Pre-steady State Electrogenic Events of Ca2+/H+ Exchange and Transport by the Ca2+-ATPase
2006; Elsevier BV; Volume: 281; Issue: 49 Linguagem: Inglês
10.1074/jbc.m606040200
ISSN1083-351X
AutoresFrancesco Tadini‐Buoninsegni, Gianluca Bartolommei, Maria Rosa Moncelli, Rolando Guidelli, Giuseppe Inesi,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoNative or recombinant SERCA (sarco(endo)plasmic reticulum Ca2+ ATPase) was adsorbed on a solid supported membrane and then activated with Ca2+ and ATP concentration jumps through rapid solution exchange. The resulting electrogenic events were recorded as electrical currents flowing along the external circuit. Current transients were observed following Ca2+ jumps in the absence of ATP and following ATP jumps in the presence of Ca2+. The related charge movements are attributed to Ca2+ reaching its binding sites in the ground state of the enzyme (E1) and to its vectorial release from the enzyme phosphorylated by ATP (E2P). The Ca2+ concentration and pH dependence as well as the time frames of the observed current transients are consistent with equilibrium and pre-steady state biochemical measurements of sequential steps within a single enzymatic cycle. Numerical integration of the current transients recorded at various pH values reveal partial charge compensation by H+ in exchange for Ca2+ at acidic (but not at alkaline) pH. Most interestingly, charge movements induced by Ca2+ and ATP vary over different pH ranges, as the protonation probability of residues involved in Ca2+/H+ exchange is lower in the E1 than in the E2P state. Our single cycle measurements demonstrate that this difference contributes directly to the reduction of Ca2+ affinity produced by ATP utilization and results in the countertransport of two Ca2+ and two H+ within each ATPase cycle at pH 7.0. The effects of site-directed mutations indicate that Glu-771 and Asp-800, within the Ca2+ binding domain, are involved in the observed Ca2+/H+ exchange. Native or recombinant SERCA (sarco(endo)plasmic reticulum Ca2+ ATPase) was adsorbed on a solid supported membrane and then activated with Ca2+ and ATP concentration jumps through rapid solution exchange. The resulting electrogenic events were recorded as electrical currents flowing along the external circuit. Current transients were observed following Ca2+ jumps in the absence of ATP and following ATP jumps in the presence of Ca2+. The related charge movements are attributed to Ca2+ reaching its binding sites in the ground state of the enzyme (E1) and to its vectorial release from the enzyme phosphorylated by ATP (E2P). The Ca2+ concentration and pH dependence as well as the time frames of the observed current transients are consistent with equilibrium and pre-steady state biochemical measurements of sequential steps within a single enzymatic cycle. Numerical integration of the current transients recorded at various pH values reveal partial charge compensation by H+ in exchange for Ca2+ at acidic (but not at alkaline) pH. Most interestingly, charge movements induced by Ca2+ and ATP vary over different pH ranges, as the protonation probability of residues involved in Ca2+/H+ exchange is lower in the E1 than in the E2P state. Our single cycle measurements demonstrate that this difference contributes directly to the reduction of Ca2+ affinity produced by ATP utilization and results in the countertransport of two Ca2+ and two H+ within each ATPase cycle at pH 7.0. The effects of site-directed mutations indicate that Glu-771 and Asp-800, within the Ca2+ binding domain, are involved in the observed Ca2+/H+ exchange. SERCA (sarco(endo)plasmic reticulum Ca2+-ATPase) is a well characterized cation transport ATPase (1Andersen J.P. Vilsen B. FEBS Lett. 1995; 359: 101-106Crossref PubMed Scopus (115) Google Scholar, 6Møller J.V. Nissen P. Sørensen T.L. Maire M. Curr. Opin. Struct. Biol. 2005; 15: 387-393Crossref PubMed Scopus (111) Google Scholar) that is obtained with vesicular fragments of sarcoplasmic reticulum (SR). 2The abbreviations used are: SR, sarcoplasmic reticulum; SSM, solid supported membrane; MOPS, 3-(N-morpholino)propanesulfonic acid; WT, wild type. Two Ca2+ are transported from the medium into the vesicles, whereas one ATP is utilized. ATPase activation requires binding of two Ca2+ per enzyme molecule (E1·2Ca2+) followed by ATP utilization and formation of a phosphoenzyme intermediate (E1-P). The free energy derived from ATP is utilized by the phosphoenzyme for a conformational transition (E1-P to E2-P) that favors translocation and release of the bound Ca2+ against its concentration gradient. The cycle is completed by hydrolytic cleavage of E2-P. Ca2+/H+ countertransport and electrogenicity were noted (7Chiesi M. Inesi G. Biochemistry. 1980; 19: 2912-2918Crossref PubMed Scopus (114) Google Scholar, 9Yamagouchi M. Kanazawa T. J. Biol. Chem. 1985; 260: 4896-4900Abstract Full Text PDF PubMed Google Scholar) with native SR vesicles, but most useful information was obtained with reconstituted proteoliposomes (10Cornelius F. Møller J.V. FEBS Lett. 1991; 284: 46-50Crossref PubMed Scopus (18) Google Scholar, 11Levy D. Gulyk A. Bluzat A. Rigaud J.L. Biochim. Biophys. Acta. 1992; 1107: 283-298Crossref PubMed Scopus (108) Google Scholar) that are not leaky to H+ or other electrolytes. It was then possible to demonstrate that, at neutral pH, the stoichiometry of Ca2+/H+ countertransport is ∼1/1, and because of uneven charge distribution, Ca2+ transport is electrogenic (12Yu X. Carroll S. Rigaud J.L. Inesi G. Biophys. J. 1993; 64: 1232-1242Abstract Full Text PDF PubMed Scopus (144) Google Scholar). The stoichiometric ratio of Ca2+/H+ exchange varies as the pH outside and inside the vesicles is changed (13Yu X. Hao L. Inesi G. J. Biol. Chem. 1994; 269: 16656-16661Abstract Full Text PDF PubMed Google Scholar), suggesting that the protonation probability of acidic protein residues involved in Ca2+/H+ exchange varies as E2-P is formed, whereby H+ acquisition favors Ca2+ dissociation. A recent contribution (14Sugita Y. Miyashita N. Ikeguchi M. Kidera A. Toyoshima C. J. Am. Chem. Soc. 2005; 127: 6150-6151Crossref PubMed Scopus (52) Google Scholar, 15Obara K. Miyashita N. Xu C. Toyoshima I. Sugita Y. Inesi G. Toyoshima C. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14489-14496Crossref PubMed Scopus (211) Google Scholar) derives from continuum electrostatic calculations based on high resolution crystal structures, where E2 or E1 ATPase states were stabilized with the high affinity inhibitors (E2-thapsigargin) or high concentrations of Ca2+ (E1·2Ca2+). The electrostatic calculations yielded estimates of H+ occupancy of specific acidic residues in the E2 and E1 states of the enzyme and suggested that a minimum of 1.7 and a maximum of 3.0 H+ are countertransported in a reaction cycle that transports two Ca2+ ions. We have described here pre-steady state charge movements induced by Ca2+ concentration jumps in the absence of ATP or by ATP concentration jumps in the presence of Ca2+ delivered to Ca2+-ATPase vesicles adsorbed onto an alkanethiol/phospholipid mixed bilayer anchored to a gold electrode (16Pintschovius J. Fendler K. Biophys. J. 1999; 76: 814-826Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). This technique has been used in studies of electrogenic transport by several membrane proteins (17Pintschovius J. Bamberg E. Fendler K. Biophys. J. 1999; 76: 827-836Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 23Bartolommei G. Tadini-Buoninsegni F. Hua S. Moncelli M.R. Inesi G. Guidelli R. J. Biol. Chem. 2006; 281: 9547-9551Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). In our experiments, the detected charge movements are attributed to the binding of activating Ca2+ to the Ca2+-ATPase in the absence of ATP and to translocation of bound Ca2+ following the addition of ATP. We have determined the pH dependence of these charge movements and demonstrated unambiguously that the observed electrogenic events correspond to sequential steps within a single enzymatic cycle. We have also demonstrated a different pH dependence of electrogenic signals originating from Ca2+ binding to the enzyme in the absence of ATP, as compared with translocation of bound Ca2+ following the addition of ATP. This difference is attributed to the variation in the stoichiometric ratio of Ca2+/H+ exchange and in the protonation probability (apparent pKa) of acidic residues in the ground (E1) and intermediate states (E2P) of the enzymatic cycle. Chemicals—Calcium and magnesium chlorides and MOPS were obtained from Merck at analytical grade. ATP (∼97%) and dithiothreitol (≥99% purity) were purchased from Fluka. Octadecanethiol (98%) from Aldrich was used without further purification. EGTA and calcimycin (calcium ionophore A23187) were obtained from Sigma. The lipid solution contained diphytanoylphosphatidylcholine (Avanti Polar Lipids) and octadecylamine (very pure; Fluka) (60:1) it was prepared at a concentration of 1.5% (w/v) in n-decane (Merck) as described by Bamberg et al. (24Bamberg E. Apell H.J. Dencher N.A. Sperling W. Stieve H. Läuger P. Biophys. Struct. Mech. 1979; 5: 277-292Crossref Scopus (116) Google Scholar). ATPase Preparations—SR vesicles were obtained from the hind leg muscles of New Zealand White rabbits as described by Eletr and Inesi (25Eletr S. Inesi G. Biochim. Biophys. Acta. 1972; 282: 174-179Crossref PubMed Scopus (308) Google Scholar). Recombinant Ca2+-ATPase was obtained from COS-1 cells infected with adenovirus vectors carrying chicken WT (26Karin N.J. Kaprielian Z. Fambrough D.M. Mol. Cell. Biol. 1989; 9: 1978-1986Crossref PubMed Scopus (71) Google Scholar) or mutant cDNA under the control of the cytomegalovirus promoter (27Strock C. Cavagna M. Peiffer W.E. Sumbilla C. Lewis D. Inesi G. J. Biol. Chem. 1998; 273: 15104-15109Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Measurement of Charge Movements—Charge movements were measured by adsorbing the SR vesicles containing the Ca2+-ATPase onto a mixed bilayer anchored to a gold electrode (the so-called solid supported membrane (SSM)). The SSM consisted of an octadecanethiol monolayer covalently linked to the gold surface via the sulfur atom with a diphytanoylphosphatidylcholine monolayer on top of it (28Seifert K. Fendler K. Bamberg E. Biophys. J. 1993; 64: 384-391Abstract Full Text PDF PubMed Scopus (121) Google Scholar). The gold electrode preparation, the whole experimental setup, as well as the solution exchange technique are described in detail elsewhere (22Tadini-Buoninsegni F. Bartolommei G. Moncelli M.R. Inesi G. Guidelli R. Biophys. J. 2004; 86: 3671-3686Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). SR vesicles, following a brief sonication in the absence of detergent, were adsorbed on the SSM (usually at an applied potential of +0.1 V, with respect to a Ag/AgCl reference electrode), and the protein was activated by the rapid injection of a solution containing a suitable substrate, e.g. Ca2+ or ATP, as follows. In Ca2+ concentration jump experiments, the "washing" solution contained 150 mm choline chloride, 25 mm MOPS (pH 7.0), 0.25 mm EGTA, 1 mm MgCl2, and 0.2 mm dithiothreitol; the "activating" solution contained, in addition, the required concentration of CaCl2. In ATP concentration jump experiments, the washing solution contained 150 mm choline chloride, 25 mm MOPS (pH 7.0), 1 mm EGTA, 1 mm MgCl2, 1.1 mm CaCl2 (100 μm free Ca2+), and 0.2 mm dithiothreitol; the activating solution contained, in addition, the required concentration of ATP (100 μm). In the case of recombinant Ca2+-ATPase, the concentration jump experiments were carried out by employing the SURFE2ROne device (IonGate Biosciences GmbH, Frankfurt am Main, Germany). Free Ca2+ concentration was calculated with the computer program WinMAXC (29Patton C. Thompson S. Epel D. Cell Calcium. 2004; 35: 427-431Crossref PubMed Scopus (329) Google Scholar). Unless otherwise stated, 1 μm calcium ionophore A23187 was used to prevent formation of a Ca2+ concentration gradient across the SR vesicles (30Hartung K. Froehlich J.P. Fendler K. Biophys. J. 1997; 72: 2503-2514Abstract Full Text PDF PubMed Scopus (17) Google Scholar). Ca2+ Binding, Enzyme Phosphorylation, and Ca2+ Transport— Ca2+ binding to the ATPase under equilibrium conditions, in the absence of ATP, was measured using a radioactive calcium tracer and EGTA-Ca2+ buffers as previously described (31Inesi G. Kurzmack M. Coan C. Lewis D.E. J. Biol. Chem. 1980; 255: 3025-3031Abstract Full Text PDF PubMed Google Scholar). Pre-steady state enzyme phosphorylation, occlusion of bound Ca2+, and Pi production were measured by adding ATP to enzyme pre-incubated with Ca2+ using rapid quench mixers (32Inesi G. Coan C. Verjovshi-Almeida S. Lewis D. Ann. N. Y. Acad. Sci. 1978; 307: 224-227Crossref PubMed Scopus (43) Google Scholar). ATP-dependent Ca2+ transport was measured by adding ATP to SR vesicles in a medium containing 20 mm pH buffer, 80 mm KCl, 2 mm MgCl2, and 50 μm CaCl2. The loaded vesicles were then vacuum-filtered (Millipore 0.45 μm) after 10-90 s of incubation, and the filters containing the loaded vesicles were washed with 1 mm LaCl3 and processed for determination of radioactive calcium by scintillation counting. Radioactive (45Ca)Ca and (γ-32P)ATP were alternatively used to provide radioactive tracers. The temperature was maintained at 22-23 °C for all of the experiments. Pre-steady state electrical measurements on an ion pump may yield direct information on charge movements within the transport cycle (16Pintschovius J. Fendler K. Biophys. J. 1999; 76: 814-826Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 33Läuger P. Electrogenic Ion Pumps. Sinauer Associates Inc., Sunderland, MA1991Google Scholar). In our experiments, we activated the pump by delivering Ca2+ and/or ATP by a rapid solution exchange procedure, and we detected electrogenic steps by measuring electrical currents along the external circuit. A diagram of our experimental system, with cartoons representing a minimal number of sequential reactions within an ATPase cycle, is shown in Fig. 1. Useful information is gained from current transients. In fact, numerical integration of each transient is related to a net charge movement, which depends upon the particular electrogenic event (i.e. following Ca2+ or ATP concentration jumps). In addition, kinetic information may be obtained by fitting the current versus time curves to a sum of exponentially decaying terms (33Läuger P. Electrogenic Ion Pumps. Sinauer Associates Inc., Sunderland, MA1991Google Scholar). A current transient induced by a Ca2+ concentration jump in the absence of ATP at pH 7 is shown by curve a in Fig. 2. This transient is due to binding of Ca2+ to the cytoplasmic side of the pump (the exterior of the vesicles), which creates a difference in potential across the vesicular membrane, positive in the direction of the metal support. To keep the applied voltage constant across the whole system, the experimental variation of potential across the vesicular membrane is compensated for by an equal and opposite potential difference across the octadecanethiolphosphatidylcholine mixed bilayer. This potential difference is produced by a flow of electrons along the external circuit toward the electrode surface. Curve b in Fig. 2 shows a current transient induced by an ATP concentration jump at pH 7 in the presence of Ca2+ (i.e. on Ca2+-ATPase vesicles pre-equilibrated with Ca2+). Here also, the release of the pre-bound Ca2+ ions into the vesicle interior (the luminal side) induced by ATP creates a potential difference, again positive toward the metal, which is compensated for by an electron flow along the external circuit. Following the first cycle, when steady state conditions are attained, the flow of Ca2+ ions out of the vesicles (favored by a Ca2+ ionophore) becomes equal to their flow into the vesicles because of continuous pumping. Under these conditions, the potential difference across the vesicular membrane becomes constant, and consequently, no further signal is detected. In fact, the current flowing along the external circuit (induced by changes in the transmembrane potential) vanishes, whereas the pump current attains its stationary value. The current recorded with the SSM technique is, therefore, different from that recorded with a bilayer lipid membrane incorporating the pump; the former is a measure of the rate of change of the transmembrane potential, whereas the latter measures the pump current. Voltage-dependent stationary pump currents were recorded by Eisenrauch and Bamberg (34Eisenrauch A. Bamberg E. FEBS Lett. 1990; 268: 152-156Crossref PubMed Scopus (13) Google Scholar) upon incorporating Ca2+-ATPase directly into a bilayer lipid membrane. In a previous work (22Tadini-Buoninsegni F. Bartolommei G. Moncelli M.R. Inesi G. Guidelli R. Biophys. J. 2004; 86: 3671-3686Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), we have shown that the SSM current vanishes within a fraction of a second time frame. Thus, jumps of ATP concentrations ranging from 1.5 to 100 μm carried out on Ca-ATPase in the presence of 100 μm free calcium generate current transients whose peak currents increase with ATP concentration following the Michaelis-Menten equation (21Bartolommei G. Tadini-Buoninsegni F. Moncelli M.R. Bioelectrochemistry. 2004; 63: 157-160Crossref PubMed Scopus (6) Google Scholar, 22Tadini-Buoninsegni F. Bartolommei G. Moncelli M.R. Inesi G. Guidelli R. Biophys. J. 2004; 86: 3671-3686Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Even though the rate of ATP binding to the pump varies to an appreciable extent, the charge obtained by the integration of these current transients remains constant and may be attributed to the overall amount of Ca2+ ions translocated by the pumps in the first cycle. The current transient induced by a simultaneous concentration jump of ATP and Ca2+ is shown by curve c in Fig. 2. Integration of transient a yields a charge (∼50 pCi) that is not particularly accurate, because the current does not vanish completely in 1 s. Integration of the current transients b and c can be performed more accurately, and yields 93 and 150 pCi, respectively. It is clear that the current transient c is practically the sum of the current transient a induced by a Ca2+ concentration jump on Ca2+-ATPase in the absence of ATP and the current transient b induced by an ATP concentration jump on Ca2+-ATPase pre-equilibrated with Ca2+. Subtracting the charge under transient b from that under transient c yields 57 pCi; this value is more accurate than that (50 pCi) obtained by direct integration of transient a and can be regarded as the charge under the current transient generated by binding of Ca2+ to the cytoplasmic sites. From a kinetic standpoint, it is also apparent that curve c includes the b component preceded by the a component. It is most important to realize that a 100 μm ATP concentration jump in the absence of Ca2+ (Fig. 2, curve d) yields no electrogenic signal. We now consider these events in greater detail. Ca2+ Concentration Jumps—A Ca2+ concentration jump (in the absence of ATP), performed with the SSM-based technique by rapid injection of Ca2+, yields a current transient (Fig. 3, Ca2+) that can be ascribed to Ca2+ binding to the ATPase. Identical current transients were obtained in the absence and in the presence of a Ca2+ ionophore, indicating that only initial binding to the cytoplasmic side of the protein is involved. Subsequent addition of a Ca2+-chelating solution (containing EGTA) produces dissociation of bound Ca2+ revealed by a reverse "off current." The off-current transients (Fig. 3, EGTA) are better defined than the on-current transients and allow a more accurate estimate of the corresponding charge by numerical integration over time. Plots of the charges under the current transients against the concentration of added Ca2+ are shown in Fig. 4 at three different pH values, yielding Ca2+ binding isotherms quite similar to those obtained by direct measurements of Ca2+ binding to SR vesicles in suspension under equilibrium conditions. The binding affinity is higher as the pH is raised from 6.0 to 8.0, as shown by the shift of the curves toward lower Ca2+ concentrations, demonstrating the same pattern in both sets of experiments. Fig. 4 was obtained by plotting the charges under the current transients at various Ca2+ concentrations normalized with reference to the maximum charge attained at saturating Ca2+ for each pH. The solid and dashed curves in Fig. 4 are fits of the plots to the Hill equation, and the plots are consistent with the known cooperativity of binding. Most importantly, we found that the net moved charge obtained with saturating Ca2+ increases from acidic pH values up to pH 7 and then remains constant up to pH 8.0, as shown by plot a in Fig. 5. This indicates that charge movement by Ca2+ binding is counterbalanced to an appreciable extent by exchange with H+ at acidic pH but not at neutral or alkaline pH. The favorable definition of the off-current transients (Fig. 3, EGTA) allows fitting to a multiexponential function, yielding two distinct relaxation rate constants of 33 and 10 s-1, which are probably related to sequential dissociation of the two bound Ca2+ ions from the cytoplasmic side of the ATPase. This is in agreement with the stacking modality of the two Ca2+ per ATPase molecule (31Inesi G. Kurzmack M. Coan C. Lewis D.E. J. Biol. Chem. 1980; 255: 3025-3031Abstract Full Text PDF PubMed Google Scholar) and with the two distinct rate constants for Ca2+ binding to the ATPase observed with a fluorescent probe (35Peinelt C. Apell H.J. Biophys. J. 2005; 89: 2427-2433Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). ATP Concentration Jumps—An ATP concentration jump in the presence of saturating Ca2+ produces a current transient due to electrogenic Ca2+ dissociation from the phosphoenzyme on the luminal side of the SR membrane. Because the Km value for ATP is 2.9 μm (22Tadini-Buoninsegni F. Bartolommei G. Moncelli M.R. Inesi G. Guidelli R. Biophys. J. 2004; 86: 3671-3686Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), we added 100 μm ATP to ensure a saturating substrate concentration. Fig. 6 shows two current transients induced by ATP jumps in the absence (curve a) and in the presence (curve b) of a Ca2+ ionophore. The current transient in curve a decays more rapidly than that in curve b, and the underlying charge is smaller. In fact, in the absence of ionophore, the high density of ATPase units in the SR vesicle (36Franzini-Armstrong C. Ferguson D.G. Biophys. J. 1985; 48: 607-615Abstract Full Text PDF PubMed Scopus (52) Google Scholar) and the limited luminal volume of the vesicle contribute to a rapid rise of luminal Ca2+ with consequent "back inhibition" and blocking of further dissociation of bound Ca2+. Conversely, the presence of ionophore prevents rapid saturation of the vesicle by Ca2+, and the pump is allowed to operate under turnover conditions. In this case, we detect the electrogenic signal generated by the first cycle undergone by the entire population of ATPase molecule. Subsequent stationary currents are not detected by the SSM technique. The time frame of the current transient produced by the addition of ATP to SR vesicles adsorbed on the SSM (Fig. 6, curve b) is comparable with that of the first cycle obtained by the addition of ATP to a suspension of SR vesicles in a rapid mixing apparatus. In fact, it is shown in Fig. 6, inset, that the first cycle triggered by simultaneous activation of the ATPase molecules upon the addition of ATP is completed in ∼150 ms, including enzyme phosphorylation (1 mol/mol), occlusion and translocation of bound Ca2+ (2 Ca2+/mole ATPase), and phosphoenzyme cleavage (1 mol Pi/ATPase). The kinetic analogy of the electrogenic and biochemical measurements is related to the use of vesicles and the addition of ATP by rapid mixing in both cases. Steady state turnover of the ATPase in these preparations is 6-8 s-1 at 20 °C, which is consistent with the time frame of the first cycle. If we compare currents obtained in the presence (Fig. 5, curve b) or in the absence of ionophore (Fig. 5, curve c), we find that the pH dependence of the translocated charge is practically identical under these two conditions but clearly different from the pH dependence of charge movements induced by Ca2+ jumps in the absence of ATP (Fig. 5, curve a). This indicates that, in both cases, the pH dependence of the current obtained by ATP jumps is related to dissociation of bound Ca2+ from the phosphoenzyme. The pH dependence of the current transients following ATP jumps indicates that charge movements related to Ca2+ release from E2P are counterbalanced by significant H+ binding at acidic and neutral pH but progressively less at alkaline pH. Comparing the diverse pH dependence of the charges under the current transients after (saturating) Ca2+ or ATP jumps (Fig. 5) and considering that the net magnitude of the electrogenic signal is dependent on the ratio of Ca2+/H+ exchange, it is apparent that, following ATP utilization, the protonation probability of the pertinent acidic residues is increased (i.e. their apparent pKa is shifted to more alkaline pH) as the enzyme undergoes the conformational change from E1P to E2P. To evaluate this effect on the ability of the ATPase to unload bound Ca2+ from E2P, we then measured the steady state levels of luminal Ca2+ that are reached by active transport at different pH values. These levels are limited by the dissociation constant of the internal phosphoenzyme sites as luminal Ca2+ increases because of active transport. It is shown in Fig. 5, curve d, that the levels of accumulated Ca2+ are significantly reduced as the pH is raised to 8, indicating that, under conditions limiting exchange with H+ following ATP utilization, Ca2+ is less likely to dissociate from E2P. Effects of Site-directed Mutations—The relatively high yield of recombinant ATPase from COS-1 cells infected with adenovirus vectors carrying WT or mutant cDNA (27Strock C. Cavagna M. Peiffer W.E. Sumbilla C. Lewis D. Inesi G. J. Biol. Chem. 1998; 273: 15104-15109Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 37Zhang Z. Lewis D. Strock C. Inesi G. Nakasako M. Nomura H. Toyoshima C. Biochemistry. 2000; 39: 8758-8767Crossref PubMed Scopus (91) Google Scholar) rendered possible studies on the effects of site-directed mutations on electrogenic signals produced by Ca2+ and ATP jumps. It is shown in Fig. 7a that the electrogenic signals obtained by concentration jumps of Ca2+ and ATP on recombinant WT ATPase display the same pattern as previously observed with native ATPase (Fig. 2). We then tried the effect of Asp-351 mutation. Asp-351 is the residue receiving the γ-phosphate of ATP at the catalytic site to form the phosphorylated intermediate. Its mutation produces catalytic inactivation, even though Ca2+ binding is retained. Accordingly, we found that the D351N mutant yields an electrogenic signal upon Ca2+ jumps but no signal at all upon ATP jumps in the presence of Ca2+ (Fig. 7b). The effects of specific mutations on Ca2+ binding were previously studied in detail (38Andersen J.P. Vilsen B. J. Biol. Chem. 1992; 267: 19383-19387Abstract Full Text PDF PubMed Google Scholar, 39Andersen J.P. Vilsen B. Acta Physiol. Scand. Suppl. 1998; 643: 45-54PubMed Google Scholar). In our experiment, we first tried the effect of a Glu-309 mutation. Glu-309 resides on Ca2+ site II, and its mutation interferes with binding of the second Ca2+, whereas non-cooperative binding of the first Ca2+ is retained (37Zhang Z. Lewis D. Strock C. Inesi G. Nakasako M. Nomura H. Toyoshima C. Biochemistry. 2000; 39: 8758-8767Crossref PubMed Scopus (91) Google Scholar). Because of a strict requirement for occupancy of site II, the enzyme is not able to utilize ATP following Glu-309 mutations. In our experiments, we found that an electrogenic signal was generated by the addition of Ca2+, although within a significantly slower time frame (Fig. 7c) as compared with WT or D351N mutant (Fig. 7, a and b). The evidently slower charge movement may be due to interference (by the E309A mutation) with Ca2+ entry into the binding cavity (40Inesi G. Ma H. Lewis D. Xu C. J. Biol. Chem. 2004; 279: 31629-31637Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). No signal was obtained with the E309A mutant following ATP jumps in the presence of Ca2+ (Fig. 7c). Finally, we tried the effect of a Glu-771 mutation. Glu-771 is located in Ca2+ site I, and because of the tight cooperativity of the Ca2+ binding mechanism, its mutation interferes with binding of both Ca2+. In fact, we observed no electrogenic signal upon the addition of Ca2+ or ATP to the E771Q mutant (Fig. 7d), demonstrating a direct relationship of the electrogenic signals with Ca2+ binding. We have reported pre-steady state charge movements within a Ca2+-ATPase cycle obtained with ATPase protein adsorbed on a SSM and subjected to Ca2+ jumps in the absence of ATP or to ATP jumps in the presence of Ca2+. In Fig. 5, the charges under the current transients, observed upon Ca2+ binding from the cytoplasmatic side in the absence of ATP and upon Ca2+ dissociation into the lumen of the vesicles following the addition of ATP, are normalized to their maximum value. This value corresponds to two Ca2+ bound and transported per cycle as determined by biochemical measurements (Fig. 6, inset). We then estimated the stoichiometry of Ca2+/H+ exchange at any given pH by using the values obtained in the same SSM experiments. The pH dependence shown in Fig. 5 indicates that, at pH 7, cooperative binding of two Ca2+ ions from the cytoplasmic side takes place without H+ exchange. This defines the E1 state, as shown in the diagram of Fig. 8, and implies that the E1/E2 equilibrium is pH-dependent. On the other hand, release of the bound Ca2+ into the lumenal side following ATP jumps is accompanied by exchange with two H+ ions. Therefore, the charge of 93 pCi under curve b in Fig. 2, generated following the addition of ATP, involves dissociation of two Ca2+ ions and exchange of two H+ ions and, hence, movement of two electronic charges. On the other hand, the charge of 57 pCi (curve a) is associated with the movement of two Ca2+ ions across a fraction x of the membrane domain of the pump with no H+ exchange (i.e. four charges). Therefore, 4x is to 57 pCi as 2 is to 93 pCi; this yields an x value of 0.31. Differently stated, these results indicate that the Ca2+ binding sites are located in the membrane domain at ∼30% of the membrane thickness from the cytoplasmic side. These conclusions agree with the three-dimensional structure of the Ca-ATPase (41Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1619) Google Scholar, 42Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (809) Google Scholar). Ca2+ binding in the absence of ATP occurs with a cooperative mechanism involving sequential occupancy of two neighboring sites (31Inesi G. Kurzmack M. Coan C. Lewis D.E. J. Biol. Chem. 1980; 255: 3025-3031Abstract Full Text PDF PubMed Google Scholar). The amino acids involved in Ca2+ binding within the membrane-bound region of the ATPase were identified by mutational analysis (43Clarke D.M. Loo T.W. Inesi G. MacLennan D.H. Nature. 1989; 339: 476-478Crossref PubMed Scopus (471) Google Scholar). High resolution structures of the Ca2+ binding sites were then obtained by crystallography (41Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1619) Google Scholar, 42Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (809) Google Scholar). Related atomic models reveal that, in addition to backbone carbonyl oxygens, two water molecules, the Thr-799 side chain, and four acidic residues (Glu-309, Glu-771, Asp-800, and Glu-908) are involved in binding two Ca2+ ions. Glu-771 and Asp-800 have very low probability of retaining protons while participating in Ca2+ complexation (14Sugita Y. Miyashita N. Ikeguchi M. Kidera A. Toyoshima C. J. Am. Chem. Soc. 2005; 127: 6150-6151Crossref PubMed Scopus (52) Google Scholar, 15Obara K. Miyashita N. Xu C. Toyoshima I. Sugita Y. Inesi G. Toyoshima C. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14489-14496Crossref PubMed Scopus (211) Google Scholar). On the other hand, Glu-309 and Glu-908 may retain their protons even in the presence of bound Ca2+, as they are involved in stabilization of local structure by hydrogen bonding. In the absence of ATP, the electrogenicity of the Ca2+ binding step increases as the pH is raised from 6.0 to 7.0 and does not increase any further beyond pH 7.0 (Fig. 5). This variation of net charge movement is related to the H+ stoichiometry available for exchange upon Ca2+ binding with an apparent pKa value within the 6-7 pH range (Fig. 5). At higher pH values, the carboxyl groups of these residues are completely deprotonated, and no exchange with H+ occurs to compensate for the charge introduced by Ca2+ binding. A pH-dependent equilibrium (Fig. 8) between E2 (with protonated acidic residues at pH 6.0) and E1 (with non-protonated acidic residues at pH 7.0) states is consistent with the requirement for enzyme phosphorylation with Pi in the absence of Ca2+ (i.e. E2 phosphorylation by Pi in the reverse direction of the pump), which is strongly dependent on acid pH (44Masuda H. de Meis L. Biochemistry. 1973; 12: 4581-4585Crossref PubMed Scopus (160) Google Scholar). In the presence of ATP, Ca2+ dissociation from the phosphoenzyme is electrogenic at pH 7.0, indicating that compensation by Ca2+/H+ exchange is not sufficient to provide complete charge balance at pH 7.0. On the other hand, whereas the signal produced by Ca2+ binding in the absence of ATP already reaches its maximal intensity at pH 7.0, the signal produced by Ca2+ release following ATP utilization doubles its intensity as the pH is raised from 7.0 to 8.0 (Fig. 5). Therefore, at pH 7.0, the protonation probability of residues involved in Ca2+/H+ exchange is increased as a consequence of enzyme phosphorylation. In fact, the moved charge increases 2-fold by raising the pH from 7.0 to 8.0 (Fig. 5) and reaches its maximum at pH 8.0. This indicates that H+ exchange is totally lacking beyond pH 8, whereby interference with Ca2+ transport is observed (Fig. 5). On the other hand, at pH 7.0, H+ exchange compensates for half the charge moved by two Ca2+ ions. Ca2+/H+ exchange was previously revealed by steady state rate measurements with proteoliposomes (13Yu X. Hao L. Inesi G. J. Biol. Chem. 1994; 269: 16656-16661Abstract Full Text PDF PubMed Google Scholar). The observed exchange was attributed to countertransport, although H+ release from nonspecific sites (i.e. calsequestrin or others) was not excluded. Presently, our pre-steady state measurements demonstrate directly a 1:1 stoichiometric ratio for Ca2+/H+ countertransport (i.e. 2.0 H+/2 Ca2) within a single cycle at pH 7.0. A 1:2 stoichiometric ratio for Ca2+/H+ countertransport is to be excluded, because such a countertransport would be electrosilent and would yield no current transient. In conclusion, our findings indicate that, at pH 7, two protons are gained from the luminal medium upon release of 2 bound Ca2+ ions from the phosphorylated enzyme intermediate. Following Pi release, the two protons are released from the enzyme into the cytoplasmic medium concomitant with the E2 to E1 transition, whereupon Ca2+ binding from the cytoplasmic side takes place (Fig. 8). We also conclude that, in the physiological transport cycle at pH 7, the protonation probability of the acidic residues participating in Ca2+/H+ exchange will be very high in E2-P (apparent pKa value between pH 7 and 8 (Fig. 5, b and c), high in E2 (15Obara K. Miyashita N. Xu C. Toyoshima I. Sugita Y. Inesi G. Toyoshima C. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14489-14496Crossref PubMed Scopus (211) Google Scholar), presumably low in E1, and very low in E1·2Ca2+ (Fig. 5a) (15Obara K. Miyashita N. Xu C. Toyoshima I. Sugita Y. Inesi G. Toyoshima C. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14489-14496Crossref PubMed Scopus (211) Google Scholar). The results of electrostatic calculations (14Sugita Y. Miyashita N. Ikeguchi M. Kidera A. Toyoshima C. J. Am. Chem. Soc. 2005; 127: 6150-6151Crossref PubMed Scopus (52) Google Scholar, 15Obara K. Miyashita N. Xu C. Toyoshima I. Sugita Y. Inesi G. Toyoshima C. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14489-14496Crossref PubMed Scopus (211) Google Scholar) may be helpful in predicting the residues involved in H+ exchange, as they indicate that protonation of the four relevant transmembrane carboxyl groups is most probable for Glu-771 and then for Asp-800, Glu-309, and Glu-908 in decreasing order of probability. It was also noted (15Obara K. Miyashita N. Xu C. Toyoshima I. Sugita Y. Inesi G. Toyoshima C. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14489-14496Crossref PubMed Scopus (211) Google Scholar) that Glu-309 and Glu-908 may retain their protons through the cycle, because of involvement in hydrogen bonding and stabilization of the Ca2+-bound conformation. Our experiments with site-directed mutations (Fig. 7) reveal electrogenic signals following Ca2+ jumps on the E309A mutant. Because Ca2+ binding on site II does not occur in this mutant, the signal must be attributed to binding on site I. Development of the electrogenic signal then indicates that, at pH 7, the Ca2+ charge movement is not neutralized by H+ release from Glu-771 or Asp-800. Therefore, these residues are dissociated at neutral pH, as the enzyme resides in the E1 state to favor Ca2+ binding (Fig. 8). It is then likely that Glu-771 and Asp-800 are involved in the acquisition of H+ upon dissociation of Ca2+, as their protonation probability increases as a consequence of conformational changes upon enzyme phosphorylation. Finally, we consider that replacement of Ca2+ with H+ as well as introduction of water molecules may have an important role in stabilizing the enzyme structure in the E2 state (14Sugita Y. Miyashita N. Ikeguchi M. Kidera A. Toyoshima C. J. Am. Chem. Soc. 2005; 127: 6150-6151Crossref PubMed Scopus (52) Google Scholar, 15Obara K. Miyashita N. Xu C. Toyoshima I. Sugita Y. Inesi G. Toyoshima C. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14489-14496Crossref PubMed Scopus (211) Google Scholar). In addition, our pre-steady state measurements demonstrate that the protonation probability (i.e. apparent pKa) of acidic residues is altered during the ATPase cycle to favor the acquisition of Ca2+ in the ground state of the enzyme and exchange of the bound Ca2+ with H+ in E2-P. These reversible shifts in H+ acquisition sustain a mechanistic role in promoting dissociation of bound Ca2+. They are produced by changes of H+ accessibility and/or alterations of the electrostatic environment resulting from long range conformational changes triggered by utilization of ATP. In fact, we show in Fig. 5 that a reduction of H+ exchange, as the pH is increased to 8, places a limit to Ca2+ dissociation from the phosphoenzyme and to the Ca2+ concentration that can be reached in the lumen of the vesicles. A sequence of partial reactions within a Ca2+-ATPase cycle, as required to explain our findings, is shown in Fig. 8. Countertransport of H+ by the Ca2+-ATPase finds an analogy in the mechanism of the Na+,K+-ATPase as well as in the structural similarity of the two enzymes (45Sweadner K.J. Donnet C. Biochem. J. 2001; 356: 685-704Crossref PubMed Scopus (181) Google Scholar). We are indebted to Prof. Chikashi Toyoshima for helpful comments and suggestions.
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