From the Laboratoire de Pharmacodynamie et de
Thérapeutique, Faculté de Médecine, Bât. GE,
808 route de Lennik, B-1070, Brussels, Belgium and the
¶ Laboratory of Physical Chemistry of Macromolecules at
Interfaces, Faculty of Sciences, Campus Plaine, Boulevard du Triomphe,
Université Libre de Bruxelles, B-1050 Brussels, Belgium
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ABSTRACT |
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The secondary structure of the purified 70-kDa
protein Na+/Ca2+ exchanger, functionally
reconstituted into asolectin lipid vesicles, was examined by Fourier
transform infrared attenuated total reflection spectroscopy. Fourier
transform infrared attenuated total reflection spectroscopy provided
evidence that the protein is composed of 44% The Na+/Ca2+ exchanger plays an important
role in Ca2+ extrusion from myocardial cells and in
Ca2+ homeostasis in cardiac muscle (1-3). Solubilization
and reconstitution of the Na+/Ca2+ exchange
activity has been carried out from different tissues (4-12). After
purification, SDS-polyacrylamide gel electrophoresis showed two major
protein bands at 160- and 120- kDa. Under reducing gel conditions and
following mild chymotrypsin treatment, the intensity of the 160- and
120-kDa band decreased while a band at 70-kDa appeared (10, 12).
Affinity-purified antibodies specific for the 3 polypeptides
cross-reacted with each other, meaning that the three proteins were
immunologically related (10).
The canine and bovine cardiac Na+/Ca2+
exchangers have been cloned and sequenced (13, 14). A hypothetical
membrane topology model was proposed based on hydropathy profile
analysis of the exchanger's amino acid sequence, in which 11 -helices, 25%
-sheets, 16%
-turns, and 15% random structures, notably the
proportion of
-helices is greater than that corresponding to the
transmembrane domains predicted by exchanger hydropathy profile.
Polarized infrared spectroscopy showed that the orientation of helices
is almost perpendicular to the membrane. Tertiary structure modifications, induced by addition of Ca2+, were evaluated
by deuterium/hydrogen exchange kinetic measurements for the
reconstituted exchanger. This approach was previously proven as a
useful tool for detection of tertiary structure modifications induced
by an interaction between a protein and its specific ligand. Deuterium/hydrogen exchange kinetic measurements indicated that, in the
absence of Ca2+, a large fraction of the protein (40%) is
inaccessible to solvent. Addition of Ca2+ increased to 55%
the inaccessibility to solvent, representing a major conformational
change characterized by the shielding of at least 93 amino acids.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical membrane-spanning segments are predicted (5 in the
NH2- and 6 in the COOH-terminal portion), separated by a
large hydrophilic cytoplasmic loop. The NH2 terminus is
extracytoplasmic and the COOH terminus intracytoplasmic (13). Beside
these predictions, the exchanger secondary and tertiary structure are
poorly understood. Topological models describing polypeptide chain
insertion in the membrane are all based on the assumption that
membrane-spanning segments would have an
-helical structure (15,
16). Recently, the transmembrane regions of porins have been described
as containing
-sheets (17). Likewise, the erythrocyte glucose
transporter might fold into membrane
-barrels (18), and the
acetylcholine receptor transmembrane domain might contain
-sheets
instead of the expected
-helices (19, 20). Taken together, these
data indicate that
-helices may not be the only secondary structure
for protein transmembrane segments. Two
Na+/Ca2+ exchange reaction mechanisms are
possible for Na+-Ca2+ countertransport across
the membrane: simultaneous or consecutive kinetics. Strong experimental
evidence favors a consecutive ion-transport scheme for the cardiac
Na+/Ca2+ exchanger (21-23). Nevertheless, a
simultaneous transport mechanism is suggested for the exchanger in
ferret red blood cells (24). Fig. 1
summarizes a consecutive reaction scheme for
Na+/Ca2+ exchange in the forward mode,
characterized by 2 different conformations (E1
and E2) (25). The secondary and tertiary
structure changes implicated in the transition from the
E1 to the E2 conformation of the exchanger have not been investigated yet, and the effect of
ion-binding on induction or stabilization of one of the conformations remains to be elucidated.
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Fig. 1.
Schematic representation of the
Na+/Ca2+ exchanger reaction scheme based on a
consecutive countertransport mechanism in the forward mode. The
exchanger exposes its ion-binding site to the extracellular side
(conformation E1) and releases Ca2+
with concomitant change in conformation to E2,
which binds 3 Na+. The binding site is then exposed to the
cytoplasmic side, Na+ ions are released, and the exchanger
returns to an E1 conformation, that binds
Ca2+, completing the transport cycle (25).
Ca2+i regulation of the Na+/Ca2+ exchange activity was studied in cardiac sarcolemmal vesicles. The cardiac Na+/Ca2+ exchanger has an intracellular regulatory site with high Ca2+ affinity, located between amino acids 445 and 455 (26). Regulation can be summarized as follows: a Ca2+i decrease inactivates Na+-Ca2+ exchange (27-29).
We have previously described the purification and characterization of
the bovine heart 70-kDa exchanger polypeptide reconstituted into
asolectin vesicles (30). The protein has a
Na+/Ca2+ exchange activity and is oriented
inside-out, i.e. with the large hydrophilic loop, between
transmembrane segments, protruding to the outside of the vesicles (30).
In the present study, the secondary structure of the 70-kDa protein is
determined by FTIR-ATR,1
which is a reliable technique to estimate membrane protein secondary structure and orientation in the lipid bilayer (31-34). Tertiary structure modifications were investigated by monitoring
2H/H exchange rate for the reconstituted exchanger in the
absence and presence of Ca2+. FTIR-ATR spectra reveal that
44% of the protein is -helical, 25%
-sheets, 16%
-turns,
and 15% random coils. 2H/H exchange measurements show that
upon Ca2+ addition, a major conformational change occurs,
characterized by a 15% reduction in solvent accessibility of the
70-kDa protein.
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EXPERIMENTAL PROCEDURES |
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Preparation of Bovine Heart Sarcolemmal Vesicles
Highly purified sarcolemmal vesicles were isolated from bovine heart left ventricle as described previously (35, 36) with minor modifications (30).
Purification and Reconstitution of the Na+/Ca2+ Exchanger Protein
We have purified and reconstituted the
Na+/Ca2+ exchanger using a previously published
protocol (10, 30). Bio-Beads SM-2 (Bio-Rad) were used for Triton X-100
removal to favor proteoliposomes formation. When reconstituted proteins
were used for ATR-IR measurements, the Bio-Beads were soaked and washed
in 0.5 mM MOPS solution (pH 7.4, 37 °C) before
incubation with the asolectin/purified protein mixture, at variance
with previously described methods (30). The protein/asolectin
preparation was loaded on top of the Bio-Beads columns and incubated
for 30 min at room temperature. Proteoliposomes were collected by
centrifugation of the columns for 1 min at 1000 × g.
Aliquots were pooled, diluted in 0.5 mM MOPS (pH 7.4, 37 °C) solution, and centrifuged at 140,000 × g for
90 min (4 °C) to pellet the reconstituted protein. This last
centrifugation step was repeated twice to ensure the complete removal
of all ion traces remaining in the supernatant from the
pelleted protein-lipid complex. Finally, proteoliposomes were
resuspended in 50 µl of the same solution and stored at 70 °C
until use.
Na+/Ca2+ Exchange Activity Measurements
Na+/Ca2+ exchange activity in reconstituted proteoliposomes was measured as Nai-dependent 45Cao uptake using two previously described protocols (35, 36). Briefly, in the first protocol, reconstituted vesicles (50 µl) were loaded with Na+ and diluted in Ca2+ uptake medium (140 mM KCl, 0.01 mM CaCl2, 0.3 µCi of 45CaCl2, 0.36 µM valinomycin) to initiate Na+i-dependent 45Ca2+o uptake. The uptake reaction was quenched after 3 s by addition of 30 µl of 140 mM KCl, 10 mM EGTA followed by the addition of 1 ml of ice-cold 140 mM KCl, 1 mM EGTA. Samples were then filtered using 0.22-µm nitrocellulose filters (Sartorius) and filters were washed with 2 × 3 ml of ice-cold 140 mM KCl, 1 mM EGTA (10). In the second protocol, a similar procedure was used except that Ca2+o uptake reaction was stopped after 3 s by addition of 5 ml of ice-cold 140 mM KCl, 0.1 mM EGTA and vesicles were harvested by filtration on Whatmann GF/A filters previously soaked in 0.3% polyethyleneimine in H2O. The filters were then washed with 2 × 5 ml of ice-cold 140 mM KCl, 0.1 mM EGTA, avoiding filter drying between rinses. In both protocols, blanks were obtained by replacing KCl by NaCl in the 45Ca2+ uptake medium. All solutions were buffered with 10 mM MOPS/Tris, pH 7.4.
Infrared Attenuated Total Reflection Spectroscopy
Sample Preparation-- 20 to 50 µl of sample was deposited on one side of the ATR germanium plate and was slowly evaporated, under a stream of nitrogen, yielding a dry thin film of reconstituted protein sample. The internal reflection element (ATR plate) is a germanium plate (50 × 20 × 2 mm, Harrick EJ 2121) with an aperture angle of 45°, yielding 25 internal reflections (37). The ATR plate was then sealed in a universal sample holder (Perkin-Elmer 186-0354).
Secondary Structure Analysis--
The sample on the ATR plate
was deuterated by flushing with a
2H2O-saturated nitrogen stream for at least
2 h. Deuterium/hydrogen (2H/H) exchange allows
differentiation of -helical secondary structures from a random one
(38) by shift of the absorption band of the former from 1,655 to
approximately 1,642 cm
1. Spectra were recorded with a
Perkin-Elmer infrared spectrophotometer FTIR 1720x equipped with a
liquid nitrogen-cooled MCT detector. The nominal resolution was 4 cm
1. The spectrophotometer recording chamber was
continuously flushed with dry air to remove atmospheric water vapor.
Each measurement corresponded to an average of 128 scans. Quantitative
assessment of the protein secondary structure (
-helices,
-sheets,
-turns, and random coils) was carried out using a combination of
resolution enhancement method and a band fitting procedure described
earlier (34, 37, 39-41).
Orientation of the Secondary Structure--
Determination of
peptide orientation by infrared ATR spectroscopy was performed as
described previously (42, 40). Briefly, spectra were recorded with a
parallel and perpendicular polarization with respect to the incidence
plane. Then, the dichroism spectrum was obtained by subtracting the
perpendicular polarized spectrum from the parallel one. A larger
absorbance of the parallel polarization indicates a dipole oriented
preferentially parallel to the normal to the ATR plate; whereas, a
larger absorbance of the perpendicular polarization indicates a dipole
orientation close to the plane of the ATR plate. It has been shown that
in -helices, amide I dipole orientation makes a 27° angle with
respect to the helix axis and that the
-sheet long axis is mainly
perpendicular to the amide C = O axis (43).
Deuteration Kinetics--
Films containing 10-20 µg of
protein were prepared on a germanium plate as described above. Nitrogen
gas was saturated with 2H2O (by bubbling
through a series of three 2H2O-containing
vials) at a flow rate of 90 ml/min (controlled by a Brooks flow-meter).
Bubbling was started at least 1 h before starting the experiment,
i.e. before connecting the tubing carrying the
2H2O-saturated nitrogen flux to the sealed
microchamber containing the sample film. Before starting
2H/H exchange measurements, 10 spectra of nondeuterated
dried sample on ATR plates were recorded to verify measurement
stability and reproducibility of band area computation. At
t = 0, the tubing was connected to the sealed
microchamber containing the studied sample. For each kinetic point, 12 spectra, with a 4 cm1 resolution, were recorded and
averaged. Spectra were recorded every 15 s for the first 2 min.
After which, the recording time interval was exponentially increased.
After 16 min, the time interval between scans was large enough to allow
the insertion of another 2H/H exchange kinetics. Thus, a
second sample, prepared on another ATR setup of the Perkin-Elmer
shuttle was analyzed with the same time sampling (with a 16-min offset)
by connecting the 2H2O-saturated nitrogen flux
in series with the first sample. Accordingly, our software changed the
shuttle position to follow both kinetics (43, 44). In our case, the
first sample was the reconstituted Na+/Ca2+
exchanger in the absence of ligands (see reconstitution protocol), and
the second sample had 0.2 mM CaCl2 added.
The spectrophotometer chamber was flushed with dry air for 20 min
before starting the experiment. However, further removal of traces of
water vapor occurred as the 2H/H exchange kinetics
proceeded, superimposing water vapor's sharp bands onto the protein
spectra (45). Atmospheric water contribution was subtracted as
described (45). Areas of the amide I, II, and II' bands were obtained
by integration between 1,702 and 1,596, 1,596 and 1,502, and 1,492 and
1,412 cm1, respectively. The amide II area was divided by
the corresponding lipid
(C = O) area which allowed us to take
into account small, but significant, variations in the overall spectra
intensity due to sample layer swelling caused by the presence of
2H2O. Indeed, ATR spectrum intensity highly
depends on the distance between sample layer and germanium plate
surface (46); and, as this distance increases by sample swelling, the
spectrum intensity decreases by a few percent for all measured bands.
The 0% deuterated sample spectra were recorded before starting the
2H/H exchange kinetics as described above, and the 100%
deuterated sample value was extrapolated by assuming a zero value for
the amide II band area. Accordingly, throughout the kinetics, the amide
II band area (normalized to the lipid (C = O) area) was expressed between 0 and 100% deuteration for each kinetic point.
Statistics and Calculations
All data are presented as mean ± S.E.
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RESULTS |
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Purification and Reconstitution of the Na+/Ca2+ Exchanger-- The bovine heart exchanger was purified using a combination of ion-exchange and affinity chromatography, and reconstituted into asolectin vesicles. The purification resulted in a 70-kDa protein showing Na+/Ca2+ exchange activity, that was inserted inside-out in the lipid vesicles, i.e. with the large cytoplasmic loop facing the extravesicular milieu. The Na+/Ca2+ exchange activity in control reconstituted sarcolemmal vesicles was 18 ± 1 nmol of Ca2+/mg protein/s at 40 µM Ca2+ and 1155 ± 81 nmol of Ca2+/mg of protein/s at the same Ca2+ concentration for the reconstituted Na+/Ca2+ exchanger, the purification factor being 64. The amount of protein in the control sample was ~1850 µg, while that in the purified sample was ~15 µg (30). The protein content of control reconstituted vesicles comprises all membrane proteins, including the exchanger; whereas, the purified samples contain only the Na+/Ca2+ exchanger protein. The purified 70-kDa polypeptide was characterized in a previous publication (30).
Secondary Structure Analysis of the Reconstituted
Exchanger--
Fig. 2 shows
spectra of purified reconstituted exchanger in the absence of ligands
(Fig. 2A) and in the presence of 0.2 mM Ca2+ (Fig. 2B). The lipid C = O band
maximal absorption was near 1,737 cm1 for ligand-free
(sample A) and Ca2+-containing (sample B) reconstituted
sample. The amide I band had a maximal absorption near 1,650 cm
1 for sample A and sample B (Fig. 2, A and
B, respectively). A quantitative evaluation of the protein
secondary structure was obtained by Fourier deconvolution and
curve-fitting analysis of the amide I region for spectra recorded in
the absence and presence of Ca2+ (42). The curve-fitting of
amide I band showing different structural components (
-helices,
-sheets,
-turns, and random) for sample A and sample B is shown
in Fig. 3, A and B,
respectively. The confirmation of these assignments is discussed by
Goormaghtigh et al. (47). Analysis of the shape of the amide
I absorption band of the reconstituted Na+/Ca2+
exchanger has been carried out as described by Goormaghtigh et al. (37). In the absence of ligands, the
Na+/Ca2+ exchanger's secondary structure was
composed of 44%
-helices, 25%
-sheets, 16%
-turns, and 15%
random. In the presence of 0.2 mM Ca2+, no
significant secondary structure change could be detected (Table
I).
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Orientation of the Na+/Ca2+ Secondary
Structure with Respect to the Lipid Membrane--
The orientation of
the reconstituted exchanger secondary structure was determined from
FTIR-ATR spectra recorded with parallel and perpendicular polarized
incident light, respectively (see "Experimental Procedures" for
details). The dichroism spectrum (Fig. 4)
shows a maximal absorption of about 1,656 cm1 in the
amide I region, characterizing an
-helices axis having a
preferential orientation near perpendicular to the lipid bilayer. No
dichroism could be detected for the
-sheet component in the spectrum.
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Deuterium/Hydrogen Exchange-- In the absence of secondary structure changes upon ligand addition, the rate of 2H/H exchange reveals tertiary structure modifications which follows interaction with Ca2+ (at constant pH and temperature). Kinetics of 2H/H exchange of the reconstituted exchanger was recorded in the absence and presence of Ca2+ to check whether any tertiary structure modifications occurred upon ligand addition.
Peptide N-H group hydrogen exchange was followed by monitoring the
amide II absorption peak decrease ((N-H) maximal absorption at 1,544 cm
1) as a function of
2H2O-saturated N2 flow exposure
time, as illustrated in Figs. 5 and
6. Spectra were recorded during the same
experiment for protein samples with no ligands and with 0.2 mM Ca2+ added (Fig. 5 and 6, respectively). The
amide II area evolution, between 0 and 100% 2H/H exchange,
was computed as described under "Experimental Procedures" and is
reported in Fig. 7. It immediately
appears in Fig. 7 that 2H/H exchange is faster for protein
sample prepared in the absence of ligands than that for sample with 0.2 mM Ca2+ added. Indeed, after 2 h of
deuteration, about 60% of the peptide N-H is exchanged for the former
sample, compared with around 45% for the latter one.
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Hydrogen atoms with different exchange rates are involved in the
exchange process. Considering that the 2H/H exchange rate
is a first-order reaction, the exchange curve can be represented by a
multiexponential decay function taking into account different amide
proton groups (i), each characterized by a time period
(Ti),
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(Eq. 1) |
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DISCUSSION |
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The aim of this work was to study the secondary structure of the purified and reconstituted 70-kDa bovine heart Na+/Ca2+ exchanger, the orientation of protein secondary structure in the lipid vesicle membrane, and possible secondary/tertiary structure changes in the presence of Ca2+ ion. Whereas the protein secondary structure is not significantly modified upon specific ligand addition, amide 2H/H exchange experiments provided information as to solvent accessibility to protein amide group, allowing detection of conformational changes and quantification of the number of amino acid residues involved. Comparison of FTIR-ATR experiments carried out on film with data obtained in solution by FTIR or NMR studies demonstrate the validity of this approach (48).
Recently, we have purified and characterized a unique 70-kDa polypeptide that retained Na+/Ca2+ exchange activity when reconstituted into asolectin vesicles and that corresponded to the NH2 terminus portion of the exchanger (30). In the reconstitution protocol, we used a lipid:protein ratio of ~7:1 to obtain a homogeneous population of reconstituted protein (inserted inside-out), which is essential for such structural studies (30, 40, 41).
Analysis of ATR-FTIR results provides information on the secondary and
tertiary structure of the reconstituted active exchanger. The estimated
secondary structure of the protein was evaluated to be 44%
-helices, 25%
-sheets, 16%
-turns, and 15% random structures (Table I).
Because the 70-kDa polypeptide, studied herein, has only the 5 NH2-terminal membrane-spanning segments compared with the
entire exchanger (30), and assuming that those 5 segments are
-helical, the 70-kDa protein should be constituted of at least 18%
-helices. The proportion of
-helices determined in this study
(44%) is markedly higher. This discrepancy between hydropathy plot
estimations and experimental secondary structure assignment has been
previously observed for other ion transporting membrane proteins (40,
41, 51, 52). A possible explanation for this difference could be that
protein topology models based on hydropathy profile analysis underestimate the proportion of transmembrane domains (assuming those
domains as exclusively
-helical) and/or that ordered
-helical structures are present outside the lipid bilayer. Table
III compares the secondary structure
proportions of the Na+/Ca2+ exchanger with
those of other proteins such as the plasma membrane Ca2+-ATPase (PMCA) (53),
Na+/K+-ATPase (54), P-glycoprotein (41),
H+-ATPase (44), and H+/K+-ATPase
(52) as determined by ATR-FTIR. A very high similarity between the
secondary structure of the 70-kDa polypeptide
Na+/Ca2+ exchanger and that of PMCA can be
observed (92% of similarity between structures, 96% for
-sheet
structures, 81% for
-turn structures, and 93% for random
structures). This high similarity may reflect a homology between the
Na+/Ca2+ exchanger and the PMCA secondary
structure (55), especially at the level of secondary structure
organization of their intracytoplasmic domains. The 70-kDa polypeptide
also shows high similarity with secondary structure proportions of
P-type ion-transporting ATPases. Interestingly, addition of
Ca2+ (0.2 mM) does not alter the
Na+/Ca2+ exchanger secondary structure (Table
I) which is an observation common to P-glycoprotein (41) and P-type
H+-ATPase upon addition of ligands (44).
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Infrared dichroism brings evidence for the presence of -helices with
a transmembrane orientation perpendicular to the lipid bilayer, in
agreement with the currently accepted model of the Na+/Ca2+ exchanger protein (13, 14). The other
secondary structures did not show any significant orientation.
2H/H exchange measurements, in the absence of ligands (0.05 mM MOPS), showed that a considerable proportion of the Na+/Ca2+ exchanger (about 40% of the total amino acid content), characterized by a slow exchange rate (half-decay T3 = 104 s), was inaccessible to the aqueous phase (Fig. 7 and Table II). This lack of accessibility could be partly due to the lipid bilayer shielding effect. Inaccessibility of solvent to membrane-spanning regions of proteins was previously demonstrated for many membrane proteins such as glycoporin, bacteriorhodopsin, P-glycoprotein, and some P-type ATPases (41, 44, 52, 56). In our case, theoretical predictions of transmembrane regions of the 70-kDa polypeptide account for only 18% of total amino acids. Accordingly, two explanations are possible for this difference: more membrane-spanning segments are present than theoretically predicted and/or a considerable proportion of the extramembrane region is organized in a highly structured manner accounting for solvent inaccessibility. Such highly organized domains could represent membrane-associated and/or membrane-embedded regions at the level of the large cytoplasmic loop.
Upon Ca2+ addition (0.2 mM), 2H/H exchange measurements showed a major conformational change characterized by a 15% reduction in solvent accessibility of the 70-kDa polypeptide total amino acids compared with solvent accessibility in the absence of ligands (Fig. 7 and Table II). Accordingly, at least 93 amino acid residues become shielded from the solvent after addition of Ca2+. Interestingly, adding Ca2+ does not alter the secondary structure of the protein; thus, the recorded conformational change is most probably due to tertiary structure changes induced by Ca2+ binding to its regulatory site (amino acids 445-455). Detailed analysis of 2H/H exchange curves indicated that Ca2+ binding to the Na+/Ca2+ exchanger leads to an increase of about 99 amino acid residues essentially of the slow and, to a smaller extent, intermediate exchanging population at the expense of the fast one (Table II). That Ca2+-induced increase of protection of a relatively large number of amino acids can be interpreted in terms of a transient protein folding or "membrane penetration" of a protein portion, resulting in a decreased solvent accessibility. A similar phenomenon of tertiary structure changes upon addition of ligands was observed in a recent study of the purified and reconstituted P-glycoprotein where addition of MgATP (which is hydrolyzed by the protein) induced an increased accessibility to at least 76 amino acid residues. But, addition of MgATP-verapamil lead to the protection of 106 amino acid residues (41). Verapamil is known to be a stimulator of P-glycoprotein MgATP hydrolysis activity (57-60). Similar important tertiary structure modifications were observed for a P-type ATPase upon ligand addition. Indeed, 2H/H exchange measurements on the H+-ATPase shows that it undergoes a major conformational change involving the additional shielding of about 175 amino acid residues (out of 920) when adding MgADP or MgATP-vanadate (vanadate is known to stimulate the H+-ATPase activity) (44, 61).
Ca2+, besides being counter-transported with Na+, is a regulatory ion having its binding site located between amino acids 445 and 455. As our 2H/H exchange measurements indicate, Ca2+ addition to the purified and reconstituted 70-kDa polypeptide medium induces important tertiary structure changes involving at least 93 amino acids. Such structural modifications can be interpreted in terms of stabilization of one of the two conformations (E1 or E2) acquired during the activity cycle (see Fig. 1). Such Ca2+-binding induction of structural changes has been observed in the case of the PMCA (62). Outstandingly, the missing COOH-terminal region in the 70-kDa polypeptide does not prevent protein-structure modifications upon Ca2+ binding. This is not surprising as the 70-kDa protein includes a large part of the cytoplasmic loop (about 400 amino acids) and retains Na+/Ca2+ exchange activity (30). Further investigations need to be carried out to elucidate the role of the COOH portion of the Na+/Ca2+ exchanger with respect to regulation.
In conclusion, the present study indicates that the secondary structure
of the 70-kDa exchanger is composed of 44% -helices, 25%
-sheets, 16%
-turns, and 15% random. Moreover, the 70-kDa polypeptide secondary structure showed high similarity to that of the
PMCA. Finally, Ca2+ binding induced a major conformational
change in the protein, with no apparent secondary structure
modifications. The Na+/Ca2+ exchanger most
probably has a consecutive ion-transport mechanism. Such transport
mechanisms are generally characterized by important conformational
changes. Tertiary structure modifications observed upon
Ca2+ binding may reflect protein folding throughout the
Na+/Ca2+ exchange catalytic cycle.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Laboratoire de Pharmacodynamie et Thérapeutique (CP 617), Faculté de Médecine, Université Libre de Bruxelles, Route de Lennik, Number 808, B-1070 Bruxelles, Belgium. Tel.: 32-2-555-60-91; Fax: 32-2-555-63-70; E-mail: rsaba{at}ulb.ac.be.
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ABBREVIATIONS |
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The abbreviations used are: FTIR-ATR, Fourier transform infrared attenuated total reflection; Cao, extracellular calcium ions; 2H/H, deuterium/hydrogen; MOPS, 4-morpholinepropanesulfonic acid; Nai, intracellular sodium ions; PMCA, plasma membrane Ca2+-ATPase.
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REFERENCES |
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