A3), NCKX1 (SLC24A1), andNCKX2 (SLC24A2) genes all share an unusually large second exon that extends from just before the initiating methionine codon into the central cytoplasmic loop (analyzed by BLAST search; also see Refs. 37Kraev A. Chumakov I. Carafoli E. Genomics. 1996; 37: 105-112Crossref PubMed Scopus (44) Google Scholar and 38Tucker J.E. Winkfein R.J. Murthy S.K. Friedman J.S. Walter M.A. Demetrick D.J. Schnetkamp P.P.M. Hum. Genet. 1998; 103: 411-414Crossref PubMed Scopus (13) Google Scholar). In contrast, the corresponding sequence of NCKX3 is split into 10 separate exons. Furthermore, the arrangement of exons encoding the C-terminal hydrophobic domain is specific for NCKX3 and differentiates it from either the arrangement conserved between NCKX1 and NCKX2 or the distinct arrangement conserved between NCX1 and NCX3. These differences suggest that, despite the sequence similarity within the α-repeat regions, NCKX3 must have its origins in a very ancient gene duplication event and subsequent divergence from other NCKX and NCX family members.The pattern of expression of NCKX3 transcripts in different tissues was examined in rat and mouse by Northern blot and in human by Multiple Tissue Expression Array dot blot analyses (Fig. 6). From these data it is clear that NCKX3 was most abundantly expressed in various brain regions. Regional distribution was examined in more detail in parasagittal sections of mouse brain using in situhybridization (Fig. 8), which revealed a very specific pattern. The highest levels of NCKX3 mRNA were found in neurons arranged in distinct nuclei of the thalamus, followed by the pyramidal CA1 neurons of the hippocampus, and large neurons of cortical layer IV. This pattern is quite different from published reports of NCX1 and NCKX2 distribution in rat brain (29Tsoi M. Rhee K.-H. Bungard D. Li X.-F. Lee S.-L. Auer R.N. Lytton J. J. Biol. Chem. 1998; 273: 4115-4162Abstract Full Text Full Text PDF Scopus (103) Google Scholar, 49Marlier L.N. Zheng T. Tang J. Grayson D.R. Brain Res. Mol. Brain Res. 1993; 20: 21-39Crossref PubMed Scopus (33) Google Scholar). Transcripts for both of these exchangers were also abundant in cortex but lacked the laminar pattern evident for NCKX3. All three exchangers were present in hippocampal pyramidal cells, but only NCKX3 was richest in CA1 neurons. In the thalamus, each exchanger had a characteristic regional distribution. NCKX3 and NCKX2 expression largely overlapped in the cerebellar molecular layer. In contrast, NCKX3 was essentially absent from the striatum, where NCKX2 was very abundant, and from the septal nuclei, where NCX1 was highly expressed.A unique feature of NCKX3 expression in comparison to other K+-dependent Na+/Ca2+exchangers was the presence of transcripts in many tissues other than brain, although at lower levels. The precise pattern appeared to vary between human and rodent, although part of this may have been due to the manner in which RNA loading was normalized (to a fixed quantity of total RNA in the case of rodent and to a varying amount of poly(A)+ mRNA, normalized to several control transcripts, in the case of the Multiple Tissue Expression Array), or due to the difference in the probes used. The tissues that had the next most abundant level of NCKX3 mRNA, after brain, were those generally rich in smooth muscle, such as aorta, uterus, and intestine. Many other tissues expressed lower levels, but only liver and kidney consistently appeared to be essentially negative for NCKX3. This rather ubiquitous pattern of expression matches more closely that of NCX1 than it does any of the other family members, which are thought to have quite restricted tissue expression patterns, NCKX1 in eye, NCKX2 in brain neurons (including eye), and NCX2 and NCX3 in brain and skeletal muscle. The selective presence of NCKX3 in specific unique cell types of the vascular wall (smooth muscle, endothelial cells, or innervating neurons) has yet to be examined. To our knowledge, however, there have been no reports of K+-dependent Na+/Ca2+ exchange activity in these tissues or cell types.At present the physiological consequences of the differential tissue-specific expression patterns for different Na+/Ca2+ exchanger family members are unclear. The situation in brain, where at least four different Na+/Ca2+ exchanger gene products (NCX1, NCX2, NCKX2, and NCKX3)3 are present at high levels, is particularly intriguing. It seems likely that unique Ca2+-handling requirements of specific tissues or cell types, combined with kinetic, thermodynamic, or regulatory differences in the function of these exchangers, will provide important clues. The difference in transport stoichiometry between NCKX and NCX families provides an opportunity for NCKX-type exchangers to maintain Ca2+ homeostasis in environments where the Na+gradient and/or the membrane potential are lower than normal. Although this argument was first used to justify the expression of NCKX1 in rod photoreceptors, there may be situations in other neurons where similar conditions exist. Another possible role for different exchanger gene products might be in the transport of Mg2+ instead of Ca2+ (50Tashiro M. Konishi M. Iwamoto T. Shigekawa M. Kurihara S. Pfluegers Arch. 2000; 440: 819-827Crossref PubMed Scopus (30) Google Scholar). We have not investigated whether NCKX3 might transport Mg2+. It is noteworthy, however, that 1 mm Mg2+ significantly inhibits NCKX3 activity (Fig. 10), although under these conditions it had no significant effect on the amplitude of either NCX1 or NCKX2 currents. 4H. Dong and J. Lytton, unpublished observations. These observations suggest that Mg2+ interacts more selectively with the transport site of NCXK3 than it does with the sites of other family members.The eventual answer to the question of unique physiological roles for the different Na+/Ca2+ exchanger family members will require the development of selective pharmacological blockers and/or of recombinant gene knock-out animals. The recent developments of an agent relatively selective for inhibition of NCX-type Na+/Ca2+ exchangers, KB-R7943 (51Iwamoto T. Watano T. Shigekawa M. J. Biol. Chem. 1996; 271: 22391-22397Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar), and mice lacking NCX1 (52Wakimoto K. Kobayashi K. Kuro O.M. Yao A. Iwamoto T. Yanaka N. Kita S. Nishida A. Azuma S. Toyoda Y. Omori K. Imahie H. Oka T. Kudoh S. Kohmoto O. Yazaki Y. Shigekawa M. Imai Y. Nabeshima Y. Komuro I. J. Biol. Chem. 2000; 275: 36991-36998Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), will help in this effort, but additional selective molecular tools are eagerly anticipated. Most notably, planning complex gene knock-out experiments will be greatly facilitated by the completion of the mouse genome sequence (due in 2002) which will set a unique stage for genetic dissection of mammalian Ca2+homeostasis. Plasma membrane Na+/Ca2+ exchangers are an important component of intracellular Ca2+ homeostasis and have been extensively studied in various cell systems (1Blaustein M.P. Lederer W.J. Physiol. Rev. 1999; 79: 763-854Crossref PubMed Scopus (1443) Google Scholar). Na+/Ca2+ exchangers are encoded by a protein superfamily present in organisms ranging from bacteria to man (2Philipson K.D. Nicoll D.A. Annu. Rev. Physiol. 2000; 62: 111-13
Using the reverse polymerase chain reaction (RT‐PCR), we have examined the expression of Na + /H + exchanger mRNA in human buffy coat preparations, lymphocytes and neutrophils. Total RNA from all cell types was reverse transcribed specifically and then amplified by PCR. The identity of the PCR products was confirmed by restriction enzyme analysis and hybridization with a specific oligonucleotide probe. The detection or low abundance Na + /H + antiporter specific transcripts by RT‐PCR in different human blood cells ex vivo should facilitate future studies on regulatory and pathophysiological aspects of Na + /H + exchanger mRNA expression in human cells and tissue samples.
Vascular smooth muscle cells (VSMC) from the spontaneously hypertensive rat (SHR) exhibit an enhanced Na+/H+ exchange activity which coincides with increased proliferation. The aim of the present work was to investigate Na+/H+ exchange activity in VSMC and thymocytes from SHR and Wistar-Kyoto (WKY) rats and to examine the steady state levels of specific Na+/H+ exchanger (isoform 1; NHE-1) mRNA transcripts in VSMC, thymocytes, heart, and kidney.VSMC from SHR proliferated significantly faster than those from WKY rats. NaVH+ exchange activity (Vmax) was significantly (p < 0.01) higher in VSMC from SHR compared to WKY (38 ± 2 vs. 22 ± 1 mmol H+ × liter–1 × min–1; mean ± SE). In contrast, Na+/H+ exchange activity was higher in thymocytes from WKY rats. By Northern blot analysis, we observed similar steady state mRNA levels for NHE-1 in tissues from SHR and WKY, respectively.We conclude that the enhanced Na+/H+ exchange activity found in VSMC from SHR is not expressed in all tissues of SHR. Furthermore, our data suggest that over-expression of NHE-1 is not responsible for the enhanced Na+/ H+ exchange activity in VSMC from SHR. Thus, alterations in cell signalling located upstream of the Na+/H+ exchanger may induce both an increased proliferation and an altered post-translational regulation of Na+/H+ exchange activity in VSMC from SHR.
It has recently been demonstrated that uremic metabolic acidosis and experimental metabolic acidosis caused by ingestion of ammonium chloride coincide with increased Na(+)-H+ exchanger (NHE-1) activity in human blood cells. In the present study, we investigated whether an increased level of NHE-1 specific mRNA in human lymphocytes during the course of an experimental metabolic acidosis could explain the enhanced transport activity during metabolic acidosis. Six healthy individuals were studied before and after 5 days of taking 15 g of ammonium chloride daily. Plasma pH and bicarbonate decreased significantly, from 7.42 +/- 0.027 to 7.28 +/- 0.05 and from 26.7 +/- 2.0 to 15.6 +/- 2.9 mM, respectively. Basal cytosolic pH (pHi) and Na(+)-H+ exchange activity were measured in lymphocytes loaded with the fluorescent pHi indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Basal pHi remained unchanged during metabolic acidosis (7.03 +/- 0.07 vs. 7.03 +/- 0.06). Ethylisopropylamiloride-sensitive pHi recovery increased from 0.046 +/- 0.007 to 0.076 +/- 0.012 dpHi/min (P < 0.0001). The transcript level of NHE-1 mRNA was measured by reverse-transcription polymerase chain reaction in comparison with a constitutively expressed reference gene (glyceraldehyde-3-phosphate dehydrogenase). NHE-1 mRNA in human lymphocytes increased 1.5-fold in metabolic acidosis. These data suggest that the increased Na(+)-H+ exchange activity in metabolic acidosis may be caused by de novo synthesis of antiport protein.
A bstract : An overview of the molecular physiology of the Na + /Ca 2+ exchanger is presented. This includes information on the variety of exchangers that have been described and their regulatory properties. Molecular insight is most detailed for the cardiac Na + /Ca 2+ exchanger (NCX1). Parts of the NCS1 molecule involved in regulation and ion transport have been elucidated, and initial information on the topology and structure is available.
NCX3 is the third isoform of a mammalian Na+-Ca2+ exchanger to be cloned. NCX3 was identified from rat brain cDNA by polymerase chain reaction (PCR) using degenerate primers derived from the sequences of two conserved regions of NCX1 and NCX2. The NCX3 PCR product was used to isolate two overlapping clones totalling 4.8 kilobases (kb) from a rat brain cDNA library. The overlapping clones were sequenced and joined at a unique Bsp106I restriction enzyme site to form a full-length cDNA clone. The NCX3 cDNA clone has an open reading frame of 2.8 kb encoding a protein of 927 amino acids. At the amino acid level, NCX3 shares 73% identity with NCX1 and 75% identity with NCX2 and is predicted to share the same membrane topology as NCX1 and NCX2. Following addition of a poly(A)+ tail to the NCX3 clone, exchanger activity could be expressed in Xenopus oocytes. NCX3 was also expressed in the mammalian BHK cell line. NCX3 transcripts are 6 kb in size and are highly restricted to brain and skeletal muscle. Linkage analysis in the mouse indicated that the NCX family of genes is dispersed, since the NCX1, NCX2, and NCX3 genes mapped to mouse chromosomes 17, 7, and 12, respectively.
Beta 2-microglobulin (beta 2M) is responsible for dialysis-associated amyloidosis. Level of beta 2M in plasma increase during chronic renal failure; however, retention does not appear to be the sole mechanism responsible. The effect of metabolic acidosis on beta 2M production was examined. Thirty-six patients with stable chronic renal insufficiency, 12 uremic patients before their first dialysis, 8 hemodialysis patients who were assigned to acetate or bicarbonate dialysate and then crossed over to the alternative regimen, and 6 normal subjects given NH4Cl to initiate metabolic acidosis were studied. In vitro studies in the human myeloid cell line U 937 were also performed. beta 2M protein was measured with ELISA, beta 2M mRNA was measured with reverse transcription polymerase chain reaction, and the U 937 cells were studied at two pH levels with FACScan flow cytometry. The cells were exposed in vitro up to 60 min in a buffered incubation medium to either pH 5.10 or pH 7.34. An inverse correlation was found between beta 2M and bicarbonate concentrations in plasma in the stable chronic renal failure patients (r = -0.54; P < 0.05) and in the uremic patients before their first dialysis (r = -0.72; P < 0.05). In hemodialysis patients, blood pH and plasma bicarbonate values were lower (P < 0.05) and beta 2M concentrations in plasma were higher (P < 0.05) with acetate than with bicarbonate dialysate. In normal men, NH4Cl resulted in an increase (P < 0.05) in beta 2M mRNA expression in lymphocytes by an average factor of 1.5 (range, 1.1 to 1.8). In U 937 cells, the cell surface expression of beta 2M and HLA Class I heavy chain assembled with beta 2M decreased at low pH compared with normal pH. Concomitantly, an increase in beta 2M release into the supernatant was observed, possibly as the result of beta 2M dissociation from cell surface HLA Class I complex. The results suggest that metabolic acidosis may enhance cellular beta 2M generation and release.
Blinatumomab is a CD19/CD3 BiTE (bispecific T-cell engager) antibody construct for the treatment of Philadelphia chromosome-negative acute B-lymphoblastic leukemia. We evaluated blinatumomab in relapsed/refractory B-cell non-Hodgkin lymphoma (NHL).This 3 + 3 design, phase I dose-escalation study determined adverse events and the maximum tolerated dose (MTD) of continuous intravenous infusion blinatumomab in patients with relapsed/refractory NHL. Blinatumomab was administered over 4 or 8 weeks at seven different dose levels (0.5 to 90 μg/m(2)/day). End points were incidence of adverse events, pharmacokinetics, pharmacodynamics, and overall response rate.Between 2004 and 2011, 76 heavily pretreated patients with relapsed/refractory NHL, who included 14 with diffuse large B-cell lymphoma, were enrolled; 42 received treatment in the formal dose-escalation phase. Neurologic events were dose limiting, and 60 μg/m(2)/day was established as the MTD. Thirty-four additional patients were recruited to evaluate antilymphoma activity and strategies for mitigating neurologic events at a prespecified MTD. Stepwise dosing (5 to 60 μg/m(2)/day) plus pentosan polysulfate SP54 (n = 3) resulted in no treatment discontinuations; single-step (n = 5) and double-step (n = 24) dosing entailed two and seven treatment discontinuations due to neurologic events, respectively. Grade 3 neurologic events occurred in 22% of patients (no grade 4/5). Among patients treated at 60 μg/m(2)/day (target dose; n = 35), the overall response rate was 69% across NHL subtypes and 55% for diffuse large B-cell lymphoma (n = 11); median response duration was 404 days (95% CI, 207 to 1,129 days).In this phase I study of relapsed/refractory NHL, continuous infusion with CD19-targeted immunotherapy blinatumomab at various doses and schedules was feasible, with an MTD of 60 μg/m(2)/day. Single-agent blinatumomab showed antilymphoma activity.
