From the Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias, Universidad de Extremadura, 06071 Badajoz, Spain, the § Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, and the ¶ Institute Curie, Section de Recherche, UMR-CNRS 168 and LRC-CEA 8,11 Rue P. et Marie Curie, 75231 Paris CEDEX, France
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ABSTRACT |
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The synaptosomal plasma membrane Ca2+-ATPase (PMCA) purified from pig brain was reconstituted with liposomes prepared by reverse phase evaporation at a lipid to protein ratio of 150/1 (w/w). ATP-dependent Ca2+ uptake and H+ ejection by the reconstituted proteoliposomes were demonstrated by following light absorption and fluorescence changes undergone by arsenazo III and 8-hydroxy-1,3,6-pyrene trisulfonate, respectively. Ca2+ uptake was increased up to 2-3-fold by the H+ ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, consistent with relief of an inhibitory transmembrane pH gradient (i.e. lumenal alkalinization) generated by H+ countertransport. The stoichiometric ratio of Ca2+/H+ countertransport was 1.0/0.6, and the ATP/Ca2+ coupling stoichiometry was 1/1 at 25° C. The electrogenic character of the Ca2+/H+ countertransport was demonstrated by measuring light absorption changes undergone by oxonol VI. It was shown that a 20 mV steady state potential (positive on the lumenal side) was formed as a consequence of net charge transfer associated with the 1/1 Ca2+/H+ countertransport. Calmodulin stimulated ATPase activity, Ca2+ uptake, and H+ ejection, demonstrating that these parameters are linked by the same mechanism of PMCA regulation.
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INTRODUCTION |
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Regulation of the intracellular free Ca2+ concentration in nerve terminals is a key factor in the mechanism of synaptic transmission (1, 2). Reduction of cytosolic Ca2+ in nerve terminals is dependent on removal by the plasmalemmal Ca2+-ATPase (PMCA)1 and sequestration by the intracellular (SERCA) Ca2+-ATPase (3). Coexistence of both ATPases, as well as the presence of various transport and permeability channels in synaptosomal preparations (4), has rendered difficult a detailed characterization of the synaptosomal PMCA with regards to Ca2+ and H+ transport and net charge transfer, as done with other Ca2+ transport systems (7-13).
We purified the PMCA from pig synaptosomes by calmodulin column chromatography (5, 6) and obtained proteoliposomal reconstitution by the method originally developed by Rigaud and coworkers (12, 14-18). The main advantages of this procedure were: (i) optimal protein incorporation and orientation, (ii) very low permeability of the proteoliposomal membrane to electrolytes, and (iii) large intravesicular volume which delays back inhibition by high concentrations of lumenal Ca2+ (8). We then studied the functional properties of the reconstituted synaptosomal PMCA, measuring ATP-dependent Ca2+-uptake activity, H+ ejection, and formations of transmembrane electrical potential.
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MATERIALS AND METHODS |
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Egg phosphatidylcholine (EPC) and egg phosphatidic acid (EPA) were obtained from Avanti Polar Lipids Inc. Calmodulin, calmodulin-agarose, C12E8, FCCP, valinomycin and A23187 were obtained from Sigma. Oxonol VI and 8-hydroxy-1,3,6-pyrene trisulfonate (pyranine) were from Molecular Probes. Bio-Beads SM-2 and the anion exchange chromatography resin AG1X8 were purchased from Bio-Rad. All other reagents were of the highest purity available.
Purification of Plasma Membrane Ca2+-ATPase--
The
pig brain plasma membrane Ca2+-ATPase was purified as
described in Salvador and Mata (5). Briefly, fresh pig brain was homogenized in 10 volumes of 10 mM Hepes/KOH, pH 7.4, 0.32 M sucrose, 0.5 mM MgSO4, 0.1 mM phenylmethylsulfonyl fluoride and 2 mM
2-mercaptoethanol. After two centrifugations at low and high speed,
respectively, the pellet was layered in a discontinuous 20-40% (w/v)
sucrose gradient and centrifuged at 63,000 × g. The
synaptosomes obtained in the interface were lysed in a hypotonic medium
containing 1 mM EDTA and centrifugated at 20,000 × g. The synaptic plasma membrane vesicles were obtained in
the pellet and solubilized in a medium containing 20 mM
Hepes/KOH, pH 7.4, 130 mM KCl, 0.5 mM
MgCl2, 50 µM CaCl2, 15%
glycerol, 2 mM 2-mercaptoethanol, and 0.6% (w/v) Triton
X-100. The solubilized fraction was applied onto a calmodulin-agarose column previously equilibrated with the same buffer except that 1 mM MgCl2 and 100 µM
CaCl2 was used. Finally the Ca2+-ATPase was
eluted in the above buffer containing 0.06% (w/v) Triton X-100 and 2 mM EDTA instead of calcium. The samples were stored at
80°C until use.
Preparation of Liposomes and Reconstitution of the Ca2+-ATPase-- EPC/EPA (9/1, w/w) liposomes were prepared by reverse phase evaporation as described previously (19) in a buffer containing 20 mM Pipes-K+, pH 7.1, 130 mM KCl (Pipes buffer, pH 7.1). After sequential extrusion through 0.8, 0.4, and 0.2 µm nucleopore filters, the liposomes were resuspended at 4 mg/ml in Pipes buffer, pH 7.1, and solubilized by 8 mg/ml C12 E8. The purified Ca2+-ATPase (0.11 mg/ml) was added to the liposomes at a lipid/protein ratio of 150/1 (w/w) and incubated for 2 min. The detergent was then removed in 3 h by subsequent additions of Bio-Beads SM-2 (80 mg/ml/h) under continuous stirring. After complete detergent removal, the proteoliposomes were pipetted off. When measurements of H+ ejection were done, the fluorescent pH indicator pyranine (200 µM) was added in the incubation medium before Bio-Beads addition. After detergent removal, the external pyranine was removed by passing the proteoliposomes through an anion exchange chromatography column (AG1X8).
Ca2+-ATPase Activity-- The enzymatic activity was measured at 25 °C by using a coupled enzyme assay following spectrophotometrically the NADH absorption change at 340 nm. The proteoliposomal protein (3 µg/ml) was incubated in 2 ml of a medium containing Pipes buffer, pH 7.1 plus 5 mM MgCl2, 10 µM CaCl2, 0.22 mM NADH, 0.42 mM phosphoenolpyruvate, 10 units of pyruvate kinase, and 28 units of lactate dehydrogenase. Additions of 0.2 mM ATP or 1 mM ATP were done to assay the activity of the reconstituted or solubilized protein, respectively.
Ca2+ Uptake-- Ca2+uptake by reconstituted proteoliposomes was measured by following the differential absorption of the metallochromic indicator arsenazo III (50 µM) as described previously (20). A 687-660-nm wavelength pair was used in a dual wavelength spectrophotometer (DW-2000, SLM-Aminco) to monitor changes in external Ca2+ concentration. The proteoliposomal protein (6 µg) was incubated at 25 °C in 2 ml of a medium containing Pipes buffer, pH 7.1, plus 0.42 µg/ml calmodulin, 5 mM MgCl2, and 50 µM arsenazo III. The absorbance signal uptake was calibrated by the addition of 10 µM final Ca2+, and the reaction was initiated by adding 0.2 mM ATP.
ATP-dependent H+ Ejection--
Lumenal
H+ ejection of the proteoliposomes was measured by
following the changes in the fluorescence intensity of the pH indicator pyranine that is entrapped during the reconstitution procedure (9).
Fluorescence intensity was measured using a exc 460 nm and
em 510 nm in a spectrofluorometer (Jasco FP-777).
Calibration of lumenal pH changes was done by subsequent additions of
0.5 mN H2SO4 to 2 ml of medium
containing 6 µg of proteoliposomal protein in Pipes buffer, pH 7.1, plus 5 mM MgCl2, 10 µM
CaCl2, and 4 µM FCCP.
Electrical Potential Assay-- The transmembrane electrical potential was measured by following the differential absorption (625-603 nm) of 2 µM oxonol VI, using dual wavelength spectrophotometry (12). The reaction mixture was as described for Ca2+ uptake except that oxonol VI was used instead of arsenazo III. The calibration was done by several additions of 130 mM KCl to the medium in the presence of 1 µM of the K+ ionophore valinomycin and in the absence of ATP. Absorption changes were standardized with electrical potential by using the Nernst equation.
Protein Determination-- The protein content was measured by the Bradford method (21). The interference of Triton X-100 was avoided by a previous treatment of the samples with Bio-Beads SM-2.
SDS-Polyacrylamide Gel Electrophoresis-- Electrophoresis was performed by the Laemmli procedure (22) in a 7.5% polyacrylamide gel.
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RESULTS |
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Purification and Reconstitution of Synaptosomal PMCA-- The pig brain PMCA was purified from solubilized synaptic vesicles by calmodulin affinity chromatography. Electrophoretic analysis of the synaptic vesicles (Fig. 1, lane 1) demonstrates the presence of several protein bands in addition to the PMCA which is hardly visible because of its very low quantity. In contrast, the purified protein eluted from the calmodulin column appears as a single band of apparent 140-kDa molecular weight (Fig. 1, lane 2). We found that the purified enzyme had an ATPase activity of 1.10 ± 0.10 IU in the presence of pure EPC, as compared with 0.063 ± 0.011 IU in the solubilized synaptosomal vesicles.
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ATP-dependent Ca2+ Uptake--
The pig
brain purified PMCA was reconstituted into EPC/EPA liposomes at a
lipid/protein ratio of 150/1 (w/w). The reconstitution procedure was
performed by detergent removal from lipid-protein-detergent micellar
solutions using Bio-Beads SM2 as detergent removing agent. The
reconstitution procedure and Ca2+ transport activity were
optimized by using different detergents and ionic conditions. The
highest ATP-dependent Ca2+ uptake was obtained
when C12E8 was used as a detergent in the reconstitution procedure, and Cl (rather than
SO42- or isothiocyanate) was used as
the prevailing anion in the reaction medium. Similar findings were
reported by Hao et al. (12) for reconstitution of the
erythrocyte PMCA.
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ATPase Activity--
We have studied under similar experimental
conditions the effects of calmodulin, H+- and
Ca2+-ionophores, and vanadate upon the
Ca2+-ATPase hydrolytic activity of the reconstituted enzyme
(Fig. 3). In the presence of 0.2 mM ATP, we observed hydrolytic rates of 0.21 ± 0.02 µmol of Pi/mg of protein·min1. This
activity was stimulated by addition of calmodulin, FCCP, and A23187.
The initial rate was increased 2-fold by calmodulin (Table I). FCCP did
not affect the initial rate, but increased the ATPase activity only
after an initial period of activity, i.e. only when a pH
gradient was present. The addition of vanadate totally inhibited the
ATPase activity. A comparison of the initial rates of Ca2+
transport and ATPase activity yields a coupling stoichiometry of 1/1 at
25 °C, independently of the presence of calmodulin and/or FCCP.
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ATP-dependent H+
Ejection--
H+ ejection from the lumen of the
proteoliposomes, associated with ATP-dependent
Ca2+ uptake was demonstrated directly by measuring changes
in lumenal pH. This was accomplished by monitoring the fluorescence
intensity of the pH indicator pyranine, which was trapped in the lumen
of the proteoliposomes during the reconstitution procedure. Upon ATP
addition (Fig. 4A), the
initial rate of H+ extrusion calculated after 30 s was
approximately 0.1 µmol of H+/mg of
protein·min1. Analogous to Ca2+ uptake,
H+ ejection was stimulated by calmodulin (see Table I and
Fig. 4B) to a rate of approximately 0.26 ± 0.05 µmol
of H+/mg of protein·min
1, reaching in 5 min
a maximal amount of 0.36 ± 0.04 µmol of H+
ejected/mg of protein. It is noteworthy that although H+
ejection was not significantly affected by valinomycin in the presence
of K+, FCCP decreased the fluorescence signal to the
original level. Therefore, H+ ejection and formation of pH
gradient are not driven by any electrical gradient, but are primary
events linked to ATP-dependent Ca2+ uptake in
the form of a Ca2+/H+ countertransport. Our
measurements indicate that the coupling stoichiometry of
Ca2+ uptake and H+ ejection is 1.0/0.6.
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Electrical Potential-- The net charge displacement associated with the observed stoichiometry of Ca2+/H+ countertransport suggests that a transmembrane electrical potential is generated across the proteoliposomal membrane as a consequence of this process. In fact, we observed the development of a transmembrane electrical potential by measuring light absorption undergone by oxonol VI. It is shown in Fig. 5 that activation of the Ca2+ pump by addition of ATP under optimal conditions produces a steady-state transmembrane potential of 19 ± 2.1 mV. Addition of valinomycin, a K+ ionophore, collapses the electrical potential by rendering the membrane permeable to K+ and thereby compensating for the uneven charge displacement.
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DISCUSSION |
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The aim of this study was a characterization of Ca2+
transport by the plasma membrane Ca2+-ATPase from pig brain
in terms of countertransport and electrogenic properties. The study of
ion fluxes was rendered possible by the use of PMCA reconstituted in
proteoliposomes with a low electrolyte permeability. The reconstituted
synaptosomal PMCA sustains a Ca2+ transport rate up to 0.4 µmol of Ca2+/mg of protein·min1, similar
to those obtained by Niggli et al. (13) and Hao et al. (12) with reconstituted erythrocyte PMCA. In addition, the low
lipid/protein ratio used for reconstitution has provided a large
lumenal volume per ATPase molecule, thereby permitting accumulation of
large amounts of Ca2+ and relatively long experimental
times before establishment of an inhibitory Ca2+
concentration in the lumen of the proteoliposomes. The 1/1
stoichiometric ratio of Ca2+ uptake and ATP hydrolysis
obtained by measurements of initial rates (Table I) is in the same
range as that reported with the reconstituted erythrocyte PMCA (12,
13), but lower than the ratio of 2/1 obtained with SERCA (9). This
difference may be related to intrinsic characteristics of the two
families of Ca2+ pumps (23-25).
The use of the proteoliposomal reconstituted system turned out to be very advantageous in demonstrating that the synaptosomal PMCA pump operates Ca2+/H+ countertransport. Collapse of the related H+ gradient by the H+ ionophore FCCP, and not by the K+ ionophore valinomycin, is consistent with direct Ca2+/H+ countertransport through the pump, rather than H+ extrusion secondary to electrical potential. The Ca2+/H+ stoichiometric ratio of 0.6 found in the synaptosomal plasma membrane Ca2+-ATPase, is similar to the ratios reported for the other Ca2+-ATPases. For example, Hao et al. (12) obtained a stoichiometric Ca2+/H+ ratio of 1 with the reconstituted erythrocyte PMCA at 12 °C temperature but a lower ratio at 30 °C. Although the Ca2+/H+ ratio may be primarily determined by the stoichiometry and pKs of acidic residues operating the Ca2+/H+ exchange (18), it is possible that higher temperatures and/or other experimental variable may produce H+ slippage (26). The uneven charge exchange produced by the observed Ca2+/H+ ratios is consistent with the development of transmembrane electrical potential following addition of ATP (Fig. 5).
The Ca2+/H+ countertransport and the net charge transfer demonstrated with purified and reconstituted brain PMCA will be useful in understanding Ca2+ regulation in synaptosomal function. Furthermore, demonstration of Ca2+/H+ countertransport in several Ca2+ transport ATPases indicates that a shift in the affinity of protein carboxylic functions for H+and Ca2+ occurs upon formation and cleavage of phosphorylated enzyme intermediate and that this is a common mechanistic feature in the coupling of ATP utilization and Ca2+ transport.
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FOOTNOTES |
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* This work was supported in part by Grant PB95-0144 from Dirección General de Investigación Científica y Técnica and Grant EIA94-29 from Junta de Extremadura, Spain (to A. M. M) and by National Institutes of Health Grant P01-HL-27867 (to G. I.).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.
Recipient of a Ph.D. Studentship from Programa Sectorial
FP-DGICYT.
To whom correspondence should be addressed. Tel./Fax:
34-924-289419; Fax: 34-924-271304; E-mail: anam{at}unex.es.
1 The abbreviations used are: PMCA, plasma membrane calcium ATPase; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; pyranine, 8-hydroxy-1,3,6-pyrene trisulfonate; EPC, egg phosphatidylcholine; EPA, egg phosphatidic acid; Pipes, 1,4-piperazinediethanesulfonic acid.
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REFERENCES |
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