Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Cytokinesis wikipedia , lookup
Cell membrane wikipedia , lookup
Magnesium transporter wikipedia , lookup
Organ-on-a-chip wikipedia , lookup
Protein phosphorylation wikipedia , lookup
Cell encapsulation wikipedia , lookup
Endomembrane system wikipedia , lookup
Western blot wikipedia , lookup
Abstract Cytosolic Ca2+ plays an important role in intracellular signaling. Na+/Ca2+ exchanger (NCX) is a plasma membrane protein and involved in cellular Ca2+ homeostasis especially in cells that require influx of Ca2+ for their physiological activities such as cardiac myocytes and neurons. NCX contains 9 transmembrane segments and a large intracellular loop (NCXIL) between membrane segments 5 and 6. The intracellular loop is an important region for regulation of NCX activity. The NCX activity is known to be regulated by Ca2+, Na+, MgATP, PKA and other modulators. Recently, ion channels such as L-type Ca2+ channel and K+ channel are found to form large complexes which are composed of kinases and phosphatases. To explore molecules which interact with NCX, we used yeast two-hybrid method to search for proteins that interact with NCXIL. The sarcomeric mitochondrial creatine kinase (CKMt2) was found to be a candidate molecule that interacts with NCXIL. CKMt2 converts ATP to phosphocreatine and its expression is restricted to cardiac and skeletal muscle. Two important phosphagens, phosphocreatine in vertebrates and phosphoarginine in invertebrates, as well as MgATP are intracellular high energy compounds. It has been reported that phosphoarginine accelerates Na+/Ca2+ exchange cycle in squid axons. There are four creatine kinase isoenzymes, CKMt1, CKMt2, CKM, and CKB. In this study, I proposed experiments to study the physiological significance of the interaction between NCX and different CK isoenzymes in mammalian cells and the mechanism involved in the regulation of NCX activity by CK isoenzymes. 1 Background 1. The role of intracellular Ca2+ Extracellular Ca2+ import through plasma membrane occurs by various types of channels including voltage-sensitive, receptor-operated (for example, glutamate receptors) and store-operated channels. The entering Ca2+ can either interact with Ca2+-binding protein or uptake into the endoplasmic reticulum (ER) or mitochondria. Calcium is known to be involved in numerous cellular processes including apoptosis (Orrenius et al., 2003a), fertilization (Stricker, 1999), exocytosis (Rettig and Neher, 2002), and the regulation of protein/protein interaction. It is known to be essential for all living cells to keep low free intracellular calcium ([Ca2+]i) levels. The concentration of free intracellular calcium, [Ca2+]i, fluctuates between 100 nM and M level (Tsien, 1981) in the neurons, cardiomyocytes, skeletal and smooth muscle cells, whereas the extracellular free calcium concentration is at mM level (Orrenius et al., 2003b). There are two sources of Ca2+ signal which induces cardiac muscle contraction. One is voltage-dependent Ca2+ channel on cardiac sarcolemma, and the other is ryanodine receptor of the sarcoplasmic reticulum through a Ca2+-induced Ca2+ release (CICR) process. Furthermore, norepinephrine binds to adrenergic receptors in the sarcolemmal membrane and stimulates adenylyl cyclase, which produces cAMP and activates cAMP-dependent protein kinase (PKA). PKA phosphorylates the sarcolemmal L-type Ca2+ channel which can induce the contractile state of the heart. The initial Ca2+ influx can also induce a delayed Ca2+ increase; approximately 20% of the total Ca2+ inducing contraction comes from extracellular sources and 80% of that is released from the sarcoplasmic reticulum (Delbridge et al., 1996). 2. The role of NCX under physiological condition There are two transport systems extrude cytoplasmic calcium out of cell and maintain the steep calcium gradient across the membrane, the sodium/calcium 2 exchanger (NCX) and calcium pump (PMCA, plasma membrane calcium/ calmodulin dependent calcium pump). PMCA has high affinity but low transport capacity of Ca2+, whereas NCX has a low affinity, but higher capacity for Ca2+ transport (Carafoli et al., 2001). The cardiac NCX has been studied extensively and has been shown to transport 10 to 15 times more calcium than the PMCA. Therefore, NCX plays a major role in calcium transport, and has an important function in excitation-contraction coupling to bring intracellular calcium back to resting levels during relaxation. 3. The role of NCX under pathological condition Protons accumulating during ischemia are extruded in exchange for sodium ions. The resulting sodium overload cannot be adequately handled by the sodium/potassium pump because it is inefficient due to ischemia-induced shortage of energy. This excess of intracellular sodium is then extruded from cells through operation of the reverse mode of sodium/calcium exchanger. It brings calcium ions in the cells allowing a dangerous calcium overload, which result in the ischemia tissue injury. 4. The genes of mammalian NCX The Na+/Ca2+ exchanger is an ubiquitously expressed membrane protein and plays an important role in cellular Ca2+ homeostasis. Three mammalian genes, NCX1, NCX2 and NCX3, have been found and cloned. NCX1 is expressed at high levels in the heart but is observed in most other tissues in varying amounts (Quednau et al., 1997). NCX2 and NCX3 are expressed primarily in brain and skeletal muscle (Li et al., 1994). 5. The structure of NCX1 The full-length of mature cardiac Na+/Ca2+ exchanger has 938 amino acids (Fig.1). It is modeled to contain 9 transmembrane segments; the fifth and sixth transmembrane segments were separated by a large intracellular loop about 550 amino acids (Philipson and Nicoll, 2000). Result from previous deletion experiments (Matsuoka et al., 1993) indicate that the large intracellular loop of NCX (NCXIL) is not essential for ion transport but is important for regulation of the exchanger activity. On the other hand, the transmembrane segments are 3 believed to participate in ion binding and translocation. 6. The direction of ion transport of NCX The NCX counter-transports three Na+ for one Ca2+ and requires the energy of the Na+ gradient produced by the Na+ pump. The NCX moves Ca2+ and is driven by the Na+ gradient, the Ca2+ gradient, or the membrane potential. Under physiological conditions, the exchanger serves well as a Ca2+ efflux mechanism. However, the direction of ion exchange can be reversed in response to an increase in internal Na+ or upon membrane depolarization. 7. The regulation of NCX Plasma membrane NCX is regulated by phosphorylation and by Na+, Ca2+, ATP, and phosphatidyl-inositol-diphosphate (PIP2). Several structure-function studies have elucidated the protein structure involved in the regulation of the exchanger activity, including Cai-regulatory site, Nai-dependent inactivation, and phosphorylation sites. A high-affinity Ca2+-binding site on the NCXIL was identified (Iwamoto et al., 2000), and intracellular regulatory Ca2+ interacts with this site to activate the exchanger. When Na+ binds to transport sites at the intracellular surface of the exchanger, NCX enters the inactive state (Hilgemann et al., 1992). The exchanger maybe directly regulated by PIP2 through its binding to a positively charged cytoplasmic regulatory domain on NCXIL (Hilgemann, 1997). The XIP, a N-terminal segment of the large intracellular loop, was reported to play an autoregulatory role in exchanger function. Synthesized peptide with XIP sequence showed that it inhibits NCX (Li et al., 1991). An endogenous XIP region is involved in the Na+-dependent inactivation of NCX (Matsuoka et al., 1997). In the case of NCX, ATP is unlikely to modulate NCX by directly binding. The nucleotide-binding sites such as the Walker motifs are not present in the NCX isoforms from different tissues and species (Iwata et al., 1996). In the presence of vanadate to inhibit the Ca2+ pump, MgATP causes a large activation of NCX in the Ca2+ efflux. Chromium-ATP (CrATP), an ATP analog, reverses the activation completely. In the presence of CrATP, MgATP is ineffective in activating the NCX in the dialysed squid axons. Application of CrATP in absence of MgATP does not stimulate NCX (DiPolo and Beauge, 1993) CrATP binds tightly to the substrate site of most kinases in competing with MgATP. In 4 addition to displacing MgATP, CrATP stops the phosphoryl transfer reaction. The result suggest that when CrATP stops the phosphoryl transfer reaction of protein kinases, MgATP can not act on the NCX to modulate the exchanger activity. Evidently, MgATP does not have an allosteric effect caused by the binding of the nucleotide to the exchanger, but it regulates the NCX through phosphorylation. Several possible mechanisms have been proposed (DiPolo and Beauge, 1999). First, MgATP binds to the protein kinase near the exchanger. The phosphate group transfers from MgATP by the kinase to the exchanger. The activity of NCX exchanges because of the phosphorylation. Second, ATP involves in the synthesis of PIPs from the phosphorylation cascade of phosphatidylinositol (PI) by endogenous lipids kinases, phosphoinoditide 4-kinase and phosphoinositide 4-phosphate 5 kinase. The PIP2 directly activates the exchanger by binding to a positive charged cytoplasmic domain on the exchanger (Hilgemann, 1997). Third, protein kinase transfer the phosphate group of the MgATP to a regulatory protein, and phosphorylated regulatory protein interacts with NCX to alter the exchanger activity. This model is developed based on a finding that a novel 13 kDa cytoplasmic soluble protein is required for the MgATP-dependent modulation of NCX in squid axons (DiPolo et al., 1997). 5 Significance Reversible phosphorylation of proteins is widely considered as an important mechanism for the control of many cellular processes. PKC has been shown to be involved in the phosphorylation-dephosphorylation of NCX. However, regulation of NCX activity by PKC remained controversial: in most cases, PKC up-regulates the NCX. For instance, PKC up-regulates NCX activity in rat aortic smooth muscle cells (Iwamoto et al., 1995), rat neonatal cardiomyocytes (Iwamoto et al., 1996), and rat hepatocytes (Ikari et al., 1998). But PKC down-regulates the NCX activity in other cell types, such as bovine chromaffin cells (Tokumura et al., 1998). Recently, macromolecular complexes have been found to regulate specific K+ channels (Marx et al., 2002) and cardiac ryanodine receptors (RyR2). In general, these complexes are composed of kinases, phosphatases, and kinase anchoring proteins. It is possible that some unfound factors participate in the regulation of NCX activity in different tissues. To search for the proteins that interact with NCX1, yeast two-hybrid screening of a human heart cDNA library was carried out by using the segment of the large cytoplasmic loop of bovine heart NCX1IL as bait. The sarcomeric mitochondrial creatine kinase (CKMt2) was found to be a candidate molecule that interacts with NCX1IL (fig.2). The shortest region of CKMt2 mRNA that contributed to the interaction between CKMt2 and NCX1IL was from 991 bp to 1458 bp, corresponding to amino acids 265-420 (Fig. 3). Specific Aims 1. To confirm the interaction between NCX1 and CKMt2 2. To examine the interaction and characterize between NCX1 and various CK isoenzymes 3. To study the physiological significance of the interaction 4. To study the mechanism involved in the creatine kinase isozymes recovery the NCX activity 5. To study the regulation of NCX1 activity in ischemic mouse model 6 Experimental Approaches Specific aim 1: To confirm the interaction between NCX1 and CKMt2 Rationale: Because the interaction between c-terminal CKMt2 and NCX1IL was found in yeast system, this interaction needed to be further examined. Proposed experiments: The direct interaction between CKMt2 and NCXIL was examined by the GST pull down assay. The results were examined by Western blot analysis. Our preliminary results show that CKMt2 could interact with NCXIL in vitro (Fig.4). Specific aim 2: To examine the interaction between NCX1 and various CK isozymes Rationale: NCX1 is a plasma membrane protein and CKMt2 was known to be expressed in intermembrane space of mitochondria. There are four creatine kinase isozymes, cytoplasmic muscle (CKM), cytoplasmic brain (CKB), ubiquitous mitochondrial (CKMt1), and sarcomeric mitochondrial (CKMt2). It is possible that NCX1 could interact with other creatine kinase isozymes. Proposed experiments: NCX1 and creatine kinase isozymes were coexpressed in HEK293T cells. The interactions between NCX1 and various CK isozymes were analyzed by coimmunoprecipitation and Western blot analysis. Our preliminary results show that in addition to CKMt2, NCX1 also interacted with CKM (fig.5) Specific aim 3: To study the physiological significance of the interaction Rationale: MgATP stimulation of the reversal exchange mode was demonstrated in guinea pig, rabbit, and mouse myocytes. ATP depletion could inhibit reverse mode of NCX activity(Condrescu et al., 1995). It was known that ATP concentration decreased during ischemia(Cave et al., 2000). However, reverse-mode NCX still have function and contribute to cell death during ischemia. It is possible that CKs may migrate to plasma membrane to interact with NCX1 and support NCX1 activity under energy depleted condition. Proposed experiments: (1) To examine the NCX1 activity in NCX1 and CKs coexpressed HEK293T cells under energy depleted condition. NCX1 and various CKs were coexpressed in HEK293T cells. After energy depletion, the NCX1 activity was examined in single cells. Our preliminary results showed that CKM and CKMt2 could recover 7 the lost NCX1 activity under energy depleted condition (Fig.6). (2) To examine the subcellular localization of NCX1 and CKs in coexpressed HEK293T cells under energy depleted condition. NCX1 and various CKs were coexpressed in HEK293T cells. After energy depletion, the subcellulr localization of NCX1 and CKs were visualized by immunocytochemistry and confocal microscopy. Our preliminary results showed CKM could recruit to plasma membrane and colocalized with NCX1 (Fig.7). (3) To examine the interaction between NCX1 and CKs under energy depleted condition. NCX1 and various CKs were coexpressed in HEK293T cells. After energy depletion, the interaction between NCX1 and CKs will be detected by co-immunoprecipitation. Specific aim 4: To study the mechanism involved in the creatine kinase isozymes recovered the NCX activity Rational: According to our preliminary results, CKM could recruit to plasma membrane and maintain NCX1 activity under energy depleted condition. It must be identified that CKM support NCX1 activity only through its binding to NCX1 or further through the produced ATP. The mutant CKM protein which can not produce ATP will be used to answer this question. Proposed experiments: A negatively charged cluster (Glu226, Glu227, Asp228) is in the active site of CKMt2 and is critical for enzymatic activity(Eder et al., 2000). Mutant CKM (E231Q, E232Q, and D233N) will be generated by mutagenesis. NCX1 and mutant CKM will be coexpressed in HEK293T cells. After energy depletion, the NCX1 activity will be examined in single cells. Specific aim 5: To study the role of CKM in regulation of NCX1 activity in ischemic mouse model Rational: Our preliminary results showed CKM could recruit to plasma membrane and maintain NCX1 activity under energy depleted condition. During ischemia, the ATP concentration was decreased. Therefore, the role of CKM in regulation of NCX1 activity in cardiomyocytes will be examined in ischemic mouse model. Proposed experiments: (1) In the mouse model of heart ischemia, the subcellular localization of CKM and NCX1 in cardiomyocytes will be examined by immunohistochmeistry and confocal microscopy. (2) In the mouse model of heart ischemia, the interaction between NCX1 and CKM 8 will be examined by co-immunoprecipitation. (3) In the mouse model of heart ischemia, the NCX1 activity will be examined in cardiomyocytes in ischemic normal mouse or CKM deficient mouse(Abraham et al., 2002). 9 Methods Yeast two-hybrid screening Yeast two-hybrid method was employed to search for NCX1IL-interacting (Vojtek and Hollenberg, 1995). Yeast two-hybrid screening was performed using the human heart cDNA library in pACT2 as the prey and plasmid pBTM116-NCX1IL as the bait. To generate pBTM116-NCX1IL, DNA sequence that encode amino acids 218-737 of bovine cardiac NCX1 intracellular loop (NCX1IL) were amplified by PCR. The pBTM116-NCX1IL and pACT2 was sequentially transformed into yeast strain L40 by high efficiency method (Gietz et al., 1995). Approximately 5109 independent cDNA clones were screened, and clones that tested positive in the screen were sequenced. X-gal agarose overlay assay To confirm the clones selected from nutrient-deficient plates were proteins that interact with NCX1IL, six positive colonies of yeasts, which containing pACT2-CKMt2 inserts and pBTM116-NCX1IL, were grown up on Trp-/Leu-/Ura-/Lys- (–KLUT) and Trp-/Leu-/Ura-/Lys-/His- (–KLUTH) plates at 30C for three days. The surface of each plate was covered with the warm X-gal assay solution (0.5 M potassium phosphate buffer, pH7.0, 6% dimethyl formamide (DMF), 5 mg/ml agarose, 0.1% SDS, 0.4 g/ml X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) and 0.5% -mercaptoethanol). The DMF and SDS permeabilize the cells and the buffer containing -mercaptoethanol to maintain the -galactosidase activity. After the agar was cooled and solidified, the plates were incubated at 30C. The reaction was developed for various times until blue color can be clearly detected. Cell Cultures HEK293T cells were maintained in DMEM with 10% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2.The myocardial ischemia induced by coronary artery ligation. Adult cardiac myocytes were enzymatically dissociated using a Langendorf perfusion-based technique (Seckin et al., 2001). The heart was sequentially perfused with Dulbecco minimum essential medium (Joklik modification with 10 mM KCl and 10 mM HEPES; Sigma Chemicals, St. Louis, MO) containing 1 mM Ca2+ and 80 mg/ml Type I collagenase (Worthington Chemicals, Lakewood, NJ) with 1% bovine serum albumin (Sigma) in 0 Ca2+. During collagenase perfusion, Ca2+ was added back in graded steps to 1 mM. The tissue was gently triturated to obtain single myocytes, which were finally washed in 10 Joklik medium with 1% albumin and 1 mM Ca2+. Immunoblotting Proteins were solubilized in sample buffer containing 5% 2-mercaptoethanol and were separated on 7.5 or 10 % polyacrylamide minigels. For immunoblotting, proteins were then transferred electrophoretically to a polyvinylidene difluoride membrane. The membrane were blocked in 1% skim milk and 1% BSA in PBST (136mM NaCl, 1.76 mM KH2PO4, 2.68 mM KCl, and 8 mM Na2HPO4-2H2O, pH 7.6, with 0.1% Tween 20) for 1 hr at room temperature and then incubated for 1 hr with appropriate primary antibody overnight at 4C. Following washing 10 min at RT for 3 times, membrane were incubated with secondary antibodies to rabbit IgG or mouse IgG conjugated to horseradish peroxidase, for 1 h at room temperature. To detect bound antibody, an enhanced chemiluminescence detection system (ECL; Amersham Biosciences) was used. GST pull-down assays Equal molecules of GST or GST-CKMt2 fusion proteins immobilized on the glutathione-agarose beads were separately incubated with 26 g of soluble His-tagged NCX1IL in GST pull-down buffer at 4C for 2 days with gentle end -over-end rotation by a wheel rotator. After washing 9 times with GST pull-down buffer, bound proteins were eluted by glutathione buffer (10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0), analyzed by western blotting using mouse antiserum against NCX1IL in blocking solution. Co-immunopricipitation Monoclonal antibody (mAb; 3 g) or poly clonal antibodies (pAb; 6 g) were incubated with 30 l protein A/G beads (Pierce) for 1 hr at 4C. The protein A/G beads (Pierce) were washed three times with 1 ml RIPA buffer (20 mM of Hepes-NaOH, pH7.8, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 50 mM NaF, 1 mM DTT, and 1 protease inhibitors (Roche)) at 4C. Proteins immunopricipitated from 3 mg of detergent-extracted total protein by incubation overnight at 4C with antibody-bound beads were then analyzed by Western blot.. Heart from 8 weeks Sprague-Dawley rats (females) were minced in RIPA buffer, then samples were homogenized with a Polytron homogenizer at 4C. The supernatant was used for immunoprecipitaton, The protein concentration was detected with the protein assay (Bio-Rad) and bovine serum albumin was used as a standard. 11 Immunocytochemstry HEK293T cells or primary cultured rat cardiac myocytes were washed with PBS 3 times and fixed for 40 min in 3.7% formaldehyde. The fixed cells on the coverslips were rinsed 5 times with PBS, permeabilized with 0.1 % Triton X-100 in PBS. After blocking nonspecific binding with 0.5 % bovine serum albumin in PBS for 1 h, the cells were incubated with primary antibodies overnight at 4C. The cells were then washed 3 times with PBS and incubated for 1 h at room temperature with fluorescence labeled secondary antibodies. The cells were then washed again in PBS, and the coverslips were mounted with Vectashield (Vector Laboratories, Burlingame, CA). Images were captured using an Olympus FluoView ™ IX70 confocal microscope. Immunohistochemistry The method of immuofluorescence of sections from ventricle was used as described previously(Walzel et al., 2002). Freshly excised rat ventricles were fixed for 3 h at room temperature in PBS containing 3% paraformaldehyde. Tissues were dehydrated and embedded in paraffin by standard techniques. Ten-m slices were cut with a microtome, paraffin was removed with xylene, and sections were washed with 70% ethanol and stored in PBS. For immunofluorescence staining, tissue sections were permeabilized first with 0.2% Triton X-100 for 15 min, then with 0.1% SDS for 30 s and subsequently washed in PBS for 30 min. The sections were blocked in 5% goat serum albumin and 1% bovine serum albumin in PBS. Primary antibodies (rabbit anti-NCXIL antibody 1:500, mouse anti-CKM (COX, Molecular Probes) 1:200, both diluted in PBS containing 2% fat-free dry milk powder), were incubated at 4 °C overnight. Subsequently, the tissue sections were washed extensively six times. Secondary antibodies (fluorescein isothiocyanate-conjugated mouse-anti-rabbit IgG 1:500 and rhodamine-conjugated goat-anti-mouse IgG, both diluted 1:500 in PBS), were incubated 1 h at room temperature in the dark. The stained sections were washed again extensively in PBS. Images were captured using an Olympus FluoView ™ IX70 confocal microscope. Mutagenesis In vitro site-directed mutagenesis is performed by overlap extension (four-primer) PCR. The 5' region of mutant CKM was generated by using the forward primer1, 5'-GCAAGAATTC ATGCCATTCGGTAACACC-3', and the reverse primer1, 5'-GAGGTGATTCTGCTGGTT -3'. The 3' region of mutant CKM will be generated by using forward primer2, 5'-AACCAGCAGAATCACCTC -3', and the reverse primer2, 5'-TTGCGCGGCCGCCTTCTGGGCGGGGATCAT -3'. Following mutagenesis reactions, the cassette DNA will be subcloned into the pEF-FLAG vector. 12 Measurement of reverse mode NCX activity HEK293T cells were cultured on 10 mm coverslips. pEF1-GFP-Myc and pEF-NCX-FLAG were co-transfected with pEF-Myc, pEF-CKMt1-Myc, pEF-CKMt2-Myc, pEF-CKM-Myc, and pEF-CKB-Myc into HEK293T cells with Lipofectamin Plus. Transfected HEK293T cells were treated with 5g/ml oligomycin and 2mM 2-deoxyglucose for 10 min at 37C. After wash three times, the cells were loaded with 5M fura-2 AM by incubation for 30 min at 37C in loading buffer (145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, 10 mM glucose). Dye-loaded cells were washed with loading buffer for 3 times and then incubated with Ca2+ -free buffer (145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, 0.1 mM ouabain, 10 M monensin and 10 M nifedipine) for 10 min at 37C. Single cell was puffed with puff buffer (145 mM NMG, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, 10 mM glucose, 0.1 mM ouabain, 10 M monensin and 10 M nifedipine). Microfluorometry was performed with a 40 oil immersion objective (numerical aperture of 1.35; UAPO 40 oil/340; Olympus Optical) and a photodiode (T.I.L.L. Photonics). 13 Reference 1. Abraham MR, Selivanov VA, Hodgson DM, Pucar D, Zingman LV, Wieringa B, Dzeja PP, Alekseev AE, Terzic A (2002) Coupling of cell energetics with membrane metabolic sensing. Integrative signaling through creatine kinase phosphotransfer disrupted by M-CK gene knock-out. J Biol Chem 277: 24427-24434. 2. Carafoli E, Santella L, Branca D, Brini M (2001) Generation, control, and processing of cellular calcium signals. Crit Rev Biochem Mol Biol 36: 107-260. 3. Cave AC, Ingwall JS, Friedrich J, Liao R, Saupe KW, Apstein CS, Eberli FR (2000) ATP synthesis during low-flow ischemia: influence of increased glycolytic substrate. Circulation 101: 2090-2096. 4. Condrescu M, Gardner JP, Chernaya G, Aceto JF, Kroupis C, Reeves JP (1995) ATP-dependent regulation of sodium-calcium exchange in Chinese hamster ovary cells transfected with the bovine cardiac sodium-calcium exchanger. J Biol Chem 270: 9137-9146. 5. Delbridge LM, Bassani JW, Bers DM (1996) Steady-state twitch Ca2+ fluxes and cytosolic Ca2+ buffering in rabbit ventricular myocytes. Am J Physiol 270: C192-C199. 6. DiPolo R, Beauge L (1993) Effects of some metal-ATP complexes on Na+-Ca2+ exchange in internally dialysed squid axons. J Physiol 462: 71-86. 7. DiPolo R, Beauge L (1999) Metabolic pathways in the regulation of invertebrate and vertebrate Na+/Ca2+ exchange. Biochim Biophys Acta 1422: 57-71. 8. DiPolo R, Berberian G, Delgado D, Rojas H, Beauge L (1997) A novel 13 kDa cytoplasmic soluble protein is required for the nucleotide (MgATP) modulation of the Na+/Ca2+ exchange in squid nerve fibers. FEBS Lett 401: 6-10. 9. Eder M, Stolz M, Wallimann T, Schlattner U (2000) A conserved negatively charged cluster in the active site of creatine kinase is critical for enzymatic activity. J Biol Chem 275: 27094-27099. 10. Gietz RD, Schiestl RH, Willems AR, Woods RA (1995) Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11: 355-360. 11. Hilgemann DW (1997) Cytoplasmic ATP-dependent regulation of ion 14 transporters and channels: mechanisms and messengers. Annu Rev Physiol 59: 193-220. 12. Hilgemann DW, Matsuoka S, Nagel GA, Collins A (1992) Steady-state and dynamic properties of cardiac sodium-calcium exchange. Sodium-dependent inactivation. J Gen Physiol 100: 905-932. 13. Ikari A, Sakai H, Takeguchi N (1998) Protein kinase C-mediated up-regulation of Na+/Ca2+-exchanger in rat hepatocytes determined by a new Na+/Ca2+-exchanger inhibitor, KB-R7943. Eur J Pharmacol 360: 91-98. 14. Iwamoto T, Pan Y, Wakabayashi S, Imagawa T, Yamanaka HI, Shigekawa M (1996) Phosphorylation-dependent regulation of cardiac Na+/Ca2+ exchanger via protein kinase C. J Biol Chem 271: 13609-13615. 15. Iwamoto T, Uehara A, Imanaga I, Shigekawa M (2000) The Na+/Ca2+ exchanger NCX1 has oppositely oriented reentrant loop domains that contain conserved aspartic acids whose mutation alters its apparent Ca2+ affinity. J Biol Chem 275: 38571-38580. 16. Iwamoto T, Wakabayashi S, Shigekawa M (1995) Growth factor-induced phosphorylation and activation of aortic smooth muscle Na+/Ca2+ exchanger. J Biol Chem 270: 8996-9001. 17. Iwata T, Kraev A, Guerini D, Carafoli E (1996) A new splicing variant in the frog heart sarcolemmal Na-Ca exchanger creates a putative ATP-binding site. Ann N Y Acad Sci 779: 37-45. 18. Li Z, Matsuoka S, Hryshko LV, Nicoll DA, Bersohn MM, Burke EP, Lifton RP, Philipson KD (1994) Cloning of the NCX2 isoform of the plasma membrane Na+-Ca2+ exchanger. J Biol Chem 269: 17434-17439. 19. Li Z, Nicoll DA, Collins A, Hilgemann DW, Filoteo AG, Penniston JT, Weiss JN, Tomich JM, Philipson KD (1991) Identification of a peptide inhibitor of the cardiac sarcolemmal Na+-Ca2+ exchanger. J Biol Chem 266: 1014-1020. 20. Marx SO, Kurokawa J, Reiken S, Motoike H, D'Armiento J, Marks AR, Kass RS (2002) Requirement of a macromolecular signaling complex for beta adrenergic receptor modulation of the KCNQ1-KCNE1 potassium channel. Science 295: 496-499. 21. Matsuoka S, Nicoll DA, He Z, Philipson KD (1997) Regulation of cardiac Na+-Ca2+ exchanger by the endogenous XIP region. J Gen Physiol 109: 273-286. 15 22. Matsuoka S, Nicoll DA, Reilly RF, Hilgemann DW, Philipson KD (1993) Initial localization of regulatory regions of the cardiac sarcolemmal Na+-Ca2+ exchanger. Proc Natl Acad Sci U S A 90: 3870-3874. 23. Orrenius S, Zhivotovsky B, Nicotera P (2003b) Regulation of cell death: the calcium-apoptosis link. Nat Rev Mol Cell Biol 4: 552-565. 24. Orrenius S, Zhivotovsky B, Nicotera P (2003a) Regulation of cell death: the calcium-apoptosis link. Nat Rev Mol Cell Biol 4: 552-565. 25. Philipson KD, Nicoll DA (2000) Sodium-calcium exchange: a molecular perspective. Annu Rev Physiol 62: 111-133. 26. Quednau BD, Nicoll DA, Philipson KD (1997) Tissue specificity and alternative splicing of the Na+/Ca2+ exchanger isoforms NCX1, NCX2, and NCX3 in rat. Am J Physiol 272: C1250-C1261. 27. Rettig J, Neher E (2002) Emerging roles of presynaptic proteins in Ca2+-triggered exocytosis. Science 298: 781-785. 28. Seckin I, Sieck GC, Prakash YS (2001) Volatile anaesthetic effects on Na+-Ca2+ exchange in rat cardiac myocytes. J Physiol 532: 91-104. 29. Stricker SA (1999) Comparative biology of calcium signaling during fertilization and egg activation in animals. Dev Biol 211: 157-176. 30. Tokumura A, Okuno M, Fukuzawa K, Houchi H, Tsuchiya K, Oka M (1998) Positive and negative controls by protein kinases of sodium-dependent Ca2+ efflux from cultured bovine adrenal chromaffin cells stimulated by lysophosphatidic acid. Biochim Biophys Acta 1389: 67-75. 31. Tsien RY (1981) A non-disruptive technique for loading calcium buffers and indicators into cells. Nature 290: 527-528. 32. Vojtek AB, Hollenberg SM (1995) Ras-Raf interaction: two-hybrid analysis. Methods Enzymol 255: 331-342. 33. Walzel B, Speer O, Zanolla E, Eriksson O, Bernardi P, Wallimann T (2002) Novel mitochondrial creatine transport activity. Implications for intracellular creatine compartments and bioenergetics. J Biol Chem 277: 37503-37511. 16 Preliminary results Fig. 1 Topological model of Na+-Ca2+ exchanger The full-length mature cardiac Na+-Ca2+ exchanger (NCX1) is 938 amino acids long. NCX1 is modeled to contain 9 transmembrane segments (TMSs) and its N terminus is glycosylated at position 9. The TMSs form two clusters separated by a large intracellular loop between TMS 5 and 6. The TMSs catalyze the ion translocation reaction and the large intracellular loop is not essential for ion transport from deletion experiment (Matsuoka et al., 1993). 17 Fig. 2 Sarcomeric mitochondrial creatine kinase (CKMt2) was found to be a candidate molecule that interacts with NCX1IL To examine the clones selected from nutrient-deficient plates contained interacting proteins, six positive colonies of yeast containing pACT2-CKMt2 inserts and pBTM116-NCXIL were grown on –KLUT and –KLUTH plates at 30C for three days. The control yeast colonies containing pACT2-CKMt2 inserts and pBTM116 were grown next to the positive colonies on the same plate. After three-day incubation, the surface of each plate was covered with warm X-gal solution. After the agar became cooled and solidified, the plates were incubated at 30C. The reaction was developed from few hours to one day until blue color can be clearly detected. 18 Fig. 3 The 991~1458 bp is the shortest region of CKMt2 needed to interact with NCXIL The CKMt2 sequences of 18 positive clones compared with the human CKMt2 mRNA (NM_001825) were shown. The shortest region of CKMt2 that contributed to the interaction between CKMt2 and NCXIL was from 991 bp to 1458 bp. The six positive clones, number 1, 2, 3, 4, 17, and 18, were used in X-gal assay. 19 Fig. 4 The interaction of GST-CKMt2 and His-tagged NCXIL was analyzed by GST pull-down assay His-tagged NCX1IL and GST-tagged CKMT2 fusion proteins were purified from E.coli. The two purified proteins were mixed and incubated with glutathione-agarose beads to pull down GST-tagged CKMt2. The western blot analysis detected the His-tagged NCX1IL. No detectable NCX1IL signal was found in control. This result show that the GST-CKMt2 could pull down the His-NCX1IL-His. 20 NCX Con V 1 2 B M kDa NCX 97 45 Fig.5 Interactions CKs between NCX and CKs were analyzed by co-immunoprecipitation pEF-NCX1-FLAG was co-transfected with one of CK isoform plasmids, pEF-CKMt1-Myc, pEF-CKMt2-Myc, pEF-CKM-Myc, or pEF-CKB-Myc into HEK293T cells. To examine the possibility that NCX1 associates with other creatine kinase isoforms, CKMt1, CKMt2, CKM, and CKB were immunoprecipitated, with mouse anti-Myc antibody and the immunoblotting was performed. The results show that NCX1 was detected in the CKMt2 and CKM immuoprecipitates, but not in the CKMt1 and CKB immunoprecipitation under our experimental conditions. 21 (a) Energy depletion Control GFP + NCX + cMyc (DMSO) GFP + NCX + cMyc (Energy depletion) 1.4 1.4 n=7 1.2 1.0 V 0.8 F340/F380 F340/F380 V n=8 1.2 1.0 0.6 0.4 0.8 0.6 0.4 0.2 0.2 0.0 0.0 0 20 40 60 80 100 120 140 160 180 0 20 40 Time (s) 140 160 180 1.4 n=8 1.2 n=8 1.2 1.0 1.0 2 0.8 0.6 F340/F380 F340/F380 120 GFP + NCX + CKMt2 (Energy depletion) 1.4 0.4 0.8 0.6 0.4 0.2 0.2 0.0 0.0 0 20 40 60 80 100 120 140 160 180 0 20 40 Time (Second) 60 80 100 120 140 160 180 160 180 Time (Second) Energy depletion Energy depletion GFP + NCX + CKB (Engery depletion) GFP + NCX + CKM (Energy depletion) 1.4 1.4 1.2 1.2 n=7 1.0 n=7 1.0 M F340/F380 F340/F380 100 Energy depletion GFP + NCX +CKMt1 (Energy depletion) B 80 Time (Second) Energy depletion 1 60 0.8 0.6 0.4 0.8 0.6 0.4 0.2 0.2 0.0 0.0 0 20 40 60 80 100 120 140 160 180 0 Time (Second) 20 40 60 80 100 120 140 Time (Second) (b) Ratio (NA/NT) 0.8 0.6 0.4 0.2 0.0 Vec CKB CKM CKMt1CKMt2 Fig. 6 Reverse mode NCX activity in transfected HEK293T cells In addition to pEF-GFP-Myc and pEF-NCX-FLAG, HEK293T cells transfected with pEF-CKMt2-Myc, pEF-CKMt1-Myc, pEF-CKM-Myc, pEF-CKB-Myc, or pEF-Myc empty vector. Transfected HEK293T cells were treated with oligomycin and 2-deoxyglucose followed by fura-2 AM loading. (a) At the 20 second, GFP expressed single cell was puffed for 30 seconds with Na+ free buffer to initiate reverse mode NCX activity. NT is the total number of cells which were tested the NCX activity. NA is the number of cells which had the response of NCX activity. The NT / NA was shown on the figure.(b) Bar graph indicated the v ratio( NT / NA). Data are meanss.e.m.; P<0.05 for the indicated comparisons. 22 NCX CKMt1 Merge NCX CKMt2 Merge NCX CKB Merge NCX CKM Merge E C E C Fig. 7 Subcellular Localization of four creatine kinase isozymes and NCX overexpressed in HEK293T cells HEK293T cells were transfected with various plasmids, pEF-CKMt1-Myc, pEF-CKMt2-Myc, pEF-CKM-Myc, pEF-CKB-Myc, and pEF-NCX-FLAG. Using mouse anti-Myc antibodies to localize CKM, CKB, CKMt2 and CKMt1. NCX was detected by rabbit anti-NCXIL antibody. E, the cells treated with oligomycin and 2-deoxyglucose that could deplete intracellular energy stores. C, the controlled cells did not be induced the energy depletion. 23 24 25