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J. Biol. Chem., Vol. 279, Issue 16, 15888-15896, April 16, 2004

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Importance of Transmembrane Segment M1 of the Sarcoplasmic Reticulum Ca²+-ATPase in Ca²+ Occlusion and Phosphoenzyme Processing^*

Anja Pernille Einholm, Bente Vilsen, and Jens Peter Andersen

From the Institute of Physiology, University of Aarhus, Ole Worms Allé 160, DK-8000 Aarhus C, Denmark

Received for publication, January 7, 2004 , and in revised form, January 27, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The functional consequences of a series of point mutations in transmembrane segment M1 of sarcoplasmic reticulum Ca²⁺-ATPase were analyzed in steady-state and transient kinetic experiments examining the partial reaction steps involved in Ca²⁺ interaction and phosphoenzyme turnover. Arginine or leucine substitution of Glu⁵¹, Glu⁵⁵, or Glu⁵⁸, located in the N-terminal third of M1, did not affect these functions. Arginine or leucine substitution of Asp⁵⁹, located right at the bend of M1 seen in the crystal structure of the thapsigargin-bound form, caused a 10-fold increase of the rate of Ca²⁺ dissociation toward the cytoplasmic side. Mutation of Leu⁶⁰ to alanine or proline and of Val⁶² to alanine also enhanced Ca²⁺ dissociation, whereas an 11-fold reduction of the rate of Ca²⁺ dissociation was observed upon alanine substitution of Leu⁶⁵, thus providing evidence for a relation of the middle part of M1 to a gating mechanism controlling the dissociation of occluded Ca²⁺ from its membranous binding sites. Moreover, phosphoenzyme processing was affected by some of the latter mutations, in particular leucine substitution of Asp⁵⁹, and alanine substitution of Leu⁶⁵ accelerated the transition to ADP-insensitive phosphoenzyme and blocked its dephosphorylation, thus demonstrating that this part of M1, besides being important in Ca²⁺ interaction, furthermore, is a critical element in the long range signaling between the transmembrane domain and the cytoplasmic catalytic site.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The Ca²⁺-ATPase¹ of sarcoplasmic reticulum is a membrane-bound enzyme that pumps Ca²⁺ from the cytosol to the lumen of the sarcoplasmic reticulum at the expense of chemical energy being released by ATP hydrolysis (1–4). It belongs to the family of P-type ATPases characterized by the formation during the enzymatic cycle of a phosphorylated enzyme intermediate, in which the -phosphoryl group of ATP has been transferred to a conserved aspartic acid side chain in the enzyme. The phosphoenzyme intermediate is fundamental to the coupling between the spatially well separated processes of ATP hydrolysis in the cytoplasmic part of the molecule and vectorial ion transport across the membrane. Phosphorylation of the enzyme by ATP is activated by Ca²⁺ binding at cytoplasmically facing high affinity sites of the E₁ form. During the subsequent conformational change, [Ca₂]E₁P Ca₂E₂P, the Ca²⁺ binding sites are reoriented toward the lumen and their affinity markedly lowered, resulting in release of the bound ions (Scheme 1). It is believed that, following their binding at the high affinity sites, the Ca²⁺ ions are contained within an "occluded" state (here indicated by brackets around the Ca²⁺ ions) with no access to the medium on either side of the membrane (5–7).

View larger version (14K): [in this window] [in a new window]   SCHEME 1. Ca2+-ATPase reaction cycle. Simplified scheme illustrating the partial reactions of the Ca2+-ATPase reaction cycle. Occluded Ca2+ ions are shown in brackets.

With respect to the specific Ca²⁺ selectivity of the Ca²⁺-ATPase, it is interesting to note that the negatively charged M1 residues Glu⁵⁵ and Glu⁵⁸ are replaced by residues with positively charged side chains in the Na⁺, K⁺-ATPase. Because transmembrane segment M3 of both ATPases contains additional negatively charged residues, which appear to be located rather close to M1 in the three-dimensional structure, the situation could be somewhat similar to voltage-gated Ca²⁺ and Na⁺ channels, where the cation selectivity filter is formed by four negatively charged side chains in the former and two negative plus one positive side chain in the latter (15, 16). Thus, the above-mentioned charged residues within the cytoplasmic part of M1 of the Ca²⁺-ATPase could possibly contribute to a Ca²⁺ selectivity filter.

These considerations on the possible function of M1 of the Ca²⁺-ATPase in Ca²⁺ migration and in long range signal transmission have prompted a detailed study of the structural and functional aspects of M1. To test the above-mentioned hypothesis about Glu⁵¹, Glu⁵⁵, Glu⁵⁸, and Asp⁵⁹ as part of a possible Ca²⁺ entry pathway and/or selectivity filter, we have mutated these amino acids individually to the neutral leucine and to the oppositely charged and rather bulky arginine, which would be expected to impose electrostatic and steric effects on Ca²⁺ migration, if positioned near the migration pathway. Moreover, we have substituted the hydrophobic amino acids Phe⁵⁷, Leu⁶⁰, Val⁶², and Leu⁶⁵ to investigate the demands for bulkiness and hydrophobicity of these side chains. Phe⁵⁷ is located on the hydrophobic surface of the bent part of M1 in the E₂-Tg structure and could be instrumental in attaching this part to the lipid bilayer. The other hydrophobic residues are embedded in the membrane, Leu⁶⁵ below the level of the Ca²⁺ ions in the Ca²⁺-bound structure. The effects of these mutations on the various partial reaction steps involved in Ca²⁺ binding and catalysis have been analyzed, using among other assays also transient kinetic methods that allow measurement of the rates of conformational changes and Ca²⁺ dissociation toward the cytoplasmic side.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Site-directed Mutagenesis and Expression—The QuikChange site-directed mutagenesis kit (Stratagene) was used to introduce the desired mutations directly into full-length cDNA encoding the rabbit fast twitch muscle Ca²⁺-ATPase (SERCA1a isoform). The template in the mutagenesis reaction consisted of SERCA1a cDNA inserted into the expression vector pMT2 (17). For expression of recombinant protein, pMT2 containing wild-type or mutant cDNA was transfected into mammalian COS-1 cells using the calcium phosphate precipitation method (18). The microsomal fraction containing expressed wild-type or mutant Ca²⁺-ATPase was isolated by differential centrifugation (19), and the expression level was quantitated by a specific enzyme-linked immunosorbent assay (20).

Functional Studies—The ability of wild-type or mutant Ca²⁺-ATPase to transport Ca²⁺ and hydrolyze ATP was determined by studying the ⁴⁵Ca²⁺ uptake into microsomal membranes and the P_i liberation, respectively (21). The molecular turnover rate was calculated as the ratio between the specific Ca²⁺-ATPase activity and the active-site concentration. The latter was determined by quantitation of phosphorylation from [-³²P]ATP at 0 °C in the presence of a saturating Ca²⁺ concentration. Samples of wild-type and mutant Ca²⁺-ATPase were examined in parallel, and the molecular turnover rate corresponding to the mutants was expressed relative to that of the wild type. Partial reactions were studied according to previously described principles (20–23), taking advantage of the acid stability of the phosphoenzyme formed from [-³²P]ATP or ³²P_i, and details are given in the figure legends. A BioLogic quenched flow module QFM-5 or SFM-400/Q (Bio-Logic Science Instruments, Claix, France) was used for rapid mixing in experiments where the reaction time was below 1 s, applying the previously described protocols (22, 23). In all phosphorylation experiments, the reaction was quenched by addition of 0.5–2 volumes of 25% (w/v) trichloroacetic acid containing 100 mM H₃PO₄. Subsequently, the acid-precipitated enzyme was washed by centrifugation, dissolved in loading buffer and subjected to separation by SDS-PAGE at pH 6.0 (24). The distribution of radioactivity associated with the gel was quantitated by electronic autoradiography of the dried gel using a Packard Cyclone^TM Storage Phosphor System.

Data Analysis—Using the Sigmaplot program (SPSS Inc.), time courses of phosphorylation and dephosphorylation were fitted according to first-order kinetics, whereas ligand concentration dependences were fitted by use of the Hill equation. Generally, the experiments were performed at least twice on different enzyme preparations, and average values are shown in the figures. Standard errors are shown in the tables except for a few cases, in which the experiment (giving a wild type-like result) was performed only once.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Site-directed Mutagenesis and Expression—Several Ca²⁺-ATPase point mutants were designed to study the functional importance of transmembrane segment M1. An overview of these mutations is given in Table I. Two different strategies were used: (i) charge reversal or removal (Glu⁵¹, Glu⁵⁵, Glu⁵⁸, and Asp⁵⁹) and (ii) hydrophobic size reduction (Phe⁵⁷, Leu⁶⁰, Val⁶², and Leu⁶⁵). The carboxylic acid residues were replaced by the positively charged and rather bulky arginine and with leucine, removing the negative charge. Phe⁵⁷, Leu⁶⁰, Val⁶², and Leu⁶⁵ were replaced by the smaller hydrophobic alanine to test the importance of the bulkiness of these side chains. In addition, Phe⁵⁷ was replaced by the more polar glutamine, and Leu⁶⁰ by proline. The expression level of all these Ca²⁺-ATPase constructs in COS cells was rather similar to that of the wild type (data not shown).

Ca²⁺ Dependence of Phosphorylation from [-³²P]ATP—The Ca²⁺-mediated activation of phosphorylation from ATP requires the binding of both calcium ions at the high affinity cytoplasmically facing sites (cf. Reactions 2 and 3 in Scheme 1). Examination of the mutational effect on Ca²⁺ titration of phosphorylation therefore provides information about the Ca²⁺-binding properties. The Ca²⁺ dependence of steady-state phosphorylation was studied at 0 °C, where phosphoenzyme accumulation is favored. Fig. 1 shows the titration curves for some of the mutants, and Table I summarizes the data for all mutants. Substitution of Glu⁵¹, Glu⁵⁵, or Glu⁵⁸ by either leucine or arginine had no significant effect on the apparent Ca²⁺ affinity, as exemplified by Glu⁵⁸ Arg in Fig. 1 (cf. Fig. 1 and Table I). In contrast, arginine substitution of Asp⁵⁹ caused a 5-fold reduction of apparent Ca²⁺ affinity (increase of K_0.5) compared with wild type, whereas the apparent affinity for Ca²⁺ was unaffected by leucine or alanine substitution of this residue. These results are in accordance with a previous study in which the Ca²⁺ dependence of Ca²⁺ transport was found normal in a cluster mutant with alanine substitutions of Glu⁵⁵, Gln⁵⁶, Glu⁵⁸, and Asp⁵⁹ (25). The mutants Phe⁵⁷ Ala, Leu⁶⁰ Ala/Pro, and Val⁶² Ala, with alterations to hydrophobic residues, displayed moderate reductions of apparent Ca²⁺ affinity (1.5- to 3-fold increase of K_0.5). On the contrary, Leu⁶⁵ Ala displayed higher affinity for Ca²⁺ than wild type, the K_0.5 being reduced to 61% of the corresponding value for the wild type (Fig. 1 and Table I).

View larger version (30K): [in this window] [in a new window]   FIG. 1. Ca2+ dependence of phosphorylation from [-32P]ATP. Phosphorylation was performed at 0 °C for 15 s in 40 mM MOPS/Tris (pH 7.0), 80 mM KCl, 5 mM MgCl2, 1 mM EGTA, 5 µM [-32P]ATP, and CaCl2 to obtain the indicated concentrations of free Ca2+. The Hill equation, EP = EPmax·[Ca2+]n/(K0.5n + [Ca2+]n), was fitted to the data, and the results were normalized separately for wild type and each mutant, taking EPmax as 100%. The (relative) K0.5 values are listed in Table I together with the corresponding values for other mutants obtained in the same way.

View larger version (27K): [in this window] [in a new window]   FIG. 2. Time course of Ca2+ dissociation toward the cytoplasmic side determined by loss of ability to phosphorylate. The kinetics of Ca2+ dissociation toward the cytoplasmic side was monitored at 25 °C by the loss of ability to undergo phosphorylation from [-32P]ATP, using a Bio-Logic quenched flow module SFM-400/Q and the previously described mixing protocol (23). Wild-type or mutant Ca2+-ATPase preincubated in 40 mM MES/Tris (pH 6.0), 80 mM KCl, 5 mM MgCl2, and 100 µM CaCl2 was mixed with an equal volume of 40 mM MES/Tris (pH 6.0), 80 mM KCl, 5 mM MgCl2, and 4 mM EGTA, followed by the addition of the double volume of 40 mM MES/Tris (pH 6.0), 80 mM KCl, 5 mM MgCl2, 2 mM EGTA, and 10 µM [-32P]ATP at the times indicated on the abscissa, and acid quenching 34 ms later. A mono- or bi-exponential function was fitted to the data and the half-life (t0.5) determined. The (relative) t0.5 values are listed in Table I together with the corresponding values for other mutants obtained in the same way.

View larger version (15K): [in this window] [in a new window]   FIG. 3. Time course of phosphorylation of Asp59 Arg and wild type following addition of [-32P]ATP and Ca2+ to Ca2+-deprived enzyme in E2 form. Rapid kinetic measurements at 25 °C were performed using Bio-Logic quenched flow module QFM-5 or SFM-400/Q according to previously described mixing protocols (22). Wild-type or mutant enzyme preincubated in 40 mM MES/Tris (pH 6.0), 80 mM KCl, and 2 mM EGTA, was mixed with an equal volume of 40 mM MES/Tris (pH 6.0), 80 mM KCl, 10 mM MgCl2, 2.2 mM CaCl2, and 10 µM [-32P]ATP, followed by acid quenching at the indicted times. A monoexponential function was fitted to the data, and the results were normalized separately for wild type and mutant, taking the phosphorylation corresponding to infinite time as 100%. The (relative) rate constant is shown in Table I together with the corresponding values for other mutants obtained in the same way.

View larger version (15K): [in this window] [in a new window]   FIG. 4. Vanadate inhibition of phosphorylation of Asp59 Arg and wild type from [-32P]ATP. Prior to phosphorylation, microsomal preparations were sequentially incubated for 1 h at 25 °C and 15 min at 0 °C in 40 mM MOPS/Tris (pH 7.0), 80 mM KCl, 5 mM MgCl2, 2 mM EGTA, and the indicated concentration of . CaCl2 (2.5 mM) was then added, and immediately after 5 µM [-32P]ATP, to phosphorylate the vanadate-free fraction of the enzyme, followed by acid quenching 15 s later. The Hill equation for inhibition, was fitted to the data, and the results were normalized separately for wild type and mutant, taking EPmax as 100%. The (relative) K0.5 value is shown in Table II together with the corresponding values for other mutants obtained in the same way.

View larger version (23K): [in this window] [in a new window]   FIG. 5. Time course of dephosphorylation of phosphoenzyme formed from [-32P]ATP. Phosphorylation was performed at 0 °C for 15 s in 40 mM MOPS/Tris (pH 7.0), 80 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.955 mM CaCl2 (giving a free Ca2+ concentration of 10 µM during phosphorylation), 10 µM calcium ionophore A23187 [GenBank] , and 5 µM [-32P]ATP. To follow the dephosphorylation, the phosphoenzyme was chased by addition of 6.7 mM EGTA with 1 mM non-radioactive MgATP followed by acid quenching at the time intervals indicated. A monoexponential decay function was fitted to the data, and the maximal phosphorylation was taken as 100%. The (relative) rate constants are listed in Table II together with the corresponding values for other mutants obtained in the same way.

View larger version (30K): [in this window] [in a new window]   FIG. 6. Time course of dephosphorylation of E2P phosphoenzyme at pH 6.0. Phosphorylation was carried out at 25 P °C for 10 min in 100 mM MES/Tris (pH 6.0), 10 mM MgCl, 2 mM EGTA, 30% (v/v) Me2SO, and 0.5 mM 32Pi. Subsequently, the phosphoenzyme was chased by a 19-fold dilution of an aliquot into a medium (kept at 25 °C) containing 100 mM MES/Tris (pH 6.0), 2 mM EGTA, 10 mM EDTA, 15% Me2SO, and 0.5 mM non-radioactive Pi, and acid quenching was performed at the indicated time intervals. A monoexponential decay function was fitted to the data, and the maximal phosphorylation was taken as 100%. Each mutant is shown together with wild-type data obtained in the same series of experiments, and the (relative) rate constants are listed in Table II together with the corresponding values for other mutants obtained in the same way.

View larger version (14K): [in this window] [in a new window]   FIG. 7. Time course of dephosphorylation of E P phosphoenzyme at pH 7.0. Experiments were conducted and analyzed as described for Fig. 6, except that the chase medium was kept at 0 °C and contained 40 mM MOPS/Tris (pH 7.0), 5 mM MgCl2, 2 mM EGTA, 80 mM KCl, and 0.5 mM non-radioactive Pi.

View larger version (14K): [in this window] [in a new window]   FIG. 8. Time course of accumulation of ADP-insensitive phosphoenzyme in Leu65 Ala and wild type. Phosphorylation was performed at 0 °C for the indicated time intervals in 40 mM TES/Tris (pH 8.0), 80 mM LiCl, 10 mM MgCl2, 50 µM CaCl2, 10 µM calcium ionophore A23187 [GenBank] , and 5 µM [-32P]ATP. At the indicated time intervals after initiation of phosphorylation, an equal volume of 40 mM TES/Tris (pH 8.0), 80 mM LiCl, 10 mM MgCl2, 10 mM EGTA, and 2 mM ADP was added to remove ADP-sensitive phosphoenzyme, followed by acid quenching 4 s later. A monoexponential function was fitted to the data, and the results were normalized separately for wild type and mutant, taking the phosphorylation corresponding to infinite time as 100%. The (relative) rate constant is shown in Table II together with the corresponding value for Asp59 Leu obtained in the same series of experiments.

The dephosphorylation kinetics of E₂P phosphoenzyme (cf. Reaction 6 in Scheme 1), formed under the conditions just described, was studied for some of the mutants by a 19-fold dilution into a chase medium, which terminated the phosphorylation from ³²P_i. Fig. 6 shows the time course of E₂P dephosphorylation at 25 °C in medium containing 15% dimethyl sulfoxide at pH 6.0 in the absence of K⁺, i.e. conditions where this reaction is rather slow and, therefore, easy to follow, and which are rather similar to those under which the K_0.5 values for P_i were obtained. The rate constants determined by fitting a monoexponential function are listed in Table II. The variation of the k_obs for E₂P dephosphorylation paralleled to a large extent the change in K_0.5 for P_i. Hence, the variation of the K_0.5 for P_i is largely due to the mutational effects on E₂P dephosphorylation. In particular, an 8-fold increase of k_obs was determined for Asp⁵⁹ Arg, whereas Asp⁵⁹ Leu showed a 4-fold decrease (Fig. 6). Leu⁶⁰ Pro showed a 5-fold enhanced k_obs, whereas Phe⁵⁷ Gln/Ala, Leu⁶⁰ Ala, and Val⁶² Ala showed minor changes relative to wild type (up to 2.3-fold). A most conspicuous, almost complete block of E₂P dephosphorylation was seen for Leu⁶⁵ Ala (Fig. 6 and Table II).

The markedly reduced k_obs values for E₂P dephosphorylation of Asp⁵⁹ Leu and Leu⁶⁵ Ala seem to explain perfectly well the accumulation of ADP-insensitive phosphoenzyme (E₂P) observed for these mutants in the phosphorylation experiments with ATP described above. These experiments were, however, performed at pH 7.0, 0 °C, and in the presence of K⁺, whereas the results presented in Fig. 6 were obtained under conditions that deliberately slowed the dephosphorylation of E₂P. This led us to study the dephosphorylation of E₂P further for selected mutants at pH 7.0, 0 °C, presence of K⁺, and the results are seen in Fig. 7 and Table II. Clearly, the dephosphorylation of E₂P was found markedly inhibited in Asp⁵⁹ Leu and Leu⁶⁵ Ala also under these conditions (10- and 33-fold, respectively, relative to wild type).

Time Course of the [Ca₂]E₁P Ca₂E₂P Transition—Because the data in Fig. 7 show that E₂P E₂ is the major rate-limiting step in the dephosphorylation of mutants Asp⁵⁹ Leu and Leu⁶⁵ Ala, even at pH 7.0, 0 °C, presence of K⁺, the results in Fig. 5 do not provide information about the rate of the [Ca₂]E₁P Ca₂E₂P transition (cf. Reaction 4, Scheme 1) in these mutants. Such information was instead obtained using an experimental design that takes advantage of the ADP-sensitivity of [Ca₂]E₁P. The enzyme was phosphorylated with [-³²P]ATP under conditions known to slow down dephosphorylation of E₂P, such that E₂P accumulates even in the wild type (0 °C, pH 8.0, presence of Li⁺ instead of K⁺, high Mg²⁺/Ca²⁺ ratio). After various phosphorylation intervals, the amount of ADP-insensitive phosphoenzyme was determined as the phosphoenzyme remaining after a 4-s incubation with ADP to remove [Ca₂]E₁P. Because the [Ca₂]E₁ [Ca₂]E₁P reaction is very rapid, the rate of appearance of ADP-insensitive phosphoenzyme corresponds to that of the [Ca₂]E₁P Ca₂E₂P transition under these conditions. Interestingly, the rates determined for Asp⁵⁹ Leu and Leu⁶⁵ Ala in this way were, respectively, 3.4- and 4.2-fold higher than that of the wild type (Fig. 8 and Table II). This constitutes a second factor, besides the block of E₂P dephosphorylation, contributing to the high steady-state concentration of ADP-insensitive phosphoenzyme in these mutants.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The present results provide the first functional evidence that transmembrane segment M1 of the Ca²⁺-ATPase is critical to Ca²⁺ interaction and to phosphoenzyme turnover. During active Ca²⁺ transport, the Ca²⁺-ATPase binds Ca²⁺ from the cytoplasmic side of the membrane, transfers the ions across the membrane, and releases them on the luminal side. It is generally believed that the translocation of Ca²⁺ across the membrane involves an "occluded" state, where the ions are inaccessible to the medium on either side of the membrane and their dissociation restricted by structural components acting as gates. In the occluded state, Ca²⁺ is bound by residues in M4, M5, M6, and M8 (8, 10–12). The Ca²⁺ ions may become occluded within the protein concomitantly with phosphorylation from ATP (5, 33). Evidence has, however, been presented that the Ca²⁺ ions are not only occluded in the phosphoenzyme, but also most of the time in the non-phosphorylated Ca²⁺-bound form (6), as indicated by the brackets in Scheme 1. This seems to agree with the crystal structure of the Ca²⁺-ATPase with bound Ca²⁺, revealing no obvious Ca²⁺ entry or exit pathways (8). Furthermore, the fact that Ca²⁺ can dissociate only very slowly from the enzyme complex with CrATP, even though the -phosphoryl group is not transferred to the enzyme in this complex, indicates that phosphorylation is not required for occlusion of Ca²⁺ (7). The present findings are consistent with the existence of a Ca²⁺-occluded non-phosphorylated state, whose stability depends on the structural properties of M1. Our measurements indicate a 10-fold increase of the rate of Ca²⁺ dissociation from this state toward the cytoplasmic side in mutants Asp⁵⁹ Arg and Asp⁵⁹ Leu, whereas Ca²⁺ dissociation was wild type-like in the mutants with arginine or leucine substitution of Glu⁵¹, Glu⁵⁵, or Glu⁵⁸ (Fig. 2 and Table I). The Phe⁵⁷ mutants also displayed wild type-like Ca²⁺ dissociation, whereas Leu⁶⁰ Ala/Pro and Val⁶² Ala caused an acceleration of Ca²⁺ dissociation, although to a lesser extent than the Asp⁵⁹ mutations. Moreover, a remarkable 11-fold reduction of the rate of Ca²⁺ dissociation toward the cytoplasmic side was observed for Leu⁶⁵ Ala. This demonstration of the role of M1 in Ca²⁺ occlusion was made feasible by the quenched flow technique that allows measurement of Ca²⁺ dissociation from the [Ca₂]E₁ form toward the cytoplasmic side, even with the relatively small amounts of enzyme that can be harvested from the cell culture. Thus, the rate of Ca²⁺ dissociation from the [Ca₂]E₁ form can be both increased and decreased by mutation within the middle part of M1 (C-terminal to the bend of M1 seen in the E₂-Tg crystal structure), and our results suggest that the middle part of M1, but not the most N-terminal part containing Glu⁵¹, Glu⁵⁵, and Glu⁵⁸, is important in control of the gates at the Ca²⁺ occlusion sites.

As regards the proposed role of Glu⁵¹, Glu⁵⁵, and Glu⁵⁸ in the Ca²⁺ entry pathway (14), the results reported here clearly argue against any involvement of these glutamate side chains in Ca²⁺ recognition and binding, and it is not likely that they contribute to a Ca²⁺ selectivity filter. Furthermore, the finding that substitution of Glu⁵⁸ by arginine left the Ca²⁺ binding properties of the Ca²⁺-ATPase unaffected questions the existence of a close interaction between Glu⁵⁸ in M1 and Glu³⁰⁹ at Ca²⁺ binding site II, such as seen in the published crystal structure of the Ca²⁺-bound enzyme (8). A possible reason for this apparent discrepancy could be a high degree of thermal mobility of the Glu⁵⁸ side chain. It is also possible that the [Ca₂]E₁ form adopted in the native state differs more profoundly from the crystal structure.

The reduced apparent affinity of mutant Asp⁵⁹ Arg for Ca²⁺ activation of phosphorylation (Fig. 1) reflects the enhanced rate of Ca²⁺ dissociation from [Ca₂]E₁ and not a displacement of the E₂-E₁ equilibrium in favor of the low affinity E₂ form. In fact, the data in Figs. 3 and 4 suggest that mutation Asp⁵⁹ Arg enhances the rate of the E₂ E₁ conformational change, leading to accumulation of E₁ with resulting low sensitivity to vanadate inhibition of phosphoenzyme formation. Because release of counter-transported protons from E₂ may be rate-limiting for the E₂ E₁ transition (27), the enhancement of this transition suggests that proton dissociation from the transport sites, like Ca²⁺ dissociation, is facilitated by the Asp⁵⁹ Arg mutation.

For mutants Leu⁶⁰ Ala/Pro and Val⁶² Ala, the enhanced rate of Ca²⁺ dissociation likewise seems to result in a reduced apparent affinity for Ca²⁺ activation of phosphorylation. In addition, there could be a contribution to the reduced apparent affinity for Ca²⁺ from displacement of the E₂-E₁ equilibrium in favor of the low affinity E₂ form in Leu⁶⁰ Ala and Val⁶² Ala, because the vanadate affinity was slightly increased in these mutants (Table II). The apparent affinity for Ca²⁺ in activation of phosphorylation was normal in Asp⁵⁹ Leu, despite the increased Ca²⁺ dissociation rate (Figs. 1 and 2). This may be accounted for by a considerably reduced rate of dephosphorylation (Figs. 5, 6, 7). As previously reported, the effect of a low rate of phosphoenzyme turnover on the apparent affinity for Ca²⁺ activation of phosphorylation can be understood on the basis of computer simulations of the Ca²⁺-ATPase reaction cycle (34). According to the computational analysis, an increased apparent affinity for Ca²⁺ is actually expected when the rate of phosphoenzyme turnover is reduced, because a lower phosphorylation rate (i.e. Ca²⁺ saturation) is required to maintain a certain level of phosphorylation under these conditions. Hence, for Asp⁵⁹ Leu, the increased rate of Ca²⁺ dissociation and the decreased rate of phosphoenzyme turnover act in opposite directions, thereby masking the effects on the apparent Ca²⁺ affinity.

The importance of the middle part of M1 in the gating at the Ca²⁺ occlusion sites may be related to the presence of a water-accessible channel leading between M1 and M3 in the crystal structure of Ca²⁺-ATPase in the Ca²⁺-free E₂-Tg form. This channel has the potential of a Ca²⁺ entry port and is apparently opened by the bending and partial unfolding of the helical structure of M1 at Asp⁵⁹ (9). A similar channel may exist in the native non-crystalline protein and function as a migration pathway for Ca²⁺ in one or more of the conformations that precede the occluded [Ca₂]E₁ form in the process of Ca²⁺ binding (E₂, E₁, and CaE₁). Because the channel leads to Glu³⁰⁹ at Ca²⁺ site II, it may provide a passage for the Ca²⁺ ion that binds at site II (i.e. the one that binds last in the sequential mechanism). In line with this assignment, the presently observed mutational effects on the rate of disappearance of ability to phosphorylate from ATP upon addition of EGTA (Fig. 2) reflect changes of the rate of dissociation of the Ca²⁺ ion that leaves first in the sequential mechanism, i.e. the one that was bound last (26, 35). The Ca²⁺ ion that binds first (at site I, cf. Ref. 12) might enter (and leave upon dissociation) by a different route. Some evidence has actually been presented suggesting the existence of a pre-binding site for Ca²⁺ in the loop between transmembrane segments M6 and M7 ("L6–7"), which could be related to the entry port for the Ca²⁺ ion binding at site I (36).

Comparison of the two crystal structures of the Ca²⁺-ATPase indicates that the transition from [Ca₂]E₁ to E₂-Tg is accompanied by a lateral and upward (toward the cytoplasmic side) movement of M1 in the membrane, and bending of the helix at Asp⁵⁹, which probably is caused by steric collision with M3 (9). In E₂-Tg, the N-terminal part of M1 containing Glu⁵¹, Glu⁵⁵, and Glu⁵⁸ is bent away from M3, and their side chains define the hydrophilic surface of the amphipathic section. These residues are therefore peripherally located with respect to the proposed Ca²⁺ entrance. A similar position of this part of M1 in the native non-crystalline protein could explain the lack of importance of the glutamate side chains for Ca²⁺ binding. Asp⁵⁹, on the other hand, is located right at the bending point of M1. Thus, the dramatic effect on Ca²⁺ dissociation of substitution of Asp⁵⁹ by leucine or arginine may result from direct interference with Ca²⁺ interaction or interference with the movement of M1 that occludes Ca²⁺, a movement that conceivably is facilitated by flexibility at Asp⁵⁹. The finding that alanine substitution of Asp⁵⁹ is tolerated stresses the requirement for a small side chain that allows movement. The negative charge of the Asp⁵⁹ side chain, which might have been expected to participate in directing Ca²⁺ to the binding sites, seems not to be required for normal Ca²⁺ interaction.

Among the hydrophobic side chains substituted in the present study, Phe⁵⁷ seems less important for the Ca²⁺-binding properties and the E₂ E₁ conformational transition than expected if it were crucial to the attachment of the bent part of M1 to the lipid bilayer and, thereby, to the positioning of this part of M1. On the other hand, the lack of a significant effect on Ca²⁺ binding of substitution of Phe⁵⁷ may be considered consistent with its location away from the proposed Ca²⁺ inlet, on the hydrophobic surface of the bent part of M1, and it is conceivable that the removal of a single anchoring side chain is not enough to disrupt the attachment to the lipid bilayer. As regards Leu⁶⁰, Val⁶², and Leu⁶⁵, which are embedded within the membrane, their bulky hydrophobic side chains appear to form an integral part of the wall lining the water-accessible channel seen in the E₂-Tg structure, with Leu⁶⁵ at the bottom. Hence, our finding of significant changes of the Ca²⁺ dissociation rate upon substitution of these residues with the smaller alanine seems to be compatible with the hypothesis that this channel serves as a migration pathway for Ca²⁺. The effects of hydrophobic size reduction in this part of M1 could simply be due to local changes in the proportions of the channel, providing a more free passage for the dissociating Ca²⁺ ions in case of Leu⁶⁰ Ala/Pro and Val⁶² Ala and collapsing the channel in case of the Leu⁶⁵ Ala mutation. Of course, it cannot be excluded that changes to M1 exert more distant effects on a Ca²⁺ migration pathway located elsewhere in the protein.

In the crystal structure of the Ca²⁺-bound enzyme, Leu⁶⁵ is below the level of the Ca²⁺ ions (i.e. closer to the luminal surface), and it seems that its bulky side chain may contribute to form a luminal gate, preventing the immediate transport of the ions to the lumen upon binding from the cytoplasm. A premature transfer of the Ca²⁺ ions to the lumen caused by a defective luminal gate in Leu⁶⁵ Ala (i.e. rapid formation of an enzyme form somewhat similar to Ca₂E₂P) could account for the rapid disappearance of ADP sensitivity of the phosphoenzyme in this mutant (Fig. 8), because the ADP sensitivity is thought to be specifically associated with the [Ca₂]E₁P form. Interestingly, also Asp⁵⁹ Leu showed an enhanced rate of disappearance of ADP sensitivity of the phosphoenzyme (Table II), which again might indicate premature Ca²⁺ transfer to luminally facing sites. In addition to the enhanced rate of disappearance of ADP sensitivity of the phosphoenzyme, Leu⁶⁵ Ala and Asp⁵⁹ Leu both showed a conspicuous block of the dephosphorylation of E₂P, the rate being reduced 33-fold and 4- to 10-fold (depending on pH), respectively (Table II). Hence, there is a preference for forms with luminally facing Ca²⁺ sites and ADP insensitivity of the phosphoenzyme (Ca₂E₂P and E₂P) in both of these mutants. A difference between these mutants is, however, that Ca²⁺ dissociation toward the cytoplasmic side is increased in Asp⁵⁹ Leu, whereas it is inhibited in Leu⁶⁵ Ala (Fig. 2).

Some of the other mutations also affected the processing of the phosphoenzyme (Reactions 4–6 in Scheme 1). Hence, Leu⁶⁰ Ala and Val⁶² Ala showed a slightly enhanced rate of dephosphorylation of phosphoenzyme formed from ATP (Fig. 5 and Table II), probably reflecting enhancement of the rate-limiting [Ca₂]E₁P Ca₂E₂P transition. The rate of E₂P dephosphorylation was markedly increased in Asp⁵⁹ Arg (8-fold) and Leu⁶⁰ Pro (4- to 5-fold) and to a lesser extent in Phe⁵⁷ Ala/Gln and Val⁶² Ala (about 2-fold, Table II). It is noteworthy that the replacement of Asp⁵⁹ with arginine and leucine led to opposite effects on E₂P dephosphorylation, which may be attributable to the difference in charge/polarity, resulting in different potentials for salt bridge formation or interaction with the lipid bilayer. The leucine may fix this part of M1 in close contact with the lipid bilayer, whereas the arginine may tend to draw it into the solvent phase. Taken together, the effects of M1 mutations on phosphoenzyme processing demonstrate that M1, besides being important for Ca²⁺ interaction, furthermore is a critical element in the long range signaling between the transmembrane domain and the catalytic site in the cytoplasmic part of the Ca²⁺-ATPase molecule. Several pieces of evidence suggest that the A-domain undergoes large movements during the catalytic cycle, being separated from the P-domain in [Ca₂]E₁ and [Ca₂]E₁P and in rather close contact with this domain in E₂ and E₂P, probably contributing to the catalytic site in these forms (9, 37–39). Because M1 is physically connected to the A-domain through a flexible hinge segment at the N-terminal end and through M2 at the other end, it is tempting to speculate that the movement of the A-domain is closely coupled with movement of M1, and that the effects of the M1 mutations on the processing of the phosphoenzyme, described here, are brought about by an altered position and/or reduced motional freedom of the A-domain, caused by changes of the position and structure of M1.

In conclusion, our results provide evidence for a relation of the middle part of M1 to a gating mechanism controlling the dissociation of Ca²⁺ from its membranous binding sites. Our results further indicate that these residues are also critical to the proper processing of the phosphoenzyme and, thus, to the long range transmission of conformational changes to the cytoplasmic catalytic site.

	FOOTNOTES

 
^* This study was funded in part by grants from the Danish Medical Research Council, the Novo Nordisk Foundation, the Lundbeck Foundation, and the Research Foundation of Aarhus University. The costs of publication of this article were defrayed in part by the payment of page charges. This 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. Tel.: 45-89-422-814; Fax: 45-86-129-065; E-mail: jpa{at}fi.au.dk.

¹ The abbreviations used are: Ca²⁺-ATPase, the sarco(endo)plasmic reticulum Ca²⁺-transporting adenosine triphosphatase (EC 3.6.1.38 [EC] ); M1–M10, transmembrane segments numbered from the N terminus; MES, 2-[N-morpholino]ethanesulfonic acid; MOPS, 3-[N-morpholino]propanesulfonic acid; TES, N-[tris(hydroxymethyl)methyl]-2-aminoethane-sulfonic acid; SERCA, sarcoplasmic-endoplasmic reticulum calcium ATPase; Tg, thapsigargin.

	ACKNOWLEDGMENTS

 
We thank Karin Kracht and Lene Jacobsen for expert technical assistance and Dr. J. D. Clausen (University of Aarhus) for discussion and help with some experiments. Dr. C. Toyoshima (University of Tokyo) is thanked for discussion on several occasions and Dr. R. J. Kaufmann (Genetics Institute, Boston) for the gift of the expression vector pMT2.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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