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
Cation/Ca(2+) exchangers are an essential component of Ca(2+) signaling pathways and function to transport cytosolic Ca(2+) across membranes against its electrochemical gradient by utilizing the downhill gradients of other cation species such as H(+), Na(+), or K(+). The cation/Ca(2+) exchanger superfamily is composed of H(+)/Ca(2+) exchangers and Na(+)/Ca(2+) exchangers, which have been investigated extensively in both plant cells and animal cells. Recently, information from completely sequenced genomes of bacteria, archaea, and eukaryotes has revealed the presence of genes that encode homologues of cation/Ca(2+) exchangers in many organisms in which the role of these exchangers has not been clearly demonstrated. In this study, we report a comprehensive sequence alignment and the first phylogenetic analysis of the cation/Ca(2+) exchanger superfamily of 147 sequences. The results present a framework for structure-function relationships of cation/Ca(2+) exchangers, suggesting unique signature motifs of conserved residues that may underlie divergent functional properties. Construction of a phylogenetic tree with inclusion of cation/Ca(2+) exchangers with known functional properties defines five protein families and the evolutionary relationships between the members. Based on this analysis, the cation/Ca(2+) exchanger superfamily is classified into the YRBG, CAX, NCX, and NCKX families and a newly recognized family, designated CCX. These findings will provide guides for future studies concerning structures, functions, and evolutionary origins of the cation/Ca(2+) exchangers.
In this study we have examined the roles of endogenous cysteine residues in the rat brain K+-dependent Na+/Ca2+ exchanger protein, NCKX2, by site-directed mutagenesis. We found that mutation of Cys-614 or Cys-666 to Ala inhibited expression of the exchanger protein in HEK-293 cells, but not in an in vitro translation system. We speculated that Cys-614 and Cys-666 might form an extracellular disulfide bond that stabilized protein structure. Such an arrangement would place the C terminus of the exchanger outside the cell, contrary to the original topological model. This hypothesis was tested by adding a hemagglutinin A epitope to the C terminus of the protein. The hemagglutinin A epitope could be recognized with a specific antibody without permeabilization of the cell membrane, supporting an extracellular location for the C terminus. Additionally, the exchanger molecule could be labeled with biotin maleimide only following extracellular application of β-mercaptoethanol. Surprisingly, mutation of Cys-395, located in the large intracellular loop, to Ala, prevented reduction-dependent labeling of the protein. The activity of wild-type exchanger, but not the Cys-395 → Ala mutant, was stimulated after application of β-mercaptoethanol. Co-immunoprecipitation experiments demonstrated self-association between wild-type and FLAG-tagged exchanger proteins that could not be inhibited by Cys-395 → Ala mutation. These results suggest that NCKX2 associates as a dimer, an interaction that does not require, but may be stabilized by, a disulfide linkage through Cys-395. This linkage, perhaps by limiting protein mobility along the dimer interface, reduces the transport activity of NCKX2. In this study we have examined the roles of endogenous cysteine residues in the rat brain K+-dependent Na+/Ca2+ exchanger protein, NCKX2, by site-directed mutagenesis. We found that mutation of Cys-614 or Cys-666 to Ala inhibited expression of the exchanger protein in HEK-293 cells, but not in an in vitro translation system. We speculated that Cys-614 and Cys-666 might form an extracellular disulfide bond that stabilized protein structure. Such an arrangement would place the C terminus of the exchanger outside the cell, contrary to the original topological model. This hypothesis was tested by adding a hemagglutinin A epitope to the C terminus of the protein. The hemagglutinin A epitope could be recognized with a specific antibody without permeabilization of the cell membrane, supporting an extracellular location for the C terminus. Additionally, the exchanger molecule could be labeled with biotin maleimide only following extracellular application of β-mercaptoethanol. Surprisingly, mutation of Cys-395, located in the large intracellular loop, to Ala, prevented reduction-dependent labeling of the protein. The activity of wild-type exchanger, but not the Cys-395 → Ala mutant, was stimulated after application of β-mercaptoethanol. Co-immunoprecipitation experiments demonstrated self-association between wild-type and FLAG-tagged exchanger proteins that could not be inhibited by Cys-395 → Ala mutation. These results suggest that NCKX2 associates as a dimer, an interaction that does not require, but may be stabilized by, a disulfide linkage through Cys-395. This linkage, perhaps by limiting protein mobility along the dimer interface, reduces the transport activity of NCKX2. Cytosolic Ca2+ ions play key second messenger roles in numerous physiological processes in virtually all types of animal cells (1Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Biol. 2000; 1: 11-21Google Scholar). Ca2+ entering the cell across the plasma membrane during calcium signaling must be quantitatively extruded to the extracellular environment to maintain long term cellular Ca2+ homeostasis. Plasma membrane Na2+/Ca2+ exchangers are a crucial component of the Ca2+ efflux process and have been extensively investigated in a wide range of tissues, particularly in the heart and brain (2Blaustein M.P. Lederer W.J. Physiol. Rev. 1999; 79: 763-854Google Scholar, 3Philipson K.D. Nicoll D.A. Annu. Rev. Physiol. 2000; 62: 111-133Google Scholar). Various functional and molecular studies have revealed the existence of two families of Na2+/Ca2+exchanger proteins that share sequence similarity in two intramolecular homologous domains known as α-repeats (4Schwarz E.M. Benzer S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10249-10254Google Scholar). One family, Na+/Ca2+ exchangers (NCX), 1The abbreviations used are: NCX, Na+/Ca2+ exchanger; AMS, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid, disodium salt; BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid; CHAPS, 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; FITC, fluorescein isothiocyanate; HA, hemagglutinin A; HEK, human embryonic kidney; IP, immunoprecipitation; β-ME, β-mercaptoethanol; MPB or biotin maleimide, 3-(N-maleimidylpropionyl)biocytin; NCKX, K+-dependent Na+/Ca2+exchanger; PBS, phosphate-buffered saline; PBSCM, PBS supplemented with 0.1 mm CaCl2 and 1 mmMgCl2; TMS, transmembrane segment. 1The abbreviations used are: NCX, Na+/Ca2+ exchanger; AMS, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid, disodium salt; BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid; CHAPS, 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; FITC, fluorescein isothiocyanate; HA, hemagglutinin A; HEK, human embryonic kidney; IP, immunoprecipitation; β-ME, β-mercaptoethanol; MPB or biotin maleimide, 3-(N-maleimidylpropionyl)biocytin; NCKX, K+-dependent Na+/Ca2+exchanger; PBS, phosphate-buffered saline; PBSCM, PBS supplemented with 0.1 mm CaCl2 and 1 mmMgCl2; TMS, transmembrane segment. are thought to catalyze the electrogenic exchange of either 3 or 4 Na+ for 1 Ca2+ (2Blaustein M.P. Lederer W.J. Physiol. Rev. 1999; 79: 763-854Google Scholar, 5Fujioka Y. Hiroe K. Matsuoka S. J. Physiol. 2000; 529: 611-623Google Scholar, 6Dong H. Dunn J. Lytton J. Biophys. J. 2002; 82: 1943-1952Google Scholar). The NCX family is made up of at least three distinct gene products: NCX1 (7Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-565Google Scholar), NCX2 (8Li 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: 17434-17439Google Scholar), and NCX3 (9Nicoll D.A. Quednau B.D. Qui Z. Xia Y.R. Lusis A.J. Philipson K.D. J. Biol. Chem. 1996; 271: 24914-24921Google Scholar). NCX1 is expressed at high levels in cardiac muscle, brain, and kidney and is also present to a lesser extent in many other tissues (10Lee S.L. Yu A.S. Lytton J. J. Biol. Chem. 1994; 269: 14849-14852Google Scholar, 11Quednau B.D. Nicoll D.A. Philipson K.D. Am. J. Physiol. 1997; 272: C1250-C1261Google Scholar). NCX2 and NCX3, in contrast, are expressed primarily in only two tissues: brain and skeletal muscle (8Li 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: 17434-17439Google Scholar, 9Nicoll D.A. Quednau B.D. Qui Z. Xia Y.R. Lusis A.J. Philipson K.D. J. Biol. Chem. 1996; 271: 24914-24921Google Scholar). All three exchangers share an overall amino acid identity of ∼70% that rises to more than 80% within the predicted transmembrane segments (TMS) (9Nicoll D.A. Quednau B.D. Qui Z. Xia Y.R. Lusis A.J. Philipson K.D. J. Biol. Chem. 1996; 271: 24914-24921Google Scholar). The second family, K+-dependent Na2+/Ca2+exchangers (NCKX), are believed to catalyze the electrogenic countertransport of 4 Na+ for 1 Ca2+ and 1 K+ (12Cervetto L. Lagnado L. Perry R.J. Robinson D.W. McNaughton P.A. Nature. 1989; 337: 740-743Google Scholar, 13Szerencsei R.T. Prinsen C.F. Schnetkamp P.P. Biochemistry. 2001; 40: 6009-6015Google Scholar, 14Dong H. Light P.E. French R.J. Lytton J. J. Biol. Chem. 2001; 276: 25919-25928Google Scholar). NCKX exchangers differ from NCX proteins in their absolute requirement for K+, lower Ca2+transport rates, and primary amino acid sequence outside the α-repeats (2Blaustein M.P. Lederer W.J. Physiol. Rev. 1999; 79: 763-854Google Scholar). NCKX1 was initially cloned from bovine rod photoreceptors and was believed to play a central and unique role in the mammalian phototransduction pathway because its ionic stoichiometry represented an adaptation to the unusual ionic environment of the vertebrate eye (15Reilander H. Achilles A. Friedel U. Maul G. Lottspeich F. Cook N.J. EMBO J. 1992; 11: 1689-1695Google Scholar, 16Schnetkamp P.P. J. Biol. Chem. 1995; 270: 13231-13239Google Scholar). However, evidence from functional measurements revealed some Na+/Ca2+ exchange processes that were dependent on K+ in tissues other than eye, for instance brain synaptic plasma membrane (17Dahan D. Spanier R. Rahamimoff H. J. Biol. Chem. 1991; 266: 2067-2075Google Scholar) and platelet (18Kimura M. Aviv A. Reeves J.P. J. Biol. Chem. 1993; 268: 6874-6877Google Scholar). This result led to the search for other putative NCKX family members. Consequently, NCKX2 was first cloned from rat brain (19Tsoi M. Rhee K.H. Bungard D. Li X.F. Lee S.L. Auer R.N. Lytton J. J. Biol. Chem. 1998; 273: 4155-4162Google Scholar) and then from chick and human cone photoreceptors (20Prinsen C.F. Szerencsei R.T. Schnetkamp P.P. J. Neurosci. 2000; 20: 1424-1434Google Scholar), and NCKX3 was recently cloned from brain and skeletal muscle (21Kraev A. Quednau B.D. Leach S. Li X.F. Dong H. Winkfein R. Perizzolo M. Cai X. Yang R. Philipson K.D. Lytton J. J. Biol. Chem. 2001; 276: 23161-23172Google Scholar). Expansion of the NCKX family suggests a wider role for K+-dependent Na+/Ca2+ exchange in maintaining cellular Ca2+ homeostasis than previously anticipated. The tissue-specific expression patterns of these known NCKX members may reflect the different Ca2+ handling properties of different tissues or cells.Cysteine accessibility studies have suggested that the initial hydropathy-based topological model of NCX1 needed to be revised so that mature NCX1 is now thought to contain nine TMSs with two re-entrant loops (22Nicoll D.A. Ottolia M. Lu L. Lu Y. Philipson K.D. J. Biol. Chem. 1999; 274: 910-917Google Scholar, 23Iwamoto T. Uehara A. Imanaga I. Shigekawa M. J. Biol. Chem. 2000; 275: 38571-38580Google Scholar, 24Qiu Z. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2001; 276: 194-199Google Scholar). The current topological model of NCKX, based solely on hydropathy analysis, is reminiscent of the original NCX model before modification. Recently, examination of the hydropathy profile for NCKX3 gave rise to a new topological model in which the C-terminal hydrophobic domain contains only five TMSs, thus placing the C terminus of the exchanger protein outside the cell (21Kraev A. Quednau B.D. Leach S. Li X.F. Dong H. Winkfein R. Perizzolo M. Cai X. Yang R. Philipson K.D. Lytton J. J. Biol. Chem. 2001; 276: 23161-23172Google Scholar), in conflict with the initially proposed NCKX model in which the C-terminal half contains six TMSs and an intracellular C terminus. Indeed, experimental determination of the topology of the Escherichia coli inner membrane protein YrbG, a putative bacterial Na+/Ca2+ exchanger, suggested the C-terminal half has five TMSs and placed the C terminus extracellularly (25Saaf A. Baars L. von Heijne G. J. Biol. Chem. 2001; 276: 18905-18907Google Scholar).Cardiac Na+/Ca2+ exchanger activity was observed to be enhanced dramatically after treatment with a combination of reducing and oxidizing reagents (26Reeves J.P. Bailey C.A. Hale C.C. J. Biol. Chem. 1986; 261: 4948-4955Google Scholar). Thiol-disulfide interchange was proposed to be the molecular mechanism underlying redox modification of exchange activity, although the precise amino acid(s) involved have not yet been identified (27Santacruz-Toloza L. Ottolia M. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2000; 275: 182-188Google Scholar). To date, experimental evidence for dynamic regulation of NCKX-type exchangers is quite limited. In this study, we have used site-directed mutagenesis to investigate the role native cysteine residues play in NCKK2 exchanger protein stability and in transport activity. A preliminary report describing some of these results was published previously in abstract form (28Cai X. Zhang K. Lytton J. Biophys. J. 2002; 82 (abstr.): 566AGoogle Scholar).EXPERIMENTAL PROCEDURESAll molecular procedures were performed according to standard protocols (29Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York2002Google Scholar, 30Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar) or the directions of reagent manufacturers, unless noted otherwise. Common chemical reagents were obtained from Fisher, Sigma, or BDH and were of analytical grade or better, unless indicated otherwise. 3-(N-Maleimidylpropionyl)biocytin (biotin maleimide, or MPB) was from Sigma or Molecular Probes. 4-Acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) was from Molecular Probes.Construction of NCKX2 MutantsThe construction of the wild-type and the FLAG-tagged full-length rat brain NCKX2 cDNA was described previously (19Tsoi M. Rhee K.H. Bungard D. Li X.F. Lee S.L. Auer R.N. Lytton J. J. Biol. Chem. 1998; 273: 4155-4162Google Scholar). Site-directed mutagenesis was performed with the polymerase chain reaction (PCR) overlap extension method using the Expand High Fidelity PCR system from Roche Molecular Biochemicals. Briefly, a pair of complementary primers in which cysteine-coding nucleotides were changed to those for alanine were synthesized. PCR fragments were generated using these mutagenic primers and external primers that flanked convenient unique restriction endonuclease sites in a two-step process. The purified fragment was digested and subcloned into the correspondingly digested full-length exchanger clone in pBluescript II SK (−) (Stratagene). The cDNA clone plasmid was then digested with KpnI and BamHI, and the ∼2.5-kilobase pair fragment containing the full-length NCKX2 clone was subcloned into the mammalian expression vector pcDNA3.1+ (Invitrogen). We made point mutations for each of the last four native cysteine residues of NCKX2, Cys-395 → Ala, Cys-614 → Ala, Cys-633 → Ala, and Cys-666 → Ala. We also generated combined cysteine to alanine mutations named according to the linear order of cysteines in NCKX2: C1–4 (Cys-16 → Ala, Cys-24 → Ala, Cys-154 → Ala, and Cys-224 → Ala), C1–5 (Cys-16 → Ala, Cys-24 → Ala, Cys-154 → Ala, Cys-224 → Ala, and Cys-395 → Ala), and C1–5,7 (Cys-16 → Ala, Cys-24 → Ala, Cys-154 → Ala, Cys-224 → Ala, Cys-395 → Ala, and Cys-633 → Ala). An additional cysteine residue was reintroduced back into the C1–5,7 construct to substitute Ser-105 in the N terminus (named C1–5,7+Ser-105 → Cys), or one residue at a time at selected sites between the putative loops in the C-terminal half. An HA epitope was inserted at the C terminus of FLAG-tagged NCKX2 as a 9-amino acid peptide extension (YPYDVPDYA) by a similar PCR overlap mutagenesis procedure as described above, and the HA-tagged construct was designated as FLAG-NCKX2-HA671. All constructs were confirmed by sequencing to ensure that no polymerase errors were introduced into the amplified segments.Antibody PreparationAffinity-purified rabbit antibody PA1–926 (Affinity Bioreagents, Inc.) was generated against a synthetic peptide corresponding to amino acid residues 90–102 (DLNDKIRDYTPQP) of the rat brain NCKX2. Polyclonal antibody F was prepared at the Southern Alberta Cancer Research Centre Hybridoma Facility by immunizing rabbits with a glutathione S-transferase fusion protein containing amino acids 384–463 of the rat brain NCKX2.Expression in HEK-293 CellsTransient expression of Qiagen-purified cDNA constructs in HEK-293 cells was performed using a standard calcium-phosphate precipitation protocol with BES buffer essentially as described previously (19Tsoi M. Rhee K.H. Bungard D. Li X.F. Lee S.L. Auer R.N. Lytton J. J. Biol. Chem. 1998; 273: 4155-4162Google Scholar). Two days following transfection, postnuclear extracts were prepared by solubilizing transfected cells for 20 min in ice-cold lysis buffer (1% Triton X-100, 0.5% deoxycholate, 0.14 m NaCl, 10 mmEDTA, 25 mm Tris-Cl, pH 7.4, 100 units/ml aproptinin, 0.1 mm phenylmethylsulfonyl fluoride) followed by centrifugation for 30 min at 16,000 × g. Protein concentration was determined by Bradford assay (reagent from Bio-Rad) using bovine γ-globulin as a standard. Immunoblotting was performed as described previously (19Tsoi M. Rhee K.H. Bungard D. Li X.F. Lee S.L. Auer R.N. Lytton J. J. Biol. Chem. 1998; 273: 4155-4162Google Scholar, 31Lytton J. Westlin M. Burk S.E. Shull G.E. MacLennan D.H. J. Biol. Chem. 1992; 267: 14483-14489Google Scholar) using PA1–926 antibody or M2 anti-FLAG monoclonal antibody (Sigma) and detected using Pierce SuperSignal Plus ECL reagents.In Vitro TranslationIn vitro translation of wild-type or mutated NCKX2 was performed essentially as described previously (21Kraev A. Quednau B.D. Leach S. Li X.F. Dong H. Winkfein R. Perizzolo M. Cai X. Yang R. Philipson K.D. Lytton J. J. Biol. Chem. 2001; 276: 23161-23172Google Scholar). In brief, cDNA constructs in the pcDNA3.1(+) vector were transcribed and translated in vitro using the TNT-T7 system (Promega) together with [35S]-methionine (Amersham Biosciences), in the presence of 0.1% Triton X-100. Following an incubation of 90 min at 30 °C, the products were resolved on an SDS-polyacrylamide gel, dried, and detected by autoradiography using Biomax MR film (Eastman Kodak Co.).Indirect ImmunofluorescenceLocation of the HA epitope was determined using immunofluorescence essentially as described previously (19Tsoi M. Rhee K.H. Bungard D. Li X.F. Lee S.L. Auer R.N. Lytton J. J. Biol. Chem. 1998; 273: 4155-4162Google Scholar) with some modifications. In brief, HEK-293 cells transfected with the FLAG-NCKX2-HA671 construct were grown on glass coverslips that had been precoated with 1 mg/ml poly-d-lysine (Sigma). HA-tagged intracellular Ca2+ release channel ryanodine receptor 3 (RyR3) construct (generous gift from Dr. W. Chen) was used as a control to demonstrate the integrity of the plasma membrane barrier. Transfected HEK-293 cells were rinsed in PBSCM (PBS supplemented with 0.1 mm CaCl2 and 1 mm MgCl2, pH 7.4) and then incubated with the rabbit anti-HA polyclonal antibody (1:500) in PBSCM containing 0.2% gelatin for 1 h at room temperature. The cells were then fixed in 4% paraformaldehyde in PBSCM and blocked with 0.2% gelatin/PBSCM for 30 min. A rhodamine-conjugated anti-rabbit second antibody (1:500) was employed in 0.2% gelatin/PBSCM for 30 min. After extensive washing with PBSCM, the cells were then treated with M2 anti-FLAG monoclonal antibody (1:500) followed by a FITC-conjugated anti-mouse second antibody. In permeabilization experiments, the cells were first fixed with 4% paraformaldehyde and then permeabilized with 0.1% Triton X-100 before consecutive application of first and second antibodies as described above. Immunofluorescence microscopy was performed using standard epifluorescence optics on a Zeiss Axioscop II through a Fluar 63× objective. Images were captured using a Spot digital camera and processed with Photoshop.Cysteine-selective Labeling of NKCX2 ExchangersBiotin maleimide labeling of the NCKX2 wild-type and mutated proteins followed methods described previously (32Seal R.P. Leighton B.H. Amara S.G. Methods Enzymol. 1998; 296: 318-331Google Scholar, 33Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Google Scholar) with modification. In brief, 2 days after transfection, HEK-293 cells on 100-mm dishes were washed three times with 10 ml of PBSCM and then incubated with 3 ml of 2% β-mercaptoethanol (β-ME) in PBSCM or 2% ethanol in PBSCM for 15 min at room temperature. Cells were washed three times with 10 ml PBSCM and treated with 2 ml of PBSCM containing 100 μm biotin maleimide (20 mm stock in Me2SO) for 15 min. After washing with 10 ml of PBSCM twice, the reaction was quenched by application of 3 ml of 2% β-ME in PBSCM for 5 min followed by washing twice with 10 ml of PBSCM. In some experiments, 2 ml of 100 μm AMS in PBSCM (20 mm stock in Me2SO) was applied before addition of biotin maleimide to block extracellular cysteine labeling. Postnuclear cell lysis and protein concentration measurements were carried out as described above.All immunoprecipitation (IP) experiments were performed at 4 °C. 1 mg of protein extract was used, and the volume of the supernatant was adjusted to 1 ml with IP buffer (lysis buffer supplemented with 0.1 mg/ml ovalbumin, 1 mm benzamidine, 2 μg/μl leupeptin, and 2 μg/μl pepstatin A). After centrifugation at 14,000 rpm for 5 min, the supernatant was precleared with protein A-Sepharose beads (Sigma) and transferred to a new tube. The supernatant was then mixed with 10 μg of anti-FLAG monoclonal antibody by rotating for 2 h and followed by 100 μl of 20% protein A beads for 30 min. The beads were washed consecutively by centrifuging at 3,000 rpm for 2 min, once each with IP buffer plus 0.5 m NaCl, IP buffer plus 0.1% SDS, and wash buffer (0.1% Triton X-100, 25 mm Tris-Cl, and 1 mm EDTA). The sample was then transferred to a fresh tube, and bounded proteins were recovered by adding 40 μl of 4× SDS sample buffer containing 8% β-ME and heating to 50 °C for 5 min.The proteins were separated on a 9% SDS-PAGE gel and transferred to nitrocellulose membranes. Biotin-labeled proteins were analyzed by incubating the membranes with PBS plus 0.1% Tween 20 containing 0.1 μg/ml horseradish peroxidase-conjugated streptavidin for 1 h after blocking with 2% bovine serum albumin for 1 h. After washing, the membranes were then developed using SuperSignal Plus ECL reagents (Pierce). To assess the level of protein present in each lane, the membranes were stripped with 0.2 m NaOH for 15 min and reprobed with rabbit anti-NCKX2 polyclonal antibody F, followed by application of horseradish peroxidase-conjugated anti-rabbit IgG antibody. The membranes were developed using ECL reagents.Calcium Imaging and Data AnalysisCalcium transport into transfected HEK-293 cells was measured by fura-2 fluorescent ratio digital imaging essentially as described previously (19Tsoi M. Rhee K.H. Bungard D. Li X.F. Lee S.L. Auer R.N. Lytton J. J. Biol. Chem. 1998; 273: 4155-4162Google Scholar, 21Kraev A. Quednau B.D. Leach S. Li X.F. Dong H. Winkfein R. Perizzolo M. Cai X. Yang R. Philipson K.D. Lytton J. J. Biol. Chem. 2001; 276: 23161-23172Google Scholar) with modification. In brief, 2 days after transfection, HEK-293 cells grown on poly-d-lysine-precoated coverslips were loaded with 5 μm fura-2 AM (Molecular Probes) and mounted in a perfusion chamber on a microscope stage. The ratio of fura-2 fluorescence was captured with excitation at 340 or 380 nm using the ImageMaster system (Photon Technology International). Several perfusion solutions were used: solution I (145 mm NaCl, 10 mmd-glucose, 0.1 mmCaCl2, 10 mm HEPES-trimethylamine, pH 7.4), solution II (in which the NaCl of solution I was replaced with 140 mm LiCl and 5 mm KCl), and solution III (in which the NaCl of solution I was substituted with 140 mmNaCl and 5 mm KCl). For testing activity of mutants, cells were initially perfused with solution I for 5 min, followed by alternating changes to solution II for 2 min. For investigating redox-dependent regulation of NCKX2, cells were first incubated with solution III for 17 min without collecting ratio imaging data. Upon changing to perfusion solution I for 2 min, fura-2 fluorescence measurements were started. Perfusion was changed successively to solution II for 2 min and solution I for 2 min. Then, the cells were incubated with either 2% β-ME or 2% ethanol in solution III for 15 min, followed by perfusion with solution III for 2 min. Finally, the cells were subjected consecutively to perfusion solutions I, II, and I for 2 min each.Imaging data were analyzed as described previously (19Tsoi M. Rhee K.H. Bungard D. Li X.F. Lee S.L. Auer R.N. Lytton J. J. Biol. Chem. 1998; 273: 4155-4162Google Scholar, 21Kraev A. Quednau B.D. Leach S. Li X.F. Dong H. Winkfein R. Perizzolo M. Cai X. Yang R. Philipson K.D. Lytton J. J. Biol. Chem. 2001; 276: 23161-23172Google Scholar) using the ImageMaster program and Excel (Microsoft). For redox experiments, all the ratio data were normalized to the height of the first peak ratio. The height of peaks (with base line subtracted) following treatment was compared with the control peak height before treatment. Data were then tested for statistical significance using one-way analysis of variance with Newman-Keuls multiple comparison.Co-immunoprecipitationThe FLAG epitope-tagged NCKX2 (the FLAG epitope is amino acids DYKDDDDK) was created by altering the extracellular sequence found at amino acids 90–97 (DLNDKIRD) in the rat brain NCKX2 as described previously (19Tsoi M. Rhee K.H. Bungard D. Li X.F. Lee S.L. Auer R.N. Lytton J. J. Biol. Chem. 1998; 273: 4155-4162Google Scholar). Consequently FLAG-tagged NCKX2 is not recognized by the PA1–926 antibody (see Fig. 5), which is directed against amino acids 90–102. Two days after co-transfection of the appropriate wild-type and FLAG-tagged constructs, HEK-293 cells were solubilized and NCKX2 was immunoprecipitated using M2 anti-FLAG monoclonal antibody as described above, except the only detergent in the lysis/IP buffer was 1% CHAPS. Protein A beads were then washed three times with IP buffer containing 0.3% CHAPS. Samples were eluted from protein A beads and analyzed by SDS-PAGE and immunoblotting using PA1–926 antibody and reprobed with anti-FLAG antibody, as describe above.DISCUSSIONIn this study, we have prepared cDNA constructs that express the plasma membrane NCKX2 exchanger protein with various single or combined mutations of cysteine residues, to investigate the role of native sulfhydryls in the expression and function of NCKX2. We demonstrated that, of eight endogenous cysteine residues, both Cys-614 and Cys-666 were critical for functional expression of NCKX2 in HEK-293 cells. In mammalian cells, the cytosol is a reducing environment, which prevents the formation of inter- or intrachain disulfide bonds between intracellularly exposed cysteine residues. Therefore, disulfide bonds of an integral membrane protein most likely exist either extracellularly, embedded internally in the protein structure, or within the lipid bilayer. Thus, we speculated that Cys-614 and Cys-666 might form a structurally and functionally important cystine disulfide bond, exposed on the extracellular side of the plasma membrane.To test this hypothesis, we examined the location of the nearby C terminus of NCKX2 with carefully controlled immunofluorescence experiments. Our data confirmed an extracellular location of the C terminus. On the basis of these data, we propose a new topology model for NCKX2 (Fig. 2C) that is consistent with both the prediction for NCKX3 (21Kraev A. Quednau B.D. Leach S. Li X.F. Dong H. Winkfein R. Perizzolo M. Cai X. Yang R. Philipson K.D. Lytton J. J. Biol. Chem. 2001; 276: 23161-23172Google Scholar) and the data on the putative bacterial Na+/Ca2+ exchanger protein, YrbG (25Saaf A. Baars L. von Heijne G. J. Biol. Chem. 2001; 276: 18905-18907Google Scholar). These findings give rise to the possibility that NCKX-type exchangers may have a different topology than NCX-type exchangers in which the C terminus is believed to be inside the cell (3Philipson K.D. Nicoll D.A. Annu. Rev. Physiol. 2000; 62: 111-133Google Scholar).NCKX-type exchangers and NCX-type exchangers share no significant similarity in their amino acid sequences outside the α-repeat regions. However, both new models for NCKX- and NCX-type exchangers place the α-repeat regions on the opposite face of the membrane (22Nicoll D.A. Ottolia M. Lu L. Lu Y. Philipson K.D. J. Biol. Chem. 1999; 274: 910-917Google Scholar). Thus, it is possible that NCX and NCKX exchangers have similar conserved structural elements formed by the α-repeat regions, surrounded by a different overall transmembrane structure. It remains an intriguing possibility that such structural differences between NCKX- and NCX-type exchangers may underlie their distinctive ion stoichiometry. The accuracy of this new NCKX-type exchanger topology will need more supportive proof from further experimental studies.Studies using cysteine-scanning mutagenesis of NCX1 have revealed a novel C-terminal structure that differs remarkably from the previous putative topological model based on hydropathy analysis (3Philipson K.D. Nicoll D.A. Annu. Rev. Physiol. 2000; 62: 111-133Google Scholar). A cysteine-labeling experiment using biotin maleimide demonstrated that the endogenous sulfhydryls of NCKX2 could not be detected under normal conditions. Therefore, cysteine residues were reintroduced, one at a time, at sites in the putative C-terminal loops. None of these reintroduced cysteine residues reacted with biotin maleimide, even after β-ME treatment, suggesting they may be buried in the membrane and hence inaccessible for labeling. Furthermore, and discussed below, β-ME-dependent labeling of NCKX2 did not involve the endogenous Cys-614 and Cys-666 proposed to have an extracellular disposition. These results may indicate that the current model for threading of the C terminus protein of NCKX2 through the membrane needs substantial revision, as demonstrated by the topological studies of NCX1 (22Nicoll D.A. Ottolia M. Lu L. Lu Y. Philipson K.D. J. Biol. Chem. 1999; 274: 910-917Google Scholar, 23Iwamoto T. Uehara A. Imanaga I. Shigekawa M. J. Biol. Chem. 2000; 275: 38571-38580Google Scholar).Furthermore, treatment of NCKX2 with reducing agent stimulated its activity, an effect that was abolished by mutation of Cys-395 to Ala. Redox signaling
Glucocorticoid-induced bone loss is a toxic effect of long-term therapy with glucocorticoids resulting in a significant increase in the risk of fracture. Here, we find that glucocorticoids reciprocally convert osteoblast-lineage cells into endothelial-like cells. This is confirmed by lineage tracing showing the induction of endothelial markers in osteoblast-lineage cells following glucocorticoid treatment. Functional studies show that osteoblast-lineage cells isolated from glucocorticoid-treated mice lose their capacity for bone formation but simultaneously improve vascular repair. We find that the glucocorticoid receptor directly targets Foxc2 and Osterix, and the modulations of Foxc2 and Osterix drive the transition of osteoblast-lineage cells to endothelial-like cells. Together, the results suggest that glucocorticoids suppress osteogenic capacity and cause bone loss at least in part through previously unrecognized osteoblast–endothelial transitions.
Ion channels are macromolecular protein complexes that open and close their pores to control rapid ion fluxes across the cell membranes (Hille, 2001). In general, a single ion-conducting pore is usually formed at the central axis by symmetrical assembly of homologous structural units, either in multimeric pore-forming subunits or internal repeats of a single pore-forming polypeptide. Based on the stoichiometry of structural units surrounding the channel pore (Unwin, 1989; Hille, 2001), most known ion channels can be categorized into classes of three, four, five and six structural units (Fig. 1A). Recent biochemical analysis and atomic force microscopy imaging suggest that P2X receptors contain three subunits. The voltage-gated Na+, K+ and Ca2+ channels, inositol 1,4,5-trisphosphate receptor and ryanodine receptor Ca2+ release channels, HCN channels, CNG channels, TRP channels, and ionotropic glutamate receptors share the four-unit stoichiometry. Some ligand-gated receptors such as nicotinic acetylcholine receptors, GABAA receptors and glycine receptors belong to the five-unit class, while the gap junction channels fit into the six-unit class. Schematic representations of stoichiometry compositions of ion channel pore-forming structural units A, three-, four-, five- and six-unit stoichiometries. In general, ion channels tend to form a single, centred pore surrounded by symmetrical assembly of three, four, five, or six pore-forming structural units. B, ion channels with multiple channel pores. In some multimeric channels, each monomeric subunit forms a functionally independent channel pore. Left, the dimeric CLC Cl− channels (two pores). Right, the tetrameric aquaporin channels (four pores). Pore-forming structural units are shown with filled grey circles, and channel pores are indicated by open circles with a thick line. In contrast, a few types of multimeric channels possess multiple ion-conducting pores, with each monomeric subunit containing a functionally independent channel pore (Fig. 1B). For instance, the dimeric CLC Cl− channels acquire two pores while the tetrameric aquaporin channels contain four water pores. Pore structure plays a critical role in determining the selective permeability of each type of ion channel. Therefore, to understand the functional properties and physiological significance of any channel protein, it is crucial to determine the stoichiometry of pore-forming subunits and the extent and nature of subunit interactions. Ca2+ release-activated Ca2+ (CRAC) channels mediate highly Ca2+ selective, store-operated currents in many cell types including lymphocytes. Recent genetic linkage analysis and genome-wide RNAi screens as well as subsequent mutagenesis studies have elegantly revealed that Orai1 or CRACM1, a four-transmembrane spanning plasma membrane protein, is the pore-forming subunit of the CRAC channel (Feske, 2007). Orai1 and its two closely related Orai family members, Orai2 and Orai3, can assemble into homo- and hetero-multimers (Lis et al. 2007). However, little is known about the stoichiometry of Orai1 subunits in forming a functional CRAC channel – does the composition of a CRAC channel fall into the common three-, four-, five-, or six-structural-units class? This question is particularly intriguing since Orai1 has no obvious channel-like domains conserved in many other types of plasma membrane ion channels and CRAC channels exhibit distinct biophysical properties and store-operated characteristics. In a recent report published in The Journal of Physiology, Shuttleworth and colleagues (Mignen et al. 2007) addressed this question by using cDNA constructs encoding tandem fusions of two to four Orai1 subunits in a single polypeptide, an approach similar to that employed in defining the subunit stoichiometry of the voltage-gated K+ channel (Liman et al. 1992). Orai tandem constructs were expressed in HEK293 cells stably expressing STIM1, a Ca2+ store sensor protein involved in store-operated Ca2+ entry. Instead of using a channel mutant with altered sensitivity for a channel blocker (Liman et al. 1992), the authors took advantage of the dominant-negative E106Q Orai1 mutant, which significantly inhibits both endogenous ICRAC in HEK293 cells and enormously increased ICRAC as a result of coexpression of wild-type Orai1 and STIM1. Consistent with previous reports (Feske, 2007), the authors show that coexpression of the E106Q mutant essentially eliminates enhanced ICRAC in HEK293 cells stably expressing both STIM1 and Orai1 monomers even though both WT and mutant Orai1 molecules target to the plasma membrane normally (their Fig. 1A). Therefore, Orai1 monomers are unlikely to be able to form functional CRAC channel pores by themselves alone and multimerization of Orai1 monomers is required for CRAC channel function. So what is the nature of subunit stoichiometry for a functional CRAC channel? The authors tested the inhibitory effect of the E106Q mutant on the capability of tandem Orai1 constructs encoding two to four linked Orai monomers in inducing macroscopic ICRAC in HEK293 cells stably expressing STIM1. Their working models (their Fig. 2) are based on the hypothesis that the E106Q mutant will diminish or completely abolish the augmented ICRAC only when coexpressed with tandem Orai1 constructs encoding fewer subunits than required for the formation of a functional channel pore. If the tandem Orai1 construct can form a functional channel pore by itself, addition of E106Q mutant will have little effect on the macroscopic ICRAC. Indeed, the authors demonstrated that coexpression of the monomeric E106Q mutant totally eliminated ICRAC induced by the trimeric Orai1 construct but without any obvious inhibitory effect of the tetrameric Orai construct (their Figs 3 and 4). The dimeric Orai1 constructs showed partial inhibition by the E106Q mutant, suggesting that dimeric Orai1 could further assemble into functional CRAC channels by itself, but the E106Q mutant might also be incorporated with the dimeric Orai1. All this evidence converges to suggest that the tetrameric Orai1 construct constitute a functional CRAC channel pore. However, is it possible that CRAC channels, like the CLC Cl− channel and the aquaporin channel, contain more than one channel pore in a tetramer, i.e. two parallel dimers? This notion is very appealing since two transmembrane segments (TMS) of Orai1, TMS1 and TMS3, form an intragenic internal repeat (Cai, 2007). TMS1 and TMS3 of Orai1 have been shown to contribute to the Ca2+ selectivity of the CRAC channel pore (Feske, 2007). In tetrameric aquaporins, two tandem repeats of a single subunit are sufficient to form a functionally independent water channel pore (Verkman, 2005). To address this question, the authors introduced a single E106Q mutation into the Orai1 tetrameric construct to form a WT–WT–WT–E106Q tetramer. If the Orai1 tetramer forms two parallel dimers and/or comprises more than one channel pore (two or four), the single E106Q mutation will likely inactivate only one of the channel pores and render the other channel pores functionally intact (unless these channel pores are somehow functionally interconnected). Indeed, this tetramer construct with a single E106Q mutation did not generate any obvious ICRAC above the background currents recorded in the control cells. Therefore, it is unlikely that the Orai1 tetramer is composed of two functional dimeric channels and possesses more than one channel pore. In conclusion, Mignen et al. (2007) provided compelling initial evidence to demonstrate the tetrameric subunit composition of functional CRAC channels. Like the majority of other four-subunit class channels, four Orai1 subunits possibly assemble symmetrically to form a CRAC channel pore at the central axis. Since functional coupling between STIM1 and Orai1 is required for CRAC channel activation, it will also be interesting to determine the stoichiometry of STIM1. STIM1 undergoes oligomerization in response to store depletion, before redistributing at distinct membrane regions. If a functional CRAC channel is composed of four Orai1 subunits, how many STIM1 monomers are present in the STIM1 oligomer? In the skeletal type excitation–contraction coupling, four skeletal muscle DHPR α1 subunits are believed to physically associate with four subunits of the skeletal muscle ryanodine receptor, RyR1. Thus, it is possible that STIM1 tetramers might also be formed to activate tetrameric CRAC channels. In addition, Orai2 and Orai3 diverged from Orai1 at different evolutionary stages with distinct characteristics (Cai, 2007). Identification of the subunit stoichiometry of CRAC channels will also help further investigation of biochemical and biophysical properties of different Orai hetero-multimers (Lis et al. 2007), and, therefore, help define the compositions of endogenous CRAC channels in native cell types. This elegant study by Shuttleworth and colleagues (Mignen et al. 2007) may serve as a useful guide for future molecular, biophysical, and biochemical analyses of function and mechanism for CRAC channels. I apologize to all colleagues whose work could not be cited here owing to space limitations. This work was supported in part by a postdoctoral fellowship from the Mid-Atlantic Affiliate of the American Heart Association (0625403U).
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a widespread and potent calcium-mobilizing messenger that is highly unusual in activating calcium channels located on acidic stores. However, the molecular identity of the target protein is unclear. In this study, we show that the previously uncharacterized human two-pore channels (TPC1 and TPC2) are endolysosomal proteins, that NAADP-mediated calcium signals are enhanced by overexpression of TPC1 and attenuated after knockdown of TPC1, and that mutation of a single highly conserved residue within a putative pore region abrogated calcium release by NAADP. Thus, TPC1 is critical for NAADP action and is likely the long sought after target channel for NAADP.
Adipose-derived cells (ADCs) from white adipose tissue are promising stem cell candidates because of their large regenerative reserves and the potential for cardiac regeneration. However, given the heterogeneity of ADC and its unsolved mechanisms of cardiac acquisition, ADC-cardiac transition efficiency remains low. In this study, we explored the heterogeneity of ADCs and the cellular kinetics of 39,432 single-cell transcriptomes along the leukemia inhibitory factor (LIF)-induced ADC-cardiac transition. We identified distinct ADC subpopulations that reacted differentially to LIF when entering the cardiomyogenic program, further demonstrating that ADC-myogenesis is time-dependent and initiates from transient changes in nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. At later stages, pseudotime analysis of ADCs navigated a trajectory with 2 branches corresponding to activated myofibroblast or cardiomyocyte-like cells. Our findings offer a high-resolution dissection of ADC heterogeneity and cell fate during ADC-cardiac transition, thus providing new insights into potential cardiac stem cells.
