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• W1990666241 abstract "K+-dependent Na+/Ca2+ exchangers (NCKX) catalyze cytosolic Ca2+ extrusion and are particularly important for neuronal Ca2+ signaling. Of the five mammalian isoforms, the detailed functional characteristics have only been reported for NCKX1 and -2. In the current study, the functional characteristics of recombinant NCKX3 and -4 expressed in HEK293 cells were determined and compared with those of NCKX2. Although the apparent affinities of the three isoforms for Ca2+ and Na+ were similar, NCKX3 and -4 displayed ∼40-fold higher affinities for K+ ions than NCKX2. Functional analysis of various NCKX2 mutants revealed that mutation of Thr-551 to Ala, the corresponding residue in NCKX4, resulted in an apparent K+ affinity shift to one similar to that of NCKX4 without a parallel shift in apparent Ca2+ affinity. In the converse situation, when Gln-476 of NCKX4 was converted to Lys, the corresponding residue in NCKX2, both the K+ and Ca2+ affinities were reduced. These results indicate that the apparently low K+ affinity of NCKX2 requires a Thr residue at position 551 that may reduce the conformational flexibility and/or K+ liganding strength of side-chain moieties on critical neighboring residues. This interaction appears to be specific to the structural context of the NCKX2 K+ binding pocket, because it was not possible to recreate the K+-specific low affinity phenotype with reciprocal mutations in NCKX4. The results of this study provide important information about the structure and function of NCKX proteins and will be critical to understanding their roles in neuronal Ca2+ signaling. K+-dependent Na+/Ca2+ exchangers (NCKX) catalyze cytosolic Ca2+ extrusion and are particularly important for neuronal Ca2+ signaling. Of the five mammalian isoforms, the detailed functional characteristics have only been reported for NCKX1 and -2. In the current study, the functional characteristics of recombinant NCKX3 and -4 expressed in HEK293 cells were determined and compared with those of NCKX2. Although the apparent affinities of the three isoforms for Ca2+ and Na+ were similar, NCKX3 and -4 displayed ∼40-fold higher affinities for K+ ions than NCKX2. Functional analysis of various NCKX2 mutants revealed that mutation of Thr-551 to Ala, the corresponding residue in NCKX4, resulted in an apparent K+ affinity shift to one similar to that of NCKX4 without a parallel shift in apparent Ca2+ affinity. In the converse situation, when Gln-476 of NCKX4 was converted to Lys, the corresponding residue in NCKX2, both the K+ and Ca2+ affinities were reduced. These results indicate that the apparently low K+ affinity of NCKX2 requires a Thr residue at position 551 that may reduce the conformational flexibility and/or K+ liganding strength of side-chain moieties on critical neighboring residues. This interaction appears to be specific to the structural context of the NCKX2 K+ binding pocket, because it was not possible to recreate the K+-specific low affinity phenotype with reciprocal mutations in NCKX4. The results of this study provide important information about the structure and function of NCKX proteins and will be critical to understanding their roles in neuronal Ca2+ signaling. Transient increases in cytosolic Ca2+ concentrations are a ubiquitous signaling mechanism in animal cells, particularly excitable cells, and subsequent Ca2+ extrusion is mediated by a variety of membrane transporter systems (1.Augustine G.J. Santamaria F. Tanaka K. Neuron. 2003; 40: 331-346Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 2.Berridge M.J. Bootman M.D. Roderick H.L. Nat. Rev. Mol. Cell. Biol. 2003; 4: 517-529Crossref PubMed Scopus (4254) Google Scholar). Major contributions to cytosolic Ca2+ clearance are made by two families of Na+/Ca2+ exchangers: those that are independent (NCX) 3The abbreviations used are: NCX, Na+/Ca2+ exchanger; NCKX, K+-dependent Na+/Ca2+ exchanger; HEK293, human embryonic kidney 293; K0.5, concentration for half-maximal Ca2+ uptake; IC50, concentration that causes 50% of maximal inhibition; TM, transmembrane segment. or dependent (NCKX) on K+ ions (3.Schnetkamp P.P. Pflugers Arch. 2004; 447: 683-688Crossref PubMed Scopus (87) Google Scholar, 4.Quednau B.D. Nicoll D.A. Philipson K.D. Pflugers Arch. 2004; 447: 543-548Crossref PubMed Scopus (99) Google Scholar, 5.Blaustein M.P. Lederer W.J. Physiol. Rev. 1999; 79: 763-854Crossref PubMed Scopus (1464) Google Scholar). Three NCX and five NCKX isoforms have been identified by molecular cloning, and each isoform displays unique tissue and cellular distribution patterns, suggesting that they play specialized, nonoverlapping roles in Ca2+ signaling. NCX1 is predominantly expressed in heart, brain, and kidney but is also present at lower levels in other tissues, whereas expression of NCX2 and -3 is limited to brain and skeletal muscle (6.Komuro I. Wenninger K.E. Philipson K.D. Izumo S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4769-4773Crossref PubMed Scopus (128) Google Scholar, 7.Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-565Crossref PubMed Scopus (628) Google Scholar, 8.Nicoll 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 (311) Google Scholar, 9.Li 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). NCKX1 is expressed exclusively in rod photoreceptor outer segments where it plays critical roles in retinal function, whereas NCKX2 expression is limited to neurons, where it has been shown play an important role in synaptic plasticity (10.Li X.F. Kiedrowski L. Tremblay F. Fernandez F.R. Perizzolo M. Winkfein R.J. Turner R.W. Bains J.S. Rancourt D.E. Lytton J. J. Biol. Chem. 2006; 281: 6273-6282Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 11.Reilander H. Achilles A. Friedel U. Maul G. Lottspeich F. Cook N.J. EMBO J. 1992; 11: 1689-1695Crossref PubMed Scopus (160) Google Scholar, 12.Tsoi M. Rhee K.H. Bungard D. Li X.F. Lee S.L. Auer R.N. Lytton J. J. Biol. Chem. 1998; 273: 4155-4162Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) and cone photoreceptors (13.Prinsen C.F. Szerencsei R.T. Schnetkamp P.P. J. Neurosci. 2000; 20: 1424-1434Crossref PubMed Google Scholar). Although they are more broadly expressed, NCKX3 and -4 are also abundant in the brain but with distinct cellular localization patterns from that of NCKX2 and NCX1 (14.Kraev 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: 23161-23172Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 15.Li X.F. Kraev A.S. Lytton J. J. Biol. Chem. 2002; 277: 48410-48417Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). NCKX5 is an exchanger of intracellular membranes that is abundantly expressed in skin and the pigmented epithelium of the eye where it appears to play an important role in pigmentation, although the mechanism is unknown (16.Lamason R.L. Mohideen M.A. Mest J.R. Wong A.C. Norton H.L. Aros M.C. Jurynec M.J. Mao X. Humphreville V.R. Humbert J.E. Sinha S. Moore J.L. Jagadeeswaran P. Zhao W. Ning G. Makalowska I. McKeigue P.M. O’Donnell D. Kittles R. Parra E.J. Mangini N.J. Grunwald D.J. Shriver M.D. Canfield V.A. Cheng K.C. Science. 2005; 310: 1782-1786Crossref PubMed Scopus (805) Google Scholar). NCX and NCKX proteins share similar overall topologies with 10 (NCX) or 11 (NCKX) transmembrane-spanning segments (TMs), the first of which (TM 0) is cleaved by a signal peptidase, and the remaining TMs divided into two clusters connected by a large cytoplasmic loop with important regulatory functions (17.Kang K. Schnetkamp P.P. Biochemistry. 2003; 42: 9438-9445Crossref PubMed Scopus (26) Google Scholar, 18.Cai X. Lytton J. Mol. Biol. Evol. 2004; 21: 1692-1703Crossref PubMed Scopus (183) Google Scholar, 19.Kinjo T.G. Szerencsei R.T. Winkfein R.J. Kang K. Schnetkamp P.P. Biochemistry. 2003; 42: 2485-2491Crossref PubMed Scopus (43) Google Scholar, 20.Cai X. Zhang K. Lytton J. J. Biol. Chem. 2002; 277: 48923-48930Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 21.Lee J.Y. Visser F. Lee J.S. Lee K.H. Soh J.W. Ho W.K. Lytton J. Lee S.H. J. Biol. Chem. 2006; 281: 39205-39216Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Amino acid sequence identity between NCX and NCKX proteins is limited to two internally homologous, oppositely oriented regions known as α-repeats 1 and 2, which are found within the N- and C-terminal clusters of TMs, respectively, and have been shown to contain amino acid residues critical for cation liganding and translocation (22.Winkfein R.J. Szerencsei R.T. Kinjo T.G. Kang K. Perizzolo M. Eisner L. Schnetkamp P.P. Biochemistry. 2003; 42: 543-552Crossref PubMed Scopus (52) Google Scholar, 23.Kang K.J. Kinjo T.G. Szerencsei R.T. Schnetkamp P.P. J. Biol. Chem. 2005; 280: 6823-6833Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 24.Kang K.J. Shibukawa Y. Szerencsei R.T. Schnetkamp P.P. J. Biol. Chem. 2005; 280: 6834-6839Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 25.Nicoll 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, 26.Ottolia M. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2005; 280: 1061-1069Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 27.Doering 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, 28.Iwamoto T. Uehara A. Imanaga I. Shigekawa M. J. Biol. Chem. 2000; 275: 38571-38580Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). An important aspect of understanding the unique physiological roles of the various NCX and NCKX isoforms is to determine their functional properties. The three NCX isoforms catalyze the exchange of 3 Na+ ions for 1 Ca2+ ion and have been demonstrated to display very similar functional properties with K0.5 values of ∼100–400 μm for Ca2+ (in the presence of 1 mm Mg2+) and ∼10–60 mm for Na+ (29.Fontana G. Rogowski R.S. Blaustein M.P. J. Physiol. 1995; 485: 349-364Crossref PubMed Scopus (42) Google Scholar, 30.Linck B. Qiu Z. He Z. Tong Q. Hilgemann D.W. Philipson K.D. Am. J. Physiol. 1998; 274: C415-C423Crossref PubMed Google Scholar). We have previously reported the electrophysiological characterization of recombinant rat brain NCKX2 expressed in HEK293 cells (31.Dong H. Light P.E. French R.J. Lytton J. J. Biol. Chem. 2001; 276: 25919-25928Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). NCKX2 was shown to exchange 4 Na+ ions for 1 Ca2+ and 1 K+ with K0.5 values of ∼1 μm or 100 μm for Ca2+ dependence, in the absence or presence of 1 mm Mg2+, respectively, 10 or 35 mm for K+ dependence, in the presence of choline+ or Li+, respectively, and 30 or 60 mm for Na+ dependence or inhibition, respectively. Although NCKX3 and -4 have been demonstrated to function as K+-dependent Na+/Ca2+ exchangers, their detailed functional characteristics are unknown (14.Kraev 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: 23161-23172Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 15.Li X.F. Kraev A.S. Lytton J. J. Biol. Chem. 2002; 277: 48410-48417Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). NCKX3 and -4 are closely related, with 71% overall amino acid sequence identity but only 40% identity with NCKX1 and -2, which may underlie functional differences between the isoforms that could have important implications for their roles in Ca2+ signaling. In the current study, functional characterization of recombinant human NCKX2, -3, and -4 was undertaken by expression in HEK293 cells loaded with fura-2 AM in a perfusion chamber using a microscope photometer. The results of these studies indicated the apparent K0.5 for K+ ions of NCKX3 and -4 was ∼40-fold lower than that of NCKX2. Site-directed mutagenesis of NCKX2 and -4 based on multiple sequence alignments of the α-repeat regions was employed to identify the molecular determinants responsible for the apparent differences in K+ affinity. These studies suggest that the identified residues may alter the conformational flexibility and/or liganding strength of key acidic residues in the K+ binding pocket. All common chemicals and reagents were purchased from Sigma, VWR, or BDH and were minimally of analytical grade. All molecular procedures were performed according to previously established protocols (32.Ausubel 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, New York, NY2006Google Scholar) or according to reagent manufacturer’s instructions unless indicated otherwise. NCKX2, -3, and -4 Expression Constructs−The cDNA corresponding to human NCKX2 was obtained by PCR amplification from a human brain Marathon cDNA library (Clontech, Palo Alto, CA) using the primers (restriction endonuclease sites underlined) hNK2–5′out (CCC GGT ACC GAT CAC CAT ATC CAC CAG AAG AC) and hNK2–3′out (CCC CTC GAG CTT CTC AAG AGG TCA AGG AGC C) followed by a second round of re-amplification using the nested primers (restriction endonuclease sites underlined) hNK2–5′in (CCC GGT ACC ATG GAT CTG CAA CAA AGC ACC AC) and hNK2–3′in (CCC CTC GAG CTA GAT GGA GAC GGG GCA TG). The resulting PCR products were directionally subcloned into the KpnI and XhoI restriction endonuclease sites of pcDNA3.1+ (Invitrogen). DNA sequencing confirmed that both the long and short splice variants (with or without resides 360–376 in the large cytoplasmic loop, respectively), were obtained and that the sequences were identical to those deposited in GenBank™ for human NCKX2 (12.Tsoi M. Rhee K.H. Bungard D. Li X.F. Lee S.L. Auer R.N. Lytton J. J. Biol. Chem. 1998; 273: 4155-4162Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 13.Prinsen C.F. Szerencsei R.T. Schnetkamp P.P. J. Neurosci. 2000; 20: 1424-1434Crossref PubMed Google Scholar). Subsequent functional analyses suggested that there were no obvious functional differences between the short and long splice variants, so all subsequent experiments were performed using the long splice variant of NCKX2. Generation of the constructs used for expression of recombinant human NCKX3 and -4 has been described previously (14.Kraev 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: 23161-23172Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 15.Li X.F. Kraev A.S. Lytton J. J. Biol. Chem. 2002; 277: 48410-48417Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). PCR was used to insert the FLAG epitope (amino acids DYKDDDDK) into the putative N-terminal extracellular loops of NCKX2 (by replacing residues 89–96), NCKX3 (by insertion after residue 66), and NCKX4 (by insertion after residue 41). Mutagenesis−Site-directed mutagenesis to achieve the desired changes in the amino acid sequences was performed using either overlap-extension PCR or megaprimer PCR according to previously established methods (33.Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6851) Google Scholar, 34.Sarkar G. Sommer S.S. BioTechniques. 1990; 8: 404-407PubMed Google Scholar). The correct sequences of all the constructs was confirmed by DNA sequencing. The amino acid numbering was based on the long splice variants of NCKX2 and NCKX3 and the short splice variant of NCKX4, which corresponds to the versions used in our functional assays. Cell Culture and Transfection−Human embryonic kidney (HEK) 293 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, and maintained in 5% CO2 at 37 °C. Transfection of Qiagen-purified expression plasmids into HEK293 cells was performed using a standard Ca2+-phosphate precipitation protocol as previously described (12.Tsoi M. Rhee K.H. Bungard D. Li X.F. Lee S.L. Auer R.N. Lytton J. J. Biol. Chem. 1998; 273: 4155-4162Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Experiments utilizing cells transfected with the parent expression plasmid, pcDNA3.1+ (Invitrogen), were used as negative controls. Photometry and Data Analysis−Two days after transfection, HEK293 cells on poly-d-lysine-coated coverslips were loaded with 5 μm fura-2 AM (Invitrogen) and mounted in a perfusion chamber on a microscope stage. The fura-2 ratio was determined by measuring fluorescence emission at 510 nm from excitations at 340 nm and 380 nm with a D-104 microscope photometer using Felix version 1.42 software (Photon Technology International). The perfusion solutions used contained 10 mm d-glucose and 10 mm HEPES-tetramethylammonium, pH 7.4, with either 145 mm NaCl or LiCl and varying concentrations of KCl and/or NaCl to a total of 145 mm, and 0.1 mm CaCl2 or varying concentrations of CaCl2 buffered with 0.5 mm EGTA to the desired final concentration for Ca2+ dependence experiments. The cells were perfused alternatively for 3 min with sodium buffer or 2 min with lithium buffer at a rate of ∼3 ml/min in a volume of ∼0.5 ml, ensuring complete solution change in ∼10 s or less. Ca2+ transport rates for reverse mode NCKX2 activity were determined by linear regression of the initial linear rate of change of the fura-2 ratio. Each coverslip was internally normalized to the average of the rates of the second and last pulses, but if they differed by >20% a correction for the change in maximal rate with time was applied, using a linear interpolation between the second and last pulses to mathematically correct the intervening pulses as illustrated in Fig. 1. All data analysis and curve-fitting were performed using GraphPad Prism version 4.0 software. The Ca2+ and K+ dependence curves and Na+ inhibition curves were fitted using nonlinear regression analysis to the equation: Y = Top – Bottom/(1–10^((log(K0.5 or IC50) – X)*Hillslope), where, with the exception of the NCKX2 K+ dependence curve, the Top was constrained to a value of 100% and the Bottom was constrained to a value of 5%. Development of a Functional Assay for Recombinant NCKX2, -3, and -4−HEK293 cells grown on coverslips and transfected with expression constructs for recombinant FLAG-tagged NCKX2, -3, or -4, or pcDNA3.1+ without insert, were loaded with fura-2 AM and mounted in a perfusion chamber on a microscope stage. The ratio of fluorescence emission at 510 nm from excitations at 340 and 380 nm, respectively, was monitored using a microscope photometer. When the perfusion solution containing 145 mm Na+ was switched to one containing 135 mm Li+ and 10 mm K+, a rapid increase in the fura-2 ratio was observed in cells transfected with NCKX constructs but not in cells transfected with pcDNA3.1+ (Fig. 1A). The rapid rise in the fura-2 fluorescence ratio required the presence of K+ ions in the perfusion solution and therefore, represented reverse mode, NCKX-mediated Ca2+ influx (data not shown). Repeated switches between Na+-containing buffer and Li+ + K+-containing buffer to a total of nine “pulses” generally resulted in initial rates of Ca2+ transport by NCKX3 and -4 that individually differed by no more than 20%, whereas NCKX2 displayed a monotonic run-down phenomenon that was linear after the first pulse and resulted in the rate of pulse nine being 40% lower than the rate of pulse two in the example presented in Fig. 1 (B and C). In all experiments, the rate of the first pulse was inconsistent with that of the following pulses and thus not included in any of the analyses. Run-down was also observed for NCKX3 and -4 in some experiments and was similarly absent in some NCKX2 experiments. Therefore, run-down phenomena were generally considered significant when the second and last pulses differed more than the variability observed in experiments where no run-down was observed (i.e. >20%). In such cases, a time-dependent correction was applied, based on linear interpolation between pulses two and nine, and used to mathematically correct the intervening pulses (Fig. 1C). It is also worth noting that the amplitude of the Ca2+ responses did not always correlate well with the initial rate of rise (this is seen in the example traces of Figs. 2 and 3). Initial rates were always used for data analysis as illustrated in Figs. 2B and 3B.FIGURE 3K+ concentration dependences of recombinant NCKX2, -3, and -4. The effects of varying K+ concentrations on the reverse-mode operation of NCKX2, -3, and -4 in the presence of 0.1 mm Ca2+ were determined and analyzed using the functional assay described in the legend to Fig. 2. A, representative data for cells expressing NCKX2, -3, and -4 or transfected with pcDNA3.1+. B, the initial rate portions of pulses two to nine from the representative traces of panel A. C, data from four to seven coverslips were normalized and averaged. Each point represents the average (± S.E.) of 4 to 14 determinations, and where the point is larger than the S.E., error bars are not shown. The K0.5 values are presented in Table 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Ca2+ and K+ Concentration Dependence for Reverse-mode Activity of NCKX2, -3, and -4−To assess the apparent affinities of various NCKX isoforms for Ca2+, the initial rates of fura-2 ratio change were determined in HEK293 cells expressing recombinant FLAG-tagged NCKX2, -3, and -4 in buffers containing 135 mm Li+,10mm K+, and varying concentrations of Ca2+. These experiments were performed using either increasing or decreasing concentrations of Ca2+, yielding identical results. Representative experiments in which decreasing concentrations were used are presented in Fig. 2A, whereas representative experiments in which increasing concentrations were used are presented in some of the subsequent figures. Cells transfected with pcDNA3.1+ did not display any notable changes in the fura-2 ratio when perfused with the various buffers. To illustrate the data analysis methods used, the portions of the representative traces shown in Fig. 2A corresponding to the initial rates of fura-2 ratio change for each pulse are presented in Fig. 2B. When the data from four to six coverslips were internally normalized, averaged, and fitted using nonlinear regression analysis, the resulting apparent K0.5 values for NCKX2, -3, and -4 were similar (1.3–1.7 μm), and the Hill slope values ranged from 0.98 to 1.2, suggesting the presence of a single class of Ca2+-binding sites (Fig. 2C and Table 1). These data were consistent with the previously reported value of 1.4 μm obtained for recombinant rat brain NCKX2 using electrophysiological methods (31.Dong H. Light P.E. French R.J. Lytton J. J. Biol. Chem. 2001; 276: 25919-25928Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar).TABLE 1Apparent affinities of NCKX2, -3, and -4 for Ca2+, K+, and Na+CationProteinApparent affinityHill slopenK0.5 (μm)Ca2+NCKX21.6 ± 0.20.98 ± 0.114NCKX31.3 ± 0.31.1 ± 0.26NCKX41.7 ± 0.21.2 ± 0.24K0.5 (mm)K+NCKX2∼40NDaND, not determined.7NCKX31.3 ± 0.41.0 ± 0.24NCKX41.1 ± 0.21.1 ± 0.27IC50 (mm)Na+NCKX234 ± 61.6 ± 0.44NCKX330 ± 41.7 ± 0.34NCKX418 ± 31.8 ± 0.54a ND, not determined. Open table in a new tab The K+ concentration dependences of the initial rates of fura-2 ratio change in HEK293 cells expressing recombinant NCKX2, -3, and -4 were determined in buffers containing 0.1 mm Ca2+ and varying concentrations of K+ (Fig. 3). In Fig. 3 (A and B) representative traces and the respective portions of each pulse corresponding to the initial rates of fura-2 ratio change are shown. For all the subsequent figures, only the representative traces are shown. When the average data from four to seven coverslips for the K+ concentration dependences were subjected to nonlinear regression analysis, NCKX2 displayed rates that did not saturate within the range of concentrations tested, whereas NCKX3 and -4 displayed apparent K0.5 values of 1.3 and 1.1 mm, respectively, and Hill slope values of 1.0 and 1.1, respectively, consistent with the presence of a single K+ binding site (Fig. 3C and Table 1). Because the range of K+ concentrations tested did not saturate NCKX2 activity, the data could not be fitted with a high degree of confidence, but an apparent K0.5 of ∼40 mm was obtained when the “Top” parameter (i.e. the maximal rate) was unconstrained in the nonlinear regression analysis. This apparent low affinity for K+ was consistent with our previously reported apparent K0.5 of ∼36 mm for recombinant rat brain NCKX2 obtained using electrophysiology (31.Dong H. Light P.E. French R.J. Lytton J. J. Biol. Chem. 2001; 276: 25919-25928Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). These data indicated that NCKX3 and -4 have ∼40-fold higher apparent affinities for K+ ions during reverse-mode operation than NCKX2. Na+ Inhibition of the Reversemode Activity of NCKX2, -3, and -4−To assess the affinities of the various NCKX isoforms for Na+ ions, we determined the initial rates of fura-2 ratio change in HEK293 cells expressing recombinant NCKX2, -3, and -4 in perfusion buffers containing 10 mm K+, 0.1 mm Ca2+, and varying concentrations of Na+ (Fig. 4A). HEK293 cells transfected with pcDNA3.1+ did not display any notable fura-2 ratio changes in the presence of the various perfusion solutions. The average data from four separate experiments for each isoform was fitted using nonlinear regression analysis and revealed apparent IC50 values of 34, 30, and 18 mm for NCKX2, -3, and -4, respectively (Fig. 4B and Table 1), which were consistent with the value of 60 mm determined for recombinant rat brain NCKX2 in the presence of 40 mm K+ and 1 mm Ca2+ using electrophysiology (31.Dong H. Light P.E. French R.J. Lytton J. J. Biol. Chem. 2001; 276: 25919-25928Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The Hill slope values ranged from 1.6 to 1.8, which was consistent with the presence of multiple classes of Na+ binding sites that are either competitive or allosteric (31.Dong H. Light P.E. French R.J. Lytton J. J. Biol. Chem. 2001; 276: 25919-25928Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 35.Reeves J.P. Sutko J.L. J. Biol. Chem. 1983; 258: 3178-3182Abstract Full Text PDF PubMed Google Scholar). These data suggested that NCKX2, -3, and -4 exhibit similar apparent affinities for Na+ ions under these conditions of assay. Multiple Sequence Alignments and Mutagenesis Strategy−In studies on NCX proteins aimed at identifying the amino acid residues responsible for the differential Ni2+ and Li+ sensitivities of NCX1, -2, and -3, extensive chimeric constructs were generated and tested, revealing that the only regions with any notable effects contained the α-repeat 1 and 2 sequences (36.Iwamoto T. Uehara A. Nakamura T.Y. Imanaga I. Shigekawa M. J. Biol. Chem. 1999; 274: 23094-23102Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Furthermore, extensive mutagenesis efforts on NCKX isoforms strongly suggested that the α-repeats contain the structural motifs responsible for cation binding and translocation (22.Winkfein R.J. Szerencsei R.T. Kinjo T.G. Kang K. Perizzolo M. Eisner L. Schnetkamp P.P. Biochemistry. 2003; 42: 543-552Crossref PubMed Scopus (52) Google Scholar, 23.Kang K.J. Kinjo T.G. Szerencsei R.T. Schnetkamp P.P. J. Biol. Chem. 2005; 280: 6823-6833Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 24.Kang K.J. Shibukawa Y. Szerencsei R.T. Schnetkamp P.P. J. Biol. Chem. 2005; 280: 6834-6839Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Therefore, to identify the molecular determinants responsible for the apparent differences in K+ affinities between NCKX2 and NCKX3 and -4, we performed multiple sequence alignments containing only the α 1 and 2 repeat regions (Fig. 5). Using this alignment, we identified residues that were different between NCKX2 and NCKX3 and -4 and constructed grouped mutations (M1–M6) that covered the entire lengths of the α-repeat regions, in which the selected residues in NCKX2 were converted to the corresponding residues in NCKX4 and vice versa (Table 2). Because NCKX3 and -4 differ by only eight amino acids in the α-repeat regions and the results presented in Table 1 suggested that these two proteins are functionally very similar, NCKX3 was not subjected to any further analyses. Immunoblotting of postnuclear extracts from HEK293 cells expressing either wild-type or mutant NCKX2 and -4 constructs using anti-FLAG monoclonal antibodies confirmed that all of the generated constructs were expressed at roughly similar levels (data not shown).TABLE 2NCKX2 mutant constructs and their reciprocal NCKX4 counterpartsMutant constructMutations forNCKX2NCKX4M1G184SS132GL193VV141LA199TT147AS201GG149SN202DD150NI205VV153IM2F218CC166FV219II167VM222VV170MA224GG172AS227AA175SM3V524II442VE526YY444EI528LL446IS531PP449SE532DD450EE533VV451EL537II455LI539FF457IM4I546VV464IL549CC467LI550MM468IT551AA469TV553LL471VK558QQ476KM5S567NN485SV569II487VI573VV491IM6T577LL495TP581GG499PL585GG503LY587QQ505Y Open table in a new tab K+ Affinity Assays for the NCKX2 and -4 Mutant Constructs−To determine if the introduced mutations resulte" @default.
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• W1990666241 date "2007-02-01" @default.
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• W1990666241 title "Analysis of Ion Interactions with the K+ -dependent Na+/Ca+ Exchangers NCKX2, NCKX3, and NCKX4" @default.
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• W1990666241 doi "https://doi.org/10.1074/jbc.m610582200" @default.
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