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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 5109 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
30C 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 30C. 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 4C. 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 4C 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 4C. 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 4C. Proteins
immunopricipitated from 3 mg of detergent-extracted total protein by incubation
overnight at 4C 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 4C. 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 4C. 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 5g/ml
oligomycin and 2mM 2-deoxyglucose for 10 min at 37C. After wash three times,
the cells were loaded with 5M fura-2 AM by incubation for 30 min at 37C 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 37C. 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
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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 30C 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 30C. 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 meanss.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