FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2073604123> ?p ?o ?g. }

• W2073604123 endingPage "23102" @default.
• W2073604123 startingPage "23094" @default.
• W2073604123 abstract "Externally applied Ni2+, which apparently competes with Ca2+ in all three isoforms of Na+/Ca2+ exchanger, inhibits exchange activity of NCX1 or NCX2 with a 10-fold higher affinity than that of NCX3, whereas stimulation of exchange by external Li+ is significantly greater in NCX2 and NCX3 than in NCX1 (Iwamoto, T., and Shigekawa, M. (1998) Am. J. Physiol. 275, C423–C430). Here we identified structural domains in the exchanger that confer differential sensitivity to Ni2+ or Li+ by measuring intracellular Na+-dependent45Ca2+ uptake in CCL39 cells stably expressing NCX1/NCX3 chimeras or mutants. We found that two segments in the exchanger corresponding mostly to the internal α-1 and α-2 repeats are individually responsible for the alteration of Ni2+sensitivity, both together accounting for ∼80% of the difference between NCX1 and NCX3. In contrast, the segment corresponding to the α-2 repeat fully accounts for the differential Li+sensitivity between the isoforms. The Ni2+ sensitivity was mimicked, respectively, by simultaneous substitution of two amino acids in the α-1 repeat (N125G/T127I in NCX1 and G159N/I161T in NCX3) and substitution of one amino acid in the α-2 repeat (V820A in NCX1 and A809V in NCX3). On the other hand, the Li+ sensitivity was mimicked by double substitution mutation in the α-2 repeat (V820A/Q826V in NCX1 and A809V/V815Q in NCX3). Single substitution mutations at Asn125 and Val820 of NCX1 caused significant alterations in the interactions of the exchanger with Ca2+ and Ni2+, and Ni2+ and Li+, respectively, although the extent of alteration varied depending on the nature of side chains of substituted residues. Since the above four important residues are mostly in the putative loops of the α repeats, these regions might form an ion interaction domain in the exchanger. Externally applied Ni2+, which apparently competes with Ca2+ in all three isoforms of Na+/Ca2+ exchanger, inhibits exchange activity of NCX1 or NCX2 with a 10-fold higher affinity than that of NCX3, whereas stimulation of exchange by external Li+ is significantly greater in NCX2 and NCX3 than in NCX1 (Iwamoto, T., and Shigekawa, M. (1998) Am. J. Physiol. 275, C423–C430). Here we identified structural domains in the exchanger that confer differential sensitivity to Ni2+ or Li+ by measuring intracellular Na+-dependent45Ca2+ uptake in CCL39 cells stably expressing NCX1/NCX3 chimeras or mutants. We found that two segments in the exchanger corresponding mostly to the internal α-1 and α-2 repeats are individually responsible for the alteration of Ni2+sensitivity, both together accounting for ∼80% of the difference between NCX1 and NCX3. In contrast, the segment corresponding to the α-2 repeat fully accounts for the differential Li+sensitivity between the isoforms. The Ni2+ sensitivity was mimicked, respectively, by simultaneous substitution of two amino acids in the α-1 repeat (N125G/T127I in NCX1 and G159N/I161T in NCX3) and substitution of one amino acid in the α-2 repeat (V820A in NCX1 and A809V in NCX3). On the other hand, the Li+ sensitivity was mimicked by double substitution mutation in the α-2 repeat (V820A/Q826V in NCX1 and A809V/V815Q in NCX3). Single substitution mutations at Asn125 and Val820 of NCX1 caused significant alterations in the interactions of the exchanger with Ca2+ and Ni2+, and Ni2+ and Li+, respectively, although the extent of alteration varied depending on the nature of side chains of substituted residues. Since the above four important residues are mostly in the putative loops of the α repeats, these regions might form an ion interaction domain in the exchanger. transmembrane helix current-voltage balanced salt solution bovine serum albumin intracellular Na+ extracellular Ca2+concentration extracellular Na+ concentration 1,2-bis-(O -aminophenoxy)ethane-N,N,N ′,N ′-tetraacetic acid The Na+/Ca2+ exchanger is an electrogenic transporter that catalyzes exchange of 3 Na+ for 1 Ca2+ across the plasma membrane of many cell types. Previous studies indicate that it plays a primary role in the extrusion of cytosolic Ca2+ from cardiomyocytes, although its contribution in the Ca2+ handling in other cell types still remains to be precisely defined (1Hilgemann D.W. Philipson K.D. Vassort G. Ann. N. Y. Acad. Sci. 1996; 779: 1-582Crossref PubMed Scopus (1) Google Scholar). The mammalian Na+/Ca2+ exchanger forms a multigene family comprising three isoforms, NCX1, NCX2, and NCX3, which share ∼70% identity in the overall amino acid sequences (2Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-565Crossref PubMed Scopus (628) Google Scholar, 3Li 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, 4Nicoll 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). On the basis of the hydropathy analysis, the mature Na+/Ca2+exchanger proteins were modeled to consist of 11 TMs1 and a large central hydrophilic loop between TM5 and TM6, with the N terminus localized on the extracellular side and the C terminus and the large central loop being on the intracellular side of the membrane (4Nicoll 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, 5Nicoll 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 (128) Google Scholar). Recent studies on the topology of the NCX1 polypeptide have produced data that are consistent with the N-terminal half of the 11 TM model (6Hryshko L.V. Nicoll D.A. Weiss J.N. Philipson K.D. Biochim. Biophys. Acta. 1993; 1151: 35-42Crossref PubMed Scopus (62) Google Scholar, 7Doering A.E. Nicoll D.A. Lu Y. Lu L. Weiss J.N. Philipson K.D. J. Biol. Chem. 1998; 273: 778-783Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 8Cook O. Low W. Rahamimoff H. Biochim. Biophys. Acta. 1998; 1371: 40-52Crossref PubMed Scopus (34) Google Scholar, 9Nicoll 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, 10Iwamoto T. Nakamura T.Y. Pan Y. Uehara A. Imanaga I. Shigekawa M. FEBS Lett. 1999; 446: 264-268Crossref PubMed Scopus (90) Google Scholar), but these data do not support the C-terminal half of the model (9Nicoll 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, 10Iwamoto T. Nakamura T.Y. Pan Y. Uehara A. Imanaga I. Shigekawa M. FEBS Lett. 1999; 446: 264-268Crossref PubMed Scopus (90) Google Scholar), indicating that the model requires revision. Na+/Ca2+ exchange occurs almost normally in a mutant exchanger deleted of the large central loop (11Matsuoka S. Nicoll D.A. Reilly R.F. Hilgemann D.W. Philipson K.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3870-3874Crossref PubMed Scopus (200) Google Scholar, 12Condrescu M. Gardner J.P. Chernaya G. Aceto J.F. Kroupis C. Reeves J.P. J. Biol. Chem. 1995; 270: 9137-9146Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 13Iwamoto T. Pan Y. Nakamura T.Y. Wakabayashi S. Shigekawa M. Biochemistry. 1998; 37: 17230-17238Crossref PubMed Scopus (94) Google Scholar), indicating that the transmembrane domain alone is sufficient to catalyze ion transport. The large central loop is localized intracellularly (8Cook O. Low W. Rahamimoff H. Biochim. Biophys. Acta. 1998; 1371: 40-52Crossref PubMed Scopus (34) Google Scholar, 14Porzing H. Li Z. Nicoll D.A. Philipson K.D. Am. J. Physiol. 1993; 265: C748-C756Crossref PubMed Google Scholar) and involved in the regulation of the exchanger by cytoplasmic Ca2+ (15Kimura J. Noma A. Irisawa H. Nature. 1986; 319: 596-597Crossref PubMed Scopus (278) Google Scholar, 16Matsuoka S. Nicoll D.A. Hryshko L.V. Levitsky D.O. Weiss J.N. Philipson K.D. J. Gen. Physiol. 1995; 105: 403-420Crossref PubMed Scopus (204) Google Scholar), cytoplasmic Na+ (17Hilgemann D.W. Matsuoka S. Nagel G.A. Collins A. J. Gen. Physiol. 1992; 100: 905-932Crossref PubMed Scopus (240) Google Scholar), ATP depletion (12Condrescu M. Gardner J.P. Chernaya G. Aceto J.F. Kroupis C. Reeves J.P. J. Biol. Chem. 1995; 270: 9137-9146Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 13Iwamoto T. Pan Y. Nakamura T.Y. Wakabayashi S. Shigekawa M. Biochemistry. 1998; 37: 17230-17238Crossref PubMed Scopus (94) Google Scholar), and protein phosphorylation by protein kinase C (13Iwamoto T. Pan Y. Nakamura T.Y. Wakabayashi S. Shigekawa M. Biochemistry. 1998; 37: 17230-17238Crossref PubMed Scopus (94) Google Scholar, 18Iwamoto T. Pan Y. Wakabayashi S. Imagawa T. Yamanaka H.I. Shigekawa M. J. Biol. Chem. 1996; 271: 13609-13615Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). In the transmembrane domain of all members of Na+/Ca2+ exchanger family, there are two highly conserved internal repeat sequences designated the α-1 and α-2 repeats that comprise most of TMs 2–3 and 8–9 and the loops connecting these TMs, respectively (5Nicoll 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 (128) Google Scholar,19Schwarz E.M. Benzer S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10249-10254Crossref PubMed Scopus (181) Google Scholar). 2For the sake of convenience, we use the 11 TM model when we refer to the putative transembrane helices of the exchanger. In such repeat sequences, the putative loop regions are more variable than the transmembrane segments. These homologous sequences may be functionally important, because mutations in the putative TMs within the α repeats cause a large reduction in exchange activity or a change in the I-V relationship in NCX1 (5Nicoll 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 (128) Google Scholar). In addition, the Thr103 to Val mutation at the cytoplasmic end of TM2 in NCX1 produces changes in the apparent affinity for intracellular Na+ and the selectivity for Li+ (7Doering A.E. Nicoll D.A. Lu Y. Lu L. Weiss J.N. Philipson K.D. J. Biol. Chem. 1998; 273: 778-783Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Therefore these repeat sequences might form part of the structure involved in the ion translocation in the exchanger molecule. We have recently found that three mammalian exchanger isoforms have distinct differences in their biochemical and pharmacological properties (20Iwamoto T. Shigekawa M. Am. J. Physiol. 1998; 275: C423-C430Crossref PubMed Google Scholar). Divalent cation Ni2+ inhibits the reverse mode of Na+/Ca2+ exchange in NCX3 with a 10-fold less affinity than in NCX1 or NCX2. On the other hand, the recently identified inhibitor KB-R7943 is 3-fold more inhibitory to NCX3 than to NCX1 or NCX2. Furthermore, stimulation of Na+/Ca2+ exchange by externally added monovalent cation Li+ is significantly greater in NCX2 and NCX3 than in NCX1, although these isoforms exhibit low affinity for Li+. Despite these differences, however, all the NCX isoforms have similar apparent affinities for extracellular transport substrates Ca2+ and Na+ (20Iwamoto T. Shigekawa M. Am. J. Physiol. 1998; 275: C423-C430Crossref PubMed Google Scholar, 21Linck B. Qiu Z. He Z. Tong Q. Hilgemann D.W. Philipson K.D. Am. J. Physiol. 1998; 274: C415-C423Crossref PubMed Google Scholar). In this study, taking advantage of high sequence identity in the NCX isoforms, we used chimeric constructs between NCX1 and NCX3 to study the structural domain(s) responsible for the difference in their sensitivity to Ni2+ or Li+. We identified four amino acid residues within the α-1 and α-2 repeats of the exchanger molecule that are predominantly responsible for the observed differential effects of Ni2+ and Li+. CCL39 cells (American Type Culture Collection) and their NCX transfectants were maintained in Dulbecco's modified Eagle's medium supplemented with 7.5% heat-inactivated fetal calf serum, 50 units/ml penicillin, and 50 μg/ml streptomycin. cDNAs of dog heart NCX1.1 and rat brain NCX3.3 were subcloned into Sac II and Hin dIII sites in pCRII (designated pCRII-NCX1 and pCRII-NCX3, respectively) (18Iwamoto T. Pan Y. Wakabayashi S. Imagawa T. Yamanaka H.I. Shigekawa M. J. Biol. Chem. 1996; 271: 13609-13615Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 20Iwamoto T. Shigekawa M. Am. J. Physiol. 1998; 275: C423-C430Crossref PubMed Google Scholar). For construction of chimeras between NCX1 and NCX3, several unique restriction enzyme sites were newly introduced into pCRII-NCX1 and pCRII-NCX3 at analogous positions in the NCX cording regions by site-directed mutagenesis (13Iwamoto T. Pan Y. Nakamura T.Y. Wakabayashi S. Shigekawa M. Biochemistry. 1998; 37: 17230-17238Crossref PubMed Scopus (94) Google Scholar). In NCX1, the sites forBam HI, Sal I, Xma I, and Nhe I were created at amino acid positions 108, 133, 192, and 787 (numbers based on Ref. 22Nicoll D.A. Philipson K.D. Ann. N. Y. Acad. Sci. U. S. A. 1991; 639: 181-188Crossref PubMed Scopus (39) Google Scholar), respectively, whereas in NCX3, the sites forBam HI, Sal I, Xma I, Nhe I, and Mlu I were inserted at positions 142, 167, 226, 776, and 818 (numbers based on Ref. 4Nicoll 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), respectively. Generation of these sites preserved the native amino acid sequences. The modified plasmids were designated pCRII-NCX1′ and pCRII-NCX3′. NCX1/NCX3 chimera, except for N1-718/787 and N3-707/776, were constructed by exchanging homologous segments from pCRII-NCX1′ and pCRII-NCX3′ using the above newly introduced restriction enzyme sites and endogenous Cla I sites (amino acid positions 445 in NCX1 and 469 in NCX3) as shown in Fig. 1. N1-718/787 and N3-707/776 chimeras were constructed after creating new unique sites forEco RV at positions 718 and 707 in pCRII-NCX1′ and pCRII-NCX3′, respectively (Fig. 1), which resulted in substitution of one amino acid (V719I in N1-718/787 and V708I in N3-707/776). Successful construction of the modified cDNAs was verified by sequencing (ABI PRISM, Perkin-Elmer). These cDNAs were transferred into Sac II and Hin dIII sites of the mammalian expression vector pKCRH (23Mishina M. Kurosaki T. Tobimatsu T. Morimoto Y. Noda M. Yamamoto T. Terao M. Lindstrom J. Takahashi T. Kuno M. Numa S. Nature. 1984; 307: 604-608Crossref PubMed Scopus (248) Google Scholar). Substitution of amino acid residues within the internal α repeat regions was performed by site-directed mutagenesis. In this procedure, DNA fragments were produced by polymerase chain reaction using pCRII-NCX1 or pCRII-NCX3 as a template and following pairs of primers: for mutation in the α-1 repeat, the sense primers contained an exogenous Bam HI site and normal NCX sequences, whereas the antisense primers contained sequences with substituted nucleotides and an exogenous Sal I site; for mutation in the α-2 repeat, the sense primers contained an exogenous Nhe I site and normal NCX sequences, while the antisense primers contained an exogenous Mlu I site and sequences with desired mutations. The polymerase chain reaction products were digested with eitherBam HI and Sal I or Nhe I andMlu I, and inserted into pCRII-NCX1′ or pCRII-NCX3′, and then the full-length mutant cDNAs were transferred into pKCRH. Successful construction was verified by sequencing. To stably express chimeric and mutant exchangers, pKCRH plasmids carrying NCX cDNAs were transfected in the presence of Lipofectin (Life Technologies, Inc.) into CCL39 fibroblasts that exhibit little endogenous Na+/Ca2+ exchange activity (13Iwamoto T. Pan Y. Nakamura T.Y. Wakabayashi S. Shigekawa M. Biochemistry. 1998; 37: 17230-17238Crossref PubMed Scopus (94) Google Scholar, 20Iwamoto T. Shigekawa M. Am. J. Physiol. 1998; 275: C423-C430Crossref PubMed Google Scholar,24Iwamoto T. Wakabayashi S. Imagawa T. Shigekawa M. Eur. J. Cell Biol. 1998; 76: 228-236Crossref PubMed Scopus (26) Google Scholar). Cell clones expressing high Na+/Ca2+exchange activity were selected by a Ca2+-killing procedure as described previously (24Iwamoto T. Wakabayashi S. Imagawa T. Shigekawa M. Eur. J. Cell Biol. 1998; 76: 228-236Crossref PubMed Scopus (26) Google Scholar). Assay of Na+i-dependent45Ca2+ uptake into cells were described in detail previously (20Iwamoto T. Shigekawa M. Am. J. Physiol. 1998; 275: C423-C430Crossref PubMed Google Scholar). Briefly, confluent NCX transfectants in 24-well dishes were loaded with Na+ by incubation at 37 °C for 30 min in 0.5 ml of BSS (10 mm Hepes/Tris (pH 7.4), 146 mm NaCl, 4 mm KCl, 2 mmMgCl2, 0.1 mm CaCl2, 10 mm glucose, and 0.1% bovine serum albumin) containing 1 mm ouabain and 10 μm monensin.45Ca2+ uptake was then initiated by switching the medium to Na+-free BSS (replacing NaCl with equimolar choline chloride) or to normal BSS, both of which contained 0.1 mm45CaCl2 (1.5 μCi/ml) and 1 mm ouabain. After a 30-s incubation,45Ca2+ uptake was terminated by washing cells four times with an ice-cold solution containing 10 mm Hepes/Tris (pH 7.4), 120 mm choline chloride, and 10 mmLaCl3. Cells were then solubilized with 0.1 nNaOH, and aliquots were taken for determination of radioactivity and protein. Outward and inward currents from NCX transfectants were measured using the whole cell voltage clamp technique as described previously (25Uehara A. Iwamoto T. Shigekawa M. Imanaga I. Pflügers Arch. 1997; 434: 335-338PubMed Google Scholar). For recording the outward current, the external solution contained 150 mm NaCl, 1 mm MgCl2, 0 or 2 mm CaCl2, 20 μm ouabain, 2 μm nicardipine, 5 μm ryanodine, and 5 mm Hepes (pH 7.2), whereas the pipette solution contained 20 mm NaCl, 90 mm CsOH, 40 mmaspartic acid, 3 mm MgCl2, 10 mmCaCl2, 5 mm MgATP, 5 mmK2CrP, 20 mm BAPTA, and 20 mm Hepes (pH 7.2). The ionized Ca2+ concentration in the pipette solution was calculated to be 0.14 μm. The outward exchange current was activated by switching the external solution from one without CaCl2 to one with CaCl2. For recording the inward current, the external solution contained 140 mm choline chloride or NaCl, 1 mmMgCl2, 20 μm ouabain, 2 μmnicardipine, 10 μm ryanodine, and 5 mm Hepes (pH 7.2), while the pipette solution contained 30 mm CsCl, 90 mm CsOH, 50 mm aspartic acid, 3 mm MgCl2, 16 mm CaCl2, 5 mm MgATP, 5 mm K2CrP, 20 mm BAPTA, and 20 mm Hepes (pH 7.2). The ionized Ca2+ concentration in the pipette solution was calculated to be 1.75 μm. The inward exchange current was induced by switching the choline chloride-containing external medium to the Na+-containing external medium. All experiments were performed at about 35 °C and the holding and test potentials were −40 mV. All data were acquired and analyzed by the pCLAMP (Axon Instrument) software. Data are expressed as mean ± S.E. of three to five independent determinations. Differences for multiple comparisons were analyzed by unpaired t test or one-way ANOVA followed by the Dunnett's test. Values ofp < 0.05 were considered statistically significant. We previously measured the whole cell current from cloned dog cardiac NCX1 expressed in CCL39 cells using a conventional patch-clamp technique (25Uehara A. Iwamoto T. Shigekawa M. Imanaga I. Pflügers Arch. 1997; 434: 335-338PubMed Google Scholar). Using the same method, we measured the outward and inward currents evoked in NCX1- or NCX3-transfected CCL39 cells by the extracellular application of 2 mm Ca2+ or 140 mm Na+(Fig. 2, A and B ). These currents were reproducibly measured; after a recovery interval, a second pulse of Ca2+ or Na+ generated the outward or inward current whose peak value was 96–102% of the corresponding first current. However, they were never observed in nontransfected CCL39 cells. Inclusion of Ni2+ in the external medium caused inhibition of the outward and inward currents (Fig. 2, A and B ). We found that the outward current in NCX1-transfected cells was approximately 10-fold more sensitive to Ni2+ than that in NCX3-transfected cells (IC50, 39 versus 310 μm) and that the inhibition in these cells reached 92 and 81%, respectively, at 3 mm Ni2+ (Fig. 2 C ). A similar difference in the sensitivity to Ni2+ was observed for the inward currents in NCX1- and NCX3-transfected cells (IC50, 25 versus >100 mm), although its inhibition by Ni2+ occurred at much greater concentrations compared with the outward current (Fig. 2, C and D ). Our previous measurement of Ni2+ dependence of Na+i-dependent45Ca2+ uptake gave IC50 values of 33 and 343 μm for Ni2+ in NCX1- and NCX3-transfected CCL39 cells, respectively (Ref. 20Iwamoto T. Shigekawa M. Am. J. Physiol. 1998; 275: C423-C430Crossref PubMed Google Scholar, see also Fig. 3,inset ), in agreement with the observed inhibitory potencies of Ni2+ on the outward current. Thus both electrophysiological and biochemical data establish that NCX1 and NCX3 exhibit a 10-fold difference in the sensitivity to Ni2+, at least when they function in the reverse exchange mode. Taking advantage of the fact that NCX1 and NCX3 exhibit high amino acid homology, we used chimeric constructs between the two isoforms to study the structural domain(s) responsible for the difference in their sensitivity to Ni2+. Using endogenous or newly introduced common restriction enzyme sites, we constructed two series of chimeric cDNAs in which one or two segments from NCX3 were incorporated into NCX1 in exchange for the homologous segment(s) in the latter (N1 series chimeras), and vice versa (N3 series chimeras) (see Fig. 1). We stably expressed these chimeras in CCL39 fibroblasts. All transfectants, after selection by a Ca2+-killing procedure, exhibited exchange activities similar to that of cells expressing the wild-type NCX1 or NCX3 (see the legend to Fig.3). We examined the effect of 0.1 mm Ni2+ on the initial rate of Na+i-dependent45Ca2+ uptake into cells expressing these chimeric exchangers (Fig. 3). Under the conditions used, Ni2+ at this concentration reduced the uptake rate of the wild-type NCX1 or NCX3 by 77 or 24%, respectively. N1 series chimeras N1-109/133, N1-788/829, and N1-109/133,788/829, which contained the homologous Bam HI-Sal I and/orNhe I-Mlu I segments from NCX3 (Fig. 1), exhibited significantly less sensitivity to inhibition by 0.1 mmNi2+ compared with the wild-type NCX1 (Fig. 3 A ). In contrast, N3 series chimeras N3-143/167, N3-777/818, and N3-143/167,777/818 containing the Bam HI-Sal I and/or Nhe I-Mlu I segments from NCX1 (Fig. 1) were more sensitive to inhibition by Ni2+ compared with the wild-type NCX3 (Fig. 3 B ). All other chimeras, however, were not significantly different from the parental NCX1 or NCX3. For some of those chimeras with an altered sensitivity to Ni2+, we determined dose-response profiles for Ni2+ by measuring the rate of Na+i-dependent45Ca2+ in the presence of 0.001 to 1 mm Ni2+. The IC50 values for this cation were 343 ± 28 μm for NCX3, 258 ± 15 μm for N1-109/133,788/829, 93 ± 5 μmfor N1-109/133, 59 ± 4 μm for N1-788/829, 65 ± 7 μm for N3-143/167,777/818, and 33 ± 3 μm for NCX1. All these results indicate that theBam HI-Sal I and Nhe I-Mlu I segments are individually responsible for the alteration of Ni2+ sensitivity and simultaneous replacement of both accounts for ∼80% of the difference in Ni2+ sensitivity between the wild-type NCX1 and NCX3. Intriguingly, the Bam HI-Sal I andNhe I-Mlu I segments correspond, respectively, to the major portions of the phylogenetically conserved regions of the Na+/Ca2+ exchanger designated the α-1 and α-2 repeats (see Fig. 1) (19Schwarz E.M. Benzer S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10249-10254Crossref PubMed Scopus (181) Google Scholar). Na+/Ca2+exchange has been shown to be highly sensitive to mutagenesis in the putative transmembrane helices within the α repeats (5Nicoll 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 (128) Google Scholar), suggesting the functional importance of these regions. Fig. 4 shows amino acid sequences of the α repeat regions from NCX1, NCX2, and NCX3. Of note, in these regions, there are only a small number of amino acids unique to each isoform, all of which are localized within theBam HI-Sal I and Nhe I-Mlu I segments. To identify the residues involved in the differential Ni2+ sensitivity, these unique residues in the α repeats were exchanged between NCX1 and NCX3 or mutated to other amino acids (Fig. 5). Three amino acids Val, Asn, and Thr in the α-1 repeat of NCX1 (corresponding to Val118, Asn125, and Thr127) were replaced with Leu, Gly, and Ile from the same region of NCX3 (corresponding to Leu152, Gly159, and Ile161), respectively, and vice versa . We found that single substitution mutants were not different from the parental NCX1 or NCX3 in their sensitivity to 0.1 mm Ni2+ (Fig. 5,A and B ). On the other hand, double substitution mutants N1-N125G/T127I and its reciprocal mutant N3-G159N/I161T showed decreased and increased sensitivities to inhibition by Ni2+, respectively, which are comparable to those seen in N1-109/133 and N3-143/167 chimeras (compare Figs. 3 and 5). Double mutants N1-V118L/N125G and N1-V118L/T127I, however, exhibited Ni2+ sensitivity similar to that of NCX1 (data not shown). Thus, Asn125 and Thr127 in the α-1 repeat of NCX1 and corresponding Gly159 and Ile161 in NCX3 are responsible for the altered responses of N1-109/133 and N3-143/167 to Ni2+. We also examined the Ni2+sensitivity of NCX1 mutants with Asn125 to Cys (N1-N125C) or Thr127 to Cys substitution (N1-T127C). Interestingly, N1-N125C, but not T127C, exhibited an increased sensitivity to inhibition by Ni2+ relative to that of the parental NCX1 (Fig. 5 A ).Figure 5Effects of Ni2+ on Na+i-dependent45Ca2+ uptake into cells expressing exchangers mutated in the α-1 and α-2 repeats. We constructed single or double substitution mutants in which the amino acid residues in the α-1 and α-2 repeats unique to each NCX isoform were exchanged between NCX1 and NCX3 or mutated to other residues. The initial rates of Na+i-dependent45Ca2+ uptake into cells expressing NCX1 mutants (A ) or NCX3 mutants (B ) were measured in the presence or absence of 0.1 mm Ni2+. The uptake rates in these mutants was 3 to 9 nmol/mg/30 s in the absence of Ni2+. Data are presented as percentage of the values obtained in the absence of Ni2+. Data are mean ± S.E. of three independent experiments. *, p < 0.05versus NCX1 (in A ); versus NCX3 (inB ).View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next exchanged Leu, Val, Thr, and Gln in the α-2 repeat of NCX1 (corresponding to Leu808, Val820, Thr823, and Gln826), respectively, with Phe, Ala, Leu, and Val (corresponding to Phe797, Ala809, Leu812, and Val815 of NCX 3). Five NCX1 and NCX3 single substitution mutants (N1-L808F, N1-T823L, N1-Q826V, N3-F797L, and N3-L812T) exhibited Ni2+sensitivity not different from the parental NCX1 or NCX3 (Fig. 5,A and B ). In contrast, N1-V820A and its reciprocal mutant N3-A809V showed decreased and increased sensitivities to inhibition by 0.1 mm Ni2+ compared with NCX1 and NCX3, respectively, whereas N3-V815Q exhibited a slightly increased inhibition by Ni2+ relative to NCX3. Double substitution mutants N1-V820A/Q826V and N3-A809V/V815Q, on the other hand, exhibited Ni2+ sensitivities similar to single mutants N1-V820A and N3-A809V, respectively. Thus Val820 of NCX1 and Ala809 of NCX3, and probably Val815 of NCX3, are involved in the determination of Ni2+ sensitivity in the α-2 repeat. We found that the Val820 to Ile substitution in NCX1 (N1-V820I) and the corresponding Ala809 to Ile substitution in NCX3 (N3-A809I) resulted in an increase in the sensitivity to inhibition by Ni2+, whereas substitution of these residues with Gly resulted in a decrease in the Ni2+ sensitivity in N1-V820G, but not in N3-A809G (Fig. 5, A and B ). Other mutants N1-Q826E and N1-Q826R and the corresponding mutants N3-V815E and N3-815R showed the Ni2+ sensitivity not different from that of the parental NCX1 or NCX3. Monovalent cation Li+ stimulates Na+i-dependent45Ca2+ uptake by all three NCX isoforms with low affinity, with the extent of stimulation being greater in NCX2 or NCX3 than in NCX1 (20Iwamoto T. Shigekawa M. Am. J. Physiol. 1998; 275: C423-C430Crossref PubMed Google Scholar). To obtain insight into the structural domains involved in the Li+-induced NCX activation, we examined the effect of Li+ on the exchange activities of the chimeras and mutants described above. Consistent with the previous result, extracellular application of Li+ caused a dose-dependent increase in the rate of Na+i-dependent Ca2+ uptake into NCX1- or NCX3-transfected cells, which reached about 145 and 270% at 146 mm Li+, respectively, of the control measured in the absence of Li+ (Fig.6, inset ). Fig. 6,A and B, show the extent of stimulation of the uptake by various chimeras and mutants at 146 mmLi+. Intriguingly, N1-788/829 and N1-109/133,788/829 containing a major portion of the α-2 repeat from NCX3 produced NCX1 mutants exhibiting Li+ sensitivity almost identical to that of the wild-type NCX3. Conversely, the reciprocal mutants N3-777/818 and N3-143/167,777/818 exhibited Li+ sensitivity similar to that in the wild-type NCX1. On the other hand, other N1 and N3 chimeras were not different from the wild-type NCX1 or NCX3, respectively" @default.
• W2073604123 created "2016-06-24" @default.
• W2073604123 creator A5003630969 @default.
• W2073604123 creator A5012157326 @default.
• W2073604123 creator A5067923638 @default.
• W2073604123 creator A5073163509 @default.
• W2073604123 creator A5081150731 @default.
• W2073604123 date "1999-08-01" @default.
• W2073604123 modified "2023-09-27" @default.
• W2073604123 title "Chimeric Analysis of Na+/Ca2+ Exchangers NCX1 and NCX3 Reveals Structural Domains Important for Differential Sensitivity to External Ni2+ or Li+" @default.
• W2073604123 cites W1507203601 @default.
• W2073604123 cites W1512575910 @default.
• W2073604123 cites W1966880718 @default.
• W2073604123 cites W1973056364 @default.
• W2073604123 cites W1980743132 @default.
• W2073604123 cites W1987476658 @default.
• W2073604123 cites W2011954551 @default.
• W2073604123 cites W2026696517 @default.
• W2073604123 cites W2031172307 @default.
• W2073604123 cites W2033507308 @default.
• W2073604123 cites W2040249443 @default.
• W2073604123 cites W2050785330 @default.
• W2073604123 cites W2053849452 @default.
• W2073604123 cites W2059201211 @default.
• W2073604123 cites W2068615953 @default.
• W2073604123 cites W2077476788 @default.
• W2073604123 cites W2081937583 @default.
• W2073604123 cites W2082208569 @default.
• W2073604123 cites W2085267754 @default.
• W2073604123 cites W2092473345 @default.
• W2073604123 cites W2093353254 @default.
• W2073604123 cites W2099654084 @default.
• W2073604123 cites W2099808541 @default.
• W2073604123 cites W2123433512 @default.
• W2073604123 cites W2128801406 @default.
• W2073604123 cites W2159160674 @default.
• W2073604123 cites W2170680714 @default.
• W2073604123 cites W2188376552 @default.
• W2073604123 cites W2196050372 @default.
• W2073604123 cites W4251242226 @default.
• W2073604123 doi "https://doi.org/10.1074/jbc.274.33.23094" @default.
• W2073604123 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10438478" @default.
• W2073604123 hasPublicationYear "1999" @default.
• W2073604123 type Work @default.
• W2073604123 sameAs 2073604123 @default.
• W2073604123 citedByCount "40" @default.
• W2073604123 countsByYear W20736041232012 @default.
• W2073604123 countsByYear W20736041232015 @default.
• W2073604123 countsByYear W20736041232017 @default.
• W2073604123 countsByYear W20736041232018 @default.
• W2073604123 countsByYear W20736041232021 @default.
• W2073604123 countsByYear W20736041232022 @default.
• W2073604123 crossrefType "journal-article" @default.
• W2073604123 hasAuthorship W2073604123A5003630969 @default.
• W2073604123 hasAuthorship W2073604123A5012157326 @default.
• W2073604123 hasAuthorship W2073604123A5067923638 @default.
• W2073604123 hasAuthorship W2073604123A5073163509 @default.
• W2073604123 hasAuthorship W2073604123A5081150731 @default.
• W2073604123 hasBestOaLocation W20736041231 @default.
• W2073604123 hasConcept C121332964 @default.
• W2073604123 hasConcept C12554922 @default.
• W2073604123 hasConcept C127413603 @default.
• W2073604123 hasConcept C178790620 @default.
• W2073604123 hasConcept C185592680 @default.
• W2073604123 hasConcept C21200559 @default.
• W2073604123 hasConcept C24326235 @default.
• W2073604123 hasConcept C2777735026 @default.
• W2073604123 hasConcept C519063684 @default.
• W2073604123 hasConcept C86803240 @default.
• W2073604123 hasConcept C93226319 @default.
• W2073604123 hasConcept C97355855 @default.
• W2073604123 hasConceptScore W2073604123C121332964 @default.
• W2073604123 hasConceptScore W2073604123C12554922 @default.
• W2073604123 hasConceptScore W2073604123C127413603 @default.
• W2073604123 hasConceptScore W2073604123C178790620 @default.
• W2073604123 hasConceptScore W2073604123C185592680 @default.
• W2073604123 hasConceptScore W2073604123C21200559 @default.
• W2073604123 hasConceptScore W2073604123C24326235 @default.
• W2073604123 hasConceptScore W2073604123C2777735026 @default.
• W2073604123 hasConceptScore W2073604123C519063684 @default.
• W2073604123 hasConceptScore W2073604123C86803240 @default.
• W2073604123 hasConceptScore W2073604123C93226319 @default.
• W2073604123 hasConceptScore W2073604123C97355855 @default.
• W2073604123 hasIssue "33" @default.
• W2073604123 hasLocation W20736041231 @default.
• W2073604123 hasOpenAccess W2073604123 @default.
• W2073604123 hasPrimaryLocation W20736041231 @default.
• W2073604123 hasRelatedWork W1977629086 @default.
• W2073604123 hasRelatedWork W1997150209 @default.
• W2073604123 hasRelatedWork W2009082369 @default.
• W2073604123 hasRelatedWork W2020589817 @default.
• W2073604123 hasRelatedWork W2525975595 @default.
• W2073604123 hasRelatedWork W2735758876 @default.
• W2073604123 hasRelatedWork W3100088800 @default.
• W2073604123 hasRelatedWork W3169802229 @default.
• W2073604123 hasRelatedWork W4247467749 @default.
• W2073604123 hasRelatedWork W184669331 @default.
• W2073604123 hasVolume "274" @default.

• next