Calcium pump of the plasma membrane.

PHYSIOLOGICAL REVIEWS Vol. 71, No. I, January 1991 Pm’nted in U.S.A.

Calcium Pump of the Plasma Membrane ERNEST0 Laboratory

of Biochemistry,

Swiss Federal

CARAFOLI Institute

of Technology

(ETH),

Zurich,

Switzerland

I. Introduction ........................................................................................... II. Plasma Membrane Ca2+ Pump ........................................................................ A. Discovery and general properties ................................................................. B. Calmodulin stimulation ........................................................................... C. Activation of Ca2+ pump by treatments different from calmodulin .............................. D. Inhibitors of Ca2+ pump ........................................................................... E. Plasma membrane Ca2+ pump in other cell types ................................................ III. Isolated Pump ......................................................................................... A. Isolation, reconstitution, and properties of purified pump ....................................... B. Proteolysis of purified pump ...................................................................... IV. Primary Structure of Pump and Identification of Some of its Functional Domains ................ V. Hormonal Regulation of Plasma Membrane Ca2+ Pump ............................................. VI. Pathological Alterations of Plasma Membrane Ca2+ Pump ..........................................

129 130 130 132 133 134 135 137 137 140 142 146 146

gradient across the plasma membrane and the large number of specific Ca2+ binding proteins, soluble or inThe role of Ca2+ in cell regulation has recently be- trinsic to membranes, that perform the task of decoding the information of Ca2+ and of controlling its cytosolic come a “hot” topic. The fact that Ca2+was fundamental concentration, respectively. The necessity of maintainto cell signaling had been shown long ago by Ringer’s ing a signaling element such as Ca2+at a very low intraclassic experiments on the role of Ca2+in muscle contraction (216) and by a number of striking findings on the cellular concentration is self-evident; the background Ca2+ triggering of functions as different as secretion, concentration of a free Ca2+ in most cytosols oscillates motility, and nervous activity. However, only a few pio- indeed between 0.1 and 0.2 PM. Although less obvious, neers had a full perception of the general importance of the convenience of the large inwardly directed Ca2+grathe observations. A reference to Heilbrunn (110) is ap- dient across the plasma membrane can be also underproriate in this context and so is the quotation of the stood. The plasma membrane has very limited and very now-famous statement by 0. Loewy: “ja, Kalzium . . . carefully controlled Ca2+ permeability. In the presence das ist alles!” Only much later, perhaps about 10 years of a large Ca2+pressure, even very minor changes of this ago, the importance of Ca2+ as a universal cell messen- permeability will result in significant swings in the inger started to become generally obvious. It is difficult to tracellular concentration of Ca2+ and would thus effitrace back the phenomenal growth of the Ca2+ field to ciently influence the modulation of Ca2+ targets. However, it must also be recognized that the large transany single development: a coincidence of events is likely to have led to it. One could list here the rapid progress in plasma membrane Ca2+ pressure, however convenient the field of Ca2+ionophores, the discovery of calmodulin dynamically, carries with it an element of danger. If the permeability barrier of the plasma membrane were to and of other intracellular Ca2+binding proteins, the dramatic advances in the area of membrane transport of fail, cellular Ca2+ overload would necessarily ensue, Ca2+, and the development of precise methods for mea- leading to a situation of emergency that could end in cell suring the intracellular concentration of Ca2+. The ex- death; inundation of the cytosol with Ca2+ is indeed a pansion of interest in the regulatory role of Ca2+ frequent and early event in cell pathology. One could thus say that eucaryotic cells have elected to live in a prompted by these developments has been autocatalytic; the number of processes known to be controlled by permanent state of controlled risk, a dynamically conveCa2+has rapidly grown to dozens. The degree of sophis- nient but nevertheless dangerous choice where the martication, structural and otherwise, in the understanding gin separating cells from Ca2+catastrophe may on occaof the mechanisms for the control of cellular Ca2’ has sion be very thin. grown in parallel. The necessity of systems ejecting Ca2+from cells to Some essential concepts in the area of cellular ho- offset its downhill penetration is an obvious consemeostasis of Ca2+ (38) are briefly discussed here as a quence. Although eucaryotic cells generally satisfy most preamble to the discussion of the plasma membrane of their Ca2+demands by extracting Ca2+from their own Ca2’ pump, i.e., the very large inwardly directed Ca2+ internal stores, it is self-evident that the long-term I.

INTRODUCTION

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maintenance of the Ca2+ gradient across the plasma membrane is the result of the concerted operation of the importing system (the Ca2+channel) and of two exporting systems (the Ca2+ pump and the Na+-Ca2+ exchanger) of the plasma membrane. The Na+-Ca2+ exchanger is a large-capacity, low-affinity carrier, which is well represented in excitable cells where it presumably ejects the bulk amounts of Ca2+ demanded by the functional cycle. The Ca2+pump is a lower capacity system, which has very high Ca2’ affinity and is ubiquitously distributed in the eucaryotic world. Its high affinity enables it to interact with Ca2+ even at the very low background intracellular concentration of resting cells. It thus presumably functions continuously, satisfying the demands for the fine tuning of intracellular Ca2+.

II.

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MEMBRANE

CA2+

A. Discove~ry and Gewral

PUMP

Properties

The existence of a Ca2+-dependent adenosinetriphosphatase (ATPase) in the erythrocyte membrane was first reported by Dunham and Glynn in 1961 (71), but it was Schatzmann (236) who connected it to the pumping of Ca2+out of the cell in 1966. The basic experiment of Schatzmann demonstrated that Ca2+“emerged” from Ca2+-and ATP-loaded ghosts at a higher rate than from cells treated in the same way except for the addition of ATP. Because Ca2’ emerged even in media containing higher Ca2+ concentrations than inside the ghosts, it was clear that an ATP-powered Ca2’ pump was at work. After this original observation, for more than 10 years studies on plasma membrane Ca2+ used the erythrocyte exclusively as an experimental subject. Only much later was the work extended to other cells, showing that the ATP-dependent pumping of Ca2+ is a general property of plasma membranes, including those from excitable cells where it was generally assumed that the Ca2+ exporting system was a Na’-Ca2+ exchanger (13, 213). The general mechanism of the pump (for review see Ref. 238) follows the general lines of all other P-type ion pumps (195,196). The cycle begins with the Ca2+-dependent transfer of the terminal phosphate of ATP to the pump, with the formation of a phosphorylated intermediate (132, 136). With the use of the erythrocyte membrane it is very easy to distinguish the Ca2+-induced phosphorylation of the pump from that of other membrane proteins, since the former is much faster [8-15 s for half-maximal phosphorylation (209)]. The a-subunit of the Na+-K+-ATPase is also phosphorylated rapidly, but its molecular weight is different: the original experiment on the erythrocyte phosphoenzyme showed that the erythrocyte pump was a protein of apparent molecular weight - 140,000, since radioactivity was incorporated in a Ca2+-dependent fashion only in a protein of

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that size. Others determined the affinity of the erythrocyte pump for Ca2+ in the phosphorylation reaction and found values between 0.2 (256) and 7 PM (209). The matter of the affinity for ATP is more complex. On the basis of biphasic activation curves a number of people have postulated that there are two ATP sites that widely differ in affinity. The high-affinity site, normally assumed to be the catalytic site, has a Michaelis constant (&J of between l-2 (179) and 2.5 PM (215). The low-affinity site has a K, of between 145 and 180 PM (179,215) and is assumed to play a role in the decomposition of the phosphorylated intermediate. The pump bearing the phosphorylated intermediate (El-P in the conventional formalism) undergoes a conformation transition promoted by Mg2+: the new conformer is called E,-P. As mentioned, its hydrolysis to E, and Pi is assumed to be accelerated by the binding of ATP to a site on the pump that has less affinity than the site that binds ATP to initiate phosphorylation (also seebelow). The steadystate phosphorylation level in the plasma membrane Ca2+-ATPase is usually much lower than in the Ca2+-ATPase of sarcoplasmic reticulum, implying that under most experimental conditions the ratio between the rates of formation and breakdown of the phosphorylated intermediate is less favorable in the plasma membrane pump. However, at variance with the sarcoplasmic reticulum pump, the well-known inhibitor La3+ (207, 231) at low concentration (between 0.05 and 0.1 mM) increases the steady-state level of the phosphoenzyme in the plasma membrane Ca2+ pump (85, 239, 255, 256, 284), an effect that could be conveniently explained with the inhibition of the hydrolysis of the phosphorylated intermediate by La3+. This is an useful observation, since it permits the detection of the phosphoenzyme, in polyacrylamide gels, with far greater sensitivity and permits one to distinguish between the phosphoenzymes of the sarcoplasmic reticulum and the plasma membrane pumps in mixed preparations. In the last step of the reaction cycle the dephosphorylated E, conformer reverts back to the starting E, conformation. It is logical to assume that the interaction of La3+ occurs at the inner side of the membrane. However, under these conditions the inhibition of Ca2+transport is complete at La3+ concentrations that inhibit the Ca2+promoted ATP hydrolysis by only -50%. In contrast, the stimulation of phosphorylation at the internal membrane side has been found to occur with an extremely high La3+ affinity [La3+ affinity constant (&J = 5 pM (256)]. As discussed below, the effects of La3+ could be traced back to the “uncoupling” of the Pump* The role of Mg2+ in the reaction cycle is complex and requires some comments. Although phosphorylation in the presence of Ca2+occurs without Mg2+ @IO), the reaction is accelerated in its presence (85,209), leading to an increase of the steady-state amount of the phosphorylated intermediate (209, 239). It may be worthwhile to note that the K, (ATP) for the phosphorylation reaction is increased in the presence of Mg2+ [from 1.6 to 6.5

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PM (209)]. Also, Mg2+ influences the dephosphorylation process, since the decay of the phosphorylated intermediate is greatly accelerated by the addition of Mg2+ after a large pulse of ATP has been allowed to occupy the low-affinity site (209). Both high ATP and Mg2+ are required for the dephosphorylation process; however, if present during the Ca2+ -promoted phosphorylation, Mg2+ can be removed by chelation before initiating the dephosphorylation without affecting the acceleration of dephosphorylation by ATP. This has been taken to indicate that ATP and Mg2+ act at different steps (85); Mg2+ would not be necessary for the hydrolysis of the phosphorylated intermediate proper but is for a preceding step, which would somehow make the phosphointermediate accessible to water (238). Orthovanadate, which is present essentially at physiological pH as [VO,(OH>1”-, is a pentacoordinate (trigonal bipyramid) stereo analogue of phosphate that is now considered as the classic inhibitor of P-type ionmotive ATPases (195,196) and has been found to inhibit the erythrocyte Ca2+ pump (23, 24, 222). It is generally assumed that vanadate inhibits by binding to the amino acid residue (an aspartyl residue in all pumps so far studied) that forms the phosphorylated intermediate at the active site. The potency of vanadate as an inhibitor greatly depends on the ionic composition of the medium and on the concentration of ATP (15). The ions Mg2+, K+, and Na+ enhance the inhibition (in fact, vanadate is almost ineffective in the absence of Mg2+) by increasing the affinity of the pump for Ca2+. Under optimal conditions, i.e., in the presence of both Mg2+ and K+, the inhibitor constant (Ki> may be as low as 2-3 PM. At the low concentrations required for the formation of the phosphoenzyme, Ca2+ does not influence the inhibition by vanadate but decreases it very markedly when used in high concentrations. Vanadate acts as a noncompetitive inhibitor against ATP at the high-affinity site (15) but is a mixed, partially competitive, inhibitor at the lowaffinity site (15). In analogy with proposals on the sarcoplasmic reticulum Ca2+ pump (111) and on the Na+K+-ATPase (129), it is generally assumed that vanadate interacts with the E, conformer of the ATPase, blocking the last step of the reaction cycle, i.e., the E, - E, transition. The enzyme is thus stabilized in the E, conformation. Experiments on erythrocyte ghosts (ZZZ), but also on other cell systems, have shown that the vanadatesensitive site is located at the cytosolic side of the plasma membrane. One aspect of the reaction cycle summarized in Figure 1 that has not been conclusively established is the step during which the translocation of Ca2+ across the hydrophobic barrier of the membrane takes place. Obviously the phosphorylation step precedes the Ca2+ translocation step; this must be because considerable evidence shows that in the phosphorylated state of the pump the Ca2+ binding site faces the interior of the cell (e.g., see Ref. 238). On the other hand, it appears logical to relate the translocation process to a conformational change of the pump protein, i.e., to the step involving the

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131

MEMBRANE

Ca2’(trans) ’ EyP FIG. 1. Reaction cycle of plasma membrane Ca2+ pump. Ca2+ translocating step is visualized here in E,P - E,P transition. As discussed in text, this identification is tentative.

E,--P - E,--P transformation, as indicated in Figure 1, or to the transformation of conformer E, into conformer E,. In both cases it is clear that the Ca2+ binding site, which is located on the interior side of the membrane step (the E, conform ation), will before the translocation be located on the external side of the plasma membrane at the end of the translocation step, i.e., the Ca2+ binding site on the E, conformer of the pump faces the exterior of the cell. The problem of the Ca2’ binding sites is related to that of the number of Ca2+ equivalents bound by the pump and translocated at each run of the reaction cycle. No conclusive information is availa ble on this. However, the observation that the Hill coefficient for the activation of the ATPase by Ca2+ significantly exceeds 1 indicates more than one site. This is supported by the experimental finding (80,151) that the saturation of the pump and its rate in erythrocytes change with the square of the concentration of cytosolic Ca2+, i.e., each pump unit would contain two high-affinity Ca2+ binding sites. However, the sigmoidal kinetics of the pump could be caused by other factors, e.g., by the Ca2+-dependent binding of the activator calmodulin (see next section). Even if one accepts that two Ca2+ equivalents are bound by each pump unit in preparation for the translocation step, whether the latter involves both of the bound Ca2+ or only one is still an open issue. Under optimal experimental conditions on erythrocyte ghosts (i.e., subtraction of Ca2+ leaks and correction for extraneous ATP hydrolysis) one normally finds a stoichiometry between Ca2+ transported and ATP hydrolyzed approaching 1 (e.g., see Ref. 237). Others have found that the Ca2+:ATP stoichiometry depends on the Ca2+ concentration, approaching 2 above 500 PM (226, 231). It was also found that concentrations of La3+ that completely block the transport of Ca2+ reduce the Ca2+-dependent ATPase only by ~50%, suggesting that the portion of ATP hydrolysis that is La3+ insensitive is not related to Ca2’ transport (207, 208, 231). This would lead to the conclusion that the real Ca2+:ATP stoichiometry of the pump would not be 1, but 2, as in the analogous pump of sarcoplasmic reticulum (109). Although it has been repeatedly claimed that water-soluble Ca2+-stimulated ATP-


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ases, presumably not involved in Ca2+ transport, are present in erythrocyte membranes and can easily be removed from them, the alternative possibility that La3+, under the experimental conditions, “uncouples” the pump must also be considered. Thermodynamically, a 1:l stoichiometry would seem more probable (238), but a 21 stoichiometry, even if less favorable, would in principle also be possible if one assumes a very tight coupling of Ca2+ transport to ATP hydrolysis and an extremely low Ca2’ leak through the membrane (238). It may be anticipated at this point that measurements in Ca2’ -tight reconstituted systems with the purified erythrocyte pump (187), i.e., under conditions where Ca2+ -ATPases not related to transport were not present, have yielded Ca2+:ATP stoichiometries approaching 1 (see sect. IIIA). The discussion of the affinity of the pump for Ca2+ would not be complete without mentioning what has been termed the ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) effect. This describes the increased Ca2+ affinity of the pump in erythrocyte ghosts measured either as ATP hydrolysis (237) or as Ca2+ transport (230) in the presence of CaEGTA buffers as compared with Ca2+ alone. In unbuffered media K, can be as high as 40 or 50 PM, whereas in the presence of EGTA it decreases to 0.5-0.7 PM. The effect apparently does not require an intact membrane, since it has also been observed on preparations of the erythrocyte membrane solubilized in Triton X-100 (4). The EGTA effect is still not well understood, although interesting hypotheses offer rationalization attempts. They are mentioned again in the next section, since the effect appears to be strictly connected with the action of calmodulin. Naturally, the ETGA effect is of great experimental importance, since the Ca2+ pump is almost always studied in the presence of EGTA. B. Calmoduh

Stimulation

The matter of the stimulation of the plasma membrane Ca2’ pump by calmodulin can be traced back to the observation (233, 237) that the specific activity of the pump was depressed in erythrocyte membranes prepared in media containing EDTA compared with membranes prepared in the presence of Ca2+. Scharff and Foder (234) thus proposed that the Ca2+ pump exists in two different conformational states, A and B. The former is induced by the presence of chelators in the medium used for the preparation of the membranes and is characterized by low Ca2+ affinity and transport rate, whereas the latter is induced by the presence of micromolar Ca2+ and has a high Ca2’ affinity and transport rate. Soon thereafter it was found that the erythrocyte cytosol contains a protein activator of the pump (ZZ), and it became clear that the transition from state B to state A is induced by the removal of the activator (78, 108,235). Then, in 1977, Gopinath and Vincenzi (98) and Jarrett and Penniston (126) showed that calmodulin

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produced the same effect on the pump as the protein activator, the expected identity of which with calmodulin was in fact established shortly thereafter (127). Of the two basic mechanisms of calmodulin activation of enzymes, direct binding or activation of calmodulin-dependent protein kinases, direct binding was shown to be the most likely by the finding (162,189) that the activation by calmodulin persisted in detergent-solubilized erythrocyte membrane preparations. The idea that calmodulin binds directly to the ATPase was also strongly suggested by the good correlation between the number of high-affinity calmodulin binding sites in erythrocyte membranes made calmodulin deficient by treatment with Ca2+ chelators and the number of pump units estimated from the amount of Ca2+-dependent phosphorylated intermediate; this phosphorylated intermediate has been calculated to correspond to -700 pump units/cell (209), whereas the number of high-affinity calmodulin binding sites on the erythrocyte membrane has been found to vary in different studies between 400 and 7,200 pump units/cell. These studies have employed 1251-labeled calmodulin (2, IOO), azido-modified 1251-labeled calmodulin (114), or kinetic titrations using Ca2+-ATPase assays in both erythrocyte ghosts (82, 125) and purified enzyme preparations (101) and have estimated a dissociation constant (Kd) for the calmodulin binding process in the presence of Ca2+ ranging between 0.3 and 14.5 nM. Although there is no precise reason to question the lowest figures quoted, i.e., 400700 pump units/cell and the Kd of 0.3 nM, according to the most recent general consensus the higher values mentioned are probably closer to reality. The assumption made in all these studies is that calmodulin and the Ca2+ pump interact with a I:1 stoichiometry. Although this appears to be the case in most studies (114), a 21 calmodulin-to-pump binding ratio has also been found in studies using the purified erythrocyte enzyme (285; see sect. IIIB). Calmodulin increases the affinity of the Ca2+ pump and its maximal transport rate. With the use of calmodulin-depleted erythrocyte membrane preparations the apparent K, (Ca”‘) of the pump has been found to decrease from values in excess of 30 PM to below 1 PM; the maximal rate of transport may increase up to 10 times (128, 147, 149, 181). The general interpretation of the effect of calmodulin on the rate of the pump is the increase of its turnover. Accordingly, calmodulin has been found to stimulate both the rate of phosphorylation of the pump and that of its dephosphorylation (128, 161, 180). As mentioned, the EGTA effect on the pump appears to be related to the action of calmodulin. Indeed, it has been found that the effect of calmodulin on the Ca2+ affinity of the pump in erythrocyte membranes is only seen in media not containing EGTA (230,268). The finding has been explained with the existence of two Ca2+ binding sites on the pump (230), with one having high Ca2+ affinity and Ca2+ specificity and the other one unable to discriminate between Ca2’ and Ca-EGTA. How-


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ever, the alternative explanation (238) that the access of Ca2+ to the binding site is facilitated by EGTA (possibly because the site is shielded by positive charges) seems more plausible. According to this alternative explanation, calmodulin would make the site more accessible in a similar way, conveniently explaining the lack of EGTA effects on the purified enzyme. C. Activat,ioTl of Ca*+ Puw~p by Treatments From Cahodulin

DiRerent

It was mentioned that EGTA, at least with the enzyme in situ, can replace calmodulin as an activator of the pump. The effect, whatever its mechanism, obviously has no physiological meaning. Other compounds and/or treatments that activate the pump in the absence of calmodulin, on the other hand, could well be physiologically meaningful. I. Lipids The fact that the Ca2’ pump is sensitive to the lipid environment was shown by early experiments using phospholipase digestion of the erythrocyte membrane in which it was found that the treatment inactivated the pump. However, Roelofsen and Schatzmann (218) found reactivation with the addition of all glycerophospholipids tested, whereas Ronner et al. (219) found a marked preference for phospholipids bearing net negative charges and could also reactivate the pump by adding long-chain polyunsaturated fatty acids. It was also found that oleic acid activated the erythrocyte enzyme (276) and competitively inhibited the binding of calmodulin, indicating that the interaction of the pump with calmodulin was influenced by the lipid ambient. More recent experiments using the purified enzyme have strongly supported the special ability of acidic phospholipids to activate the pump as an alternative to calmodulin and are discussed in detail in section IIIA and IV. The special role of acidic phospholipids now appears to be generally accepted. The divergent results on the reactivation of the enzyme in situ have been rationalized (238) with the different degree of calmodulin depletion induced by the phospholipase treatments in the membranes used. However, it is difficult to see how membrane preparations in which the pump environment has been made calmodulin deficient could be reactivated by all phospholipids, including those not bearing net negative charges. A corollary of these points on acidic phospholipids and calmodulin is the percent fraction of the Ca2+ pump that is free of calmodulin in the erythrocyte membrane in vivo. Conversely, it would be important to establish whether the phospholipid environment in the membrane provides for some activation of the pump in vivo. The large excess of calmodulin with respect to the plasma membrane Ca2+ pump in eucaryotic cells (at

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least in erythrocytes) has been generally taken to indicate that there is no ATPase free of calmodulin in the membrane (see Ref. 264). However, optimal calmodulin saturation with Ca2+ only occurs at Ca2+ concentrations somewhat higher than those presumably present in most cytosols; this could decrease the efficiency of calmodulin interaction with the pump. On the other hand, acidic phospholipids do occur in plasma membranes, particularly in its inner leaflet where the active site(s) of the pump is located. Calculations on the purified enzyme (see sect. IIIA) have led to the conclusion that the acidic phospholipid ambient of the plasma membrane is in principle adequate to support -50% of maximal activation. 2. Proteolysis The activation of the Ca2+ pump in the plasma membrane by proteolysis was first shown by Taverna and Hanahan (257), using chymotrypsin and trypsin on isolated erythrocyte membranes. A detailed study of the phenomenon was performed shortly thereafter on inverted erythrocyte vesicles by Sarkadi and co-workers (76,228), who established that trypsin mimicked the effects of calmodulin and showed that the activated pump was no longer stimulated by it. Of interest in light of the work on the proteolysis of the purified enzyme discussed in section IIIB (285) was the finding that the treatment removed from the cytosolic side of the enzyme a fragment of apparent molecular weight --30,000 (76), which was suggested to contain the regulatory domain of the pump. However, the treatment also reduced the steadystate level of the phosphorylation in the absence of Mg2+. The work on trypsin and chymotrypsin, however interesting and important in the definition of the functional domain of the pump (see sect. III&, obviously has no physiological meaning. Recent work on the effects of the intracellular Ca2+-dependent protease calpain could, on the other hand, be physiologically relevant (8, 122, 274). Calpain increases the basal activity of the pump in the erythrocyte membrane but does so much more slowly than the extracellular proteases, reaching the maximal levels attainable with calmodulin in 1 or 2 h. The treatment reduces the apparent molecular weight of the enzyme by ~12,000, leaving in the membrane a component of apparent molecular weight of -1124,000, which still binds calmodulin in gel-overlay experiments with 1251-calmodulin during the first phase of proteolysis. In one of the studies (274), probably because of the somewhat more drastic digestion conditions, calpain proteolysis also produced other fragments of lower molecular weight. In the same study, calmodulin was found to delay the digestion and to decrease the activation by calpain. The work by James et al. (122) has shown that the component of molecular weight -1124,000 gradually loses the ability to bind calmodulin as the basal activity of the pump reaches the level attained with saturating


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calmodulin. It has now been found that calpain digests the calmodulin binding domain in two successive steps, the second of which completely removes it from the main body of the enzyme (see sect. IV).

conditions have now shown that the pump molecule does indeed become phosphorylated during the reactivation process (183). This eliminates the necessity of postulating a regulatory protein different from the ATPase as the target of the phosphorylation reaction. Stimulation of the Ca2’ pump in inverted human 3. Other activati72g treatmeu2ts erythrocyte vesicles (but also of the purified pump) by protein kinase C was recently reported by Smallwood et I) PROTEINS. Mauldin and Roufogalis (168) have de- al. (245). The stimulation required the presence of either scribed an erythrocyte protein that activates the Ca2+ phorbol ester or diolein and was seen on both the Ca2+-stimulated ATPase and the transport of Ca2+ The pump. The protein is membrane bound and in the original report was described to have an apparent molecular effect was a five- to sevenfold increase in the maximum weight of 63,000. It could be extracted from erythrocyte velocity ( Vmax)of the enzyme, without effecting its Ca2+ membranes with EDTA and was originally claimed to affinity (however, it was smaller on the purified endiffer from calmodulin. More recently, after a sugges- zyme). The stimulation was additive with that produced tion (9) that the activator protein was aggregated cal- by calmodulin; whether the pump molecule proper is the modulin, it was found that the activator protein indeed substrate of the protein kinase C phosphorylation or contained calmodulin bound in a Ca2+-independent way whether an accessory protein linked to the pump is the substrate of protein kinase C is still an open question. to a membrane protein (225). Another protein that activates the Ca2+ pump was described in extracts of dog The guanosine 3’,5’-cyclic monophosphate (cGMP)-deheart sarcolemma (211). The activator protein has an pendent protein kinase has also been claimed to stimuapparent molecular weight of -60,000 and activates the late the plasma membrane Ca2’ pump in vascular pump (purified from erythrocytes) also in the presence smooth muscle (83,138,204,205,232,253). This has been of calmodulin. Very recently a bacterial protein, termed used to explain the relaxing action of agents that stimubacteriomodulin, was found to activate the erythrocyte late the cGMP-dependent protein kinase in smooth Ca2+ pump (3). The protein has an apparent molecular muscle. Although it is possible that the G kinase indeed weight of 19,000 and apparently contains a domain that stimulates the plasma membrane Ca2+pump in vascular resembles calmodulin. It activates the Ca2+ pump in a smooth muscle, possibly by phosphorylating phosphatiCa2+-dependent manner. dylinositol (267), recent work has shown that the pump A protein activator that activates a Ca2+-dependent molecule per se is not the substrate of the phosphorylaATPase of the hepatocyte plasma membrane has been tion process (14). Again, an accessory protein phosphorisolated from liver cytosol (158). Its role, and that of all ylated by the G kinase could be involved. these protein activators in the regulation of the pump and their relationship to the well-established regulation D. Inhibitors of Ca2+Pump by calmodulin, is still obscure. II) KINASE-MEDIATED PHOSPHORYLATION. Stimulation of the Ca2+-stimulated ATPase and of the assoIn addition to vanadate (which inhibits all P-type ciated transport of Ca2+ by a phosphorylation reaction transport ATPases) and to lanthanides (see sect. IIA), a was first reported by Caroni and Carafoli (42) in 1981. number of nonspecific reagents inhibit the plasma The original observation was made on heart cell plasma membrane Ca2+pump, among them ruthenium red (275) membranes (sarcolemma) and required the pretreatand sulfhydryl reagents (21, 203). Of interest is the obment of the membranes with a protein phosphatase to servation that N-ethylmaleimide inhibits to a different dephosphorylate sarcolemmal proteins. The latter degree, depending on the presence of effecters of the treatment inactivated the Ca2+-dependent ATPase and pump (ATP, Ca2+, Mg2+) (214). This suggests that the the pumping of Ca2+,and the inhibition was reversed by effecters induce a conformational change of the pump, incubation with ATP, Ca2+, and Mg2+. Evidently, the resulting in a different degree of exposure of a sensitive heart sarcolemma membrane contains a protein kinase SH group(s) in the active site. Other nonspecific inhibithat phosphorylates the pump (or some other regulators are quercetin (279), suramin sodium (238), vinblastory protein associated with it); the kinase is most likely tine (94), and the inhibitors of the band 3 anion channel the adenosine 3’,5’-cyclic monophosphate (cAMP)-de4,4’-diisothiocyanostilbene-2,2’-disulfonic acid (DIDS) pendent kinase, because the reactivation process was and N-(4-azido-Z-nitrophenyl)-2-aminoethylsulfonate blocked by the inhibitor of the CAMP-dependent protein (NAP-taurine) (171, 190, 269,270). The latter inhibition kinase. However, exogenously added phosphorylase B was attributed to the blockage of the transport of kinase was also able to reactivate the pump after de- charge-compensating anions through band 3 (269). Howphosphorylation, leaving the problem of the nature of ever, the inhibition by DIDS and NAP-taurine took the kinase active in the reactivation process in vivo still place on the inner side of the membrane, whereas band 3 unsolved. Early attempts to demonstrate incorporation inhibition by DIDS and NAP-taurine takes place at the of phosphate in the pump molecule were unsuccessful outer side. Inhibition studies on the purified pump have (43, 44), but recent experiments under more favorable conclusively documented that the inhibition by DIDS


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THE

and NAP-taurine is due to direct interaction with the pump (1). The problem of the phenothiazine neuroleptics (and of other anticalmodulin drugs) deserves a special comment; these compounds are normally assumed to be anticalmodulin reagents, which would only inhibit the calmodulin-stimulated ATPase. When used in low concentrations, this is indeed the case. However, at higher concentrations these compounds also inhibit the basal ATPase activity of the pump and the activity stimulated by acidic phospholipids and limited proteolysis, i.e., under the experimental conditions, they interact directly with the pump molecule (I, 263). The most effective anticalmodulin compound is the antimycotic myconazole derivative calmidazolium (93), which removes the stimulation of the ATPase by calmodulin with a Ki of ~0.5 PM. Together with the almost equally potent compound 48/80 (91), it also appears to be the most selective, i.e., the difference in the optimal concentrations needed to remove the stimulation by calmodulin and to inhibit the basal activity of the pump or the activity stimulated by acidic phospholipids or by proteolysis is maximal with respect to all other anticalmodulin drugs, which now comprise a large number of diverse compounds (224). The inhibitors described here have helped clarify several aspects of the function of the Ca2+ pump, and some have become essential experimental tools. However, none of them has a physiological function. Physiological (protein) inhibitors of the pump might, however, exist, i.e., proteins have been described in erythrocytes and in other cells that inhibit the Ca2+ pump. One has been extracted from bovine brain (273) as an inhibitor of the CAMP phosphodiesterase. The inhibitor combines with calmodulin in a Ca2+-dependent way, thus removing the activation of phosphodiesterase by calmodulin. It is thus not surprising that the inhibitor also antagonizes the activation of the pump in erythrocyte membranes by calmodulin, leaving the basal activity unaffected (148). The brain inhibitor is thus apparently an anticalmodulin agent and cannot be considered as a Ca2+ pump inhibitor in a strict sense. Although other described proteins (7, 10, 11, 278) also interfere with calmodulin, they could also have direct effects on the pump. In particular, the liver protein described by Loterztajn et al. (158) could be a real Ca2+pump inhibitor. The proteins described by Au and co-workers (7, IO, II) were either extracted from the pig erythrocyte membrane and shown to be associated with calmodulin or purified to homogeneity 1) from human erythrocyte cytosol as a protein of apparent molecular weight 19,000 and Z) from rat brain cytosol as a protein of apparent molecular weight 6,000. Although the inhibition was apparently due to the decrease of the affinity of the pump for Ca2+at all calmodulin concentrations, the effect was overcome by high concentrations of calmodulin. The liver inhibitor was shown to inhibit a Ca2+-activated ATPase in hepatocyte plasma membranes (158). This ATPase has peculiar properties that differentiate it somewhat from the classic Ca2+ pump of other plasma

PLASMA

MEMBRANE

135

membranes, including the apparent lack of calmodulin sensitivity (see sect. IIE). In summary, the matter of natural Ca2+pump inhibitors in erythrocytes and other cells is obscure, and the differences in properties according to tissue and laboratory are bewildering, but it appears probable that an essential ingredient in their mechanism of action is the interaction with calmodulin. This has relevance to the problem of the fraction of pump that is “free” of calmodulin and thus available to other activators in the plasma membranes. E. Plasma Membrane Ca2+Pump in Other Cell Types As mentioned, for a number of years the erythrocyte pump was universally adopted as the object of study. It was not only because of the experimental advantages offered by the erythrocyte system. Somehow the system responsible for the general extrusion of Ca2+ from cells, particularly excitable cells, was rather assumed to be the Na’-Ca2+ exchanger. It thus came as a particular unexpected finding that an ATP-dependent, Na+-independent system was apparently extruding Ca2+ also from classically excitable cells, such as the squid giant axon (68) and the mammalian heart (40). In both cases the conclusion that an ATP-powered pump was involved was unambiguous. In the squid axon, all Na’ could be removed from the medium, effectively ruling out the Na+-Ca2+ exchanger as a participant in the ATPdependent transport of Ca2+. In the heart sarcolemma system the Ca2+that had been induced to penetrate into the vesicles in an ATP-dependent manner was discharged by externally added Na+, showing that the ATP-dependent system had loaded Ca2+into a vesicular space that was available to the Na+-Ca2+ exchanger, that is, in the sarcolemma vesicles and not in contaminating (sarcoplasmic reticulum) vesicular structures. Indirect evidence that an ATP-powered system was extruding Ca2+ from cells different from erythrocytes, including excitable cells, had in fact already appeared in the literature (48, 70, 217). However, the studies by DiPolo (68) and Caroni and Carafoli (40), together with a study on kidney basolateral vesicles by Gmaj et al. (96), provided the first definitive evidence for the existence of Ca2+pumps in a number of diverse excitable and nonexcitable plasma membranes, thus suggesting that the system could be a general property of eucaryotic plasma membranes. These studies also established that the system had the expected high affinity for Ca2+:K,s of ~0.5 PM were reported. High-affinity ATP-dependent Ca2’ transport was also found in a study by Morcos and Drummond (175) on heart sarcolemma. The fact that a Ca2+pump of the erythrocyte type could be at work also in the plant world was suggested by a study on vesicles from various plant sources (167) in which the ATP-driven Ca2+uptake was shown to be sensitive to calmodulin. The system was thus homologous to the plasma membrane Ca2+ pump rather than to the Ca2+pump in


136

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pump, namely, the stimulation by calmodulin and the apparent molecular weight of -140,000. As Table 1 shows, at least one plasma membrane contains a Ca2+ Approximate pump that apparently lacks calmodulin stimulation, i.e., Calmodulin Molecular that of liver. The information on the liver pump is still Cell Type Sensitivity Weight Reference controversial, but considerable evidence suggests that + 140,000 169, 170,254 the pump could indeed have peculiar properties. For this Skeletal muscle + ND 34,111 Sarcolemma reason, the liver pump is discussed in more detail later T-tubules in this section. + 140,000 40,175 Heart Some of the aspects mentioned for the enzymes in 42,143, 146,258 Table 1 are worth emphasizing. The pump in rat intesSmooth muscle + 140,000 176, 204, 281, 282, 284 tinal mucosa has been reported to be stimulated by the Kidney tubules + 140,000 63-66,86, 95-97, administration of 1,25-dihydroxyvitamin D, to the ani174 mals (89), a property suggesting the interaction of the Nervous cells ND ND 68 vitamin D-dependent Ca2+-binding protein calbindin Squid axon + 140,000 107,194,247 Synaptosomes + ND 51 (which shows structural similarities to calmodulin) Optical nerve + ND 52 with the pump. In fact, stimulation of the pump in intesNeurohypophysis tinal mucosa plasma membrane vesicles by calbindin Intestinal epithelium + 115,000-130,000 58, 87, 88, 112, has been reported (272). However, the stimulation re182,258,271 quired special conditions, e.g., the absence of EGTA + ND 139,199 Endocrine pancreas + 100,000 6, 16, 117 Exocrine pancreas from the reaction medium, an observation that could + ND 198,200,201 Adipocytes possibly be related to the effects of EDTA on the Ca2+ + ND 242 Osteoblasts affinity of the pump discussed (230,270). Additional in+ Leukocytes formation is required to settle the matter of the effects 150,000 152, 177,229,241 Lymphocytes + ND 192,206,265 of calbindin. Monocytes + Neutrophils One other interesting aspect of the intestinal mu+ 132,500 150,240 Macrophages cosa Ca2+ pump is its anatomic distribution. In intes+ ND 134,248 Ehrlich ascites cells tinal mucosa the Ca2+ pumping activity decreases from + ND 67,104, 167, 184 Plant cells the duodenum to the jejunum and ileum (89, 178), and Liver 70,000-105,000 12, 45, 118, 119, 140,157 this correlates with the formation of the 140,000-mol wt phosphorylated intermediate (271). Moreover, the pump A series of studies by Kumar, Penniston, and associates have is apparently inhomogeneously distributed along the made use of monoclonal antibodies raised against human erythrocyte villar-crypt axis of the enterocytes, showing maximal pump to document presence of Ca2’ pump in a number of cell types. activities near the villar tip and decreasing toward the Antibody reactivity has been observed in human osteoblasts (30), mammalian choroid plexus (28), eel gill chloride cells (29), and placenbase and the crypt (259). Furthermore, within single ental trophoblasts (27). Indications for presence of ATP-powered Ca2+ terocytes the pump appears to be located in the basopumps have been provided also for plasma membranes of other eulateral domain of the plasma membrane, which faces caryotic cells, e.g., thyroid (130), spermatozoon (35), and human neuthe blood stream, and not in the brush border (58, 88, trophil (145). However, information on these systems is still unsuffi112,182). This is in keeping with the accepted role of the ciently advanced for inclusion here. The matter of platelets is more complex. Although some authors have ruled out the presence of a Ca2+ enterocyte plasma membrane in mediating the absorppump in plasma membrane (249), others have provided evidence in its tion and/or active extrusion of Ca2+in intestine. The rat favor (73, 75, 212). Procaryotes normally eject Ca2+ using a Ca2+-H+ and the human kidney pumps are apparently also inhoantiporter (36), but Strepfococcus~aecalis apparently possesses a Ca2+ mogeneously distributed, as inferred from studies using pump (220). ND, not determined. monoclonal antibodies directed toward the human erythrocyte pump (31, 32, 142). Antibody reactivity has (endo)sarcoplasmic reticulum. After these initial obser- been observed only in the distal tubules. As in the case vations, numerous other reports have documented the of enterocytes, in the kidney the pump is also located in existence of ATP-driven Ca2+ pumps in the plasma the basolateral domain of the plasma membrane. The membranes of other cells, showing that the pump inmatter of the inhomogenous distribution of the pump in deed is a necessary attribute of eucaryotic plasma mem- different plasma membrane domains of the same cell branes. Most of the studies have documented the essen- has also been studied in hepatocytes, where the enzyme tial properties of the erythrocyte system, chiefly, cal- apparently displays peculiar properties. Here the situamodulin sensitivity and high Ca” affinity. Whenever tion is still somewhat confused, since the pump has been tested, vanadate sensitivity has also been observed. assigned predominantly either to the canalicular doThe lists of cells in the plasma membranes of which main facing the bile canaliculi (12) or to the basolateral a Ca2+ pump has been described is long and growing domain facing the neighboring hepatocytes. The differrapidly. Table 1 groups the observations made until re- ences may reflect the difficulty of obtaining suitably cently and emphasizes the two essential parameters enriched and functionally competent preparations of that define the “conventional” plasma membrane Ca2+ the various domains of the hepatocyte plasma memTABLE 1. Calcium pumps in plasma membrane of elucary ot fit cells


Jmmry

1991

CALCIUM

PUMP

OF

THE

brane, which also contains, in addition to the basolatera1 and canalicular domains, the sinusoidal domain facing the blood. Recent developments actually indicate the latter domain as the probable locale of the pump. It has been found that the concentration of Ca2+ is lower in the bile than in the liver perfusate (113), suggesting passive absorption from the former and active extrusion into the latter (i.e., into the blood). Recent work in my laboratory offers strong support to the idea that the pump is preferentially located in the sinusoidal domain. With the use of monoclonal antibodies raised against the human erythrocyte Ca2’ -ATPase, the pump has been directly and preferentially visualized in the sinusoidal domain of rat hepatocytes. Results on various liver plasma membrane preparations point to properties of the hepatocyte pump that differ in some significant aspects from those of the erythrocyte enzyme. The requirement for exceedingly low (i.e. PM) Mg2+ concentrations and the inhibition by the usual concentrations required to show Ca2+ pumping activity in other plasma membranes (119,154,157), the unusually high affinity for Ca2+ [below the PM range (119,140,154,157)], the relative lack of nucleotide specificity (140, 153, 157) (however, only seen when measuring ATP hydrolysis, not when measuring Ca2+ transport), and the absence of calmodulin stimulation (12, 119,140,153,157) have all been taken as evidence for the peculiar nature of the pump. A Ca2+-dependent phosphoenzyme of molecular weight 105,000 has been reported in rat liver plasma membranes (45) under conditions that would presumably inhibit the proteolytic degradation of a larger pump protein. The presence of both an endogenous protein activator (157) and inhibitor (158), as well as the peculiar sensitivity to hormones, is another indication that the liver enzyme may be special. The absence of calmodulin reactivity has prevented the use of the calmodulin affinity-chromatography column, successfully employed by Niggli et al. (188) for the erythrocyte enzyme, to purify the hepatocyte pump. As a result, far less ideal methods had to be used to isolate the liver enzyme, e.g., lectin chromatography. Purified preparations have nevertheless been described (153,157), but very little is known on their molecular properties apart from a reported apparent molecular weight of 200,000 on gel filtration, reduced to 70,000 in denaturing polyacrylamide gels (153). More recent work in my laboratory (133a) has significantly advanced the understanding of the hepatocyte pump. Evident cross-reactivity with monoclonal antibodies raised against the human erythrocyte pump has been detected predominantly in the sinusoidal domain of the rat liver plasma membrane. The antibody reacts with a protein, the apparent molecular weight of which is slightly higher than that of the erythrocyte pump (in the 150,000 region). Of great interest is the observation that the band that crossreacts with the erythrocyte pump antibody can, under specified experimental conditions, interact with calmodulin in blotted gel-overlay experiments. In fact, in the gel-overlay experiment the liver protein seems to inter-

PLASMA

137

MEMBRANE

act with calmodulin even better than the erythrocyte enzyme. Clearly, the matter of the Ca2+ pump in the hepatocyte plasma membrane is far from settled; further work will possibly show that the previously suggested differences with the erythrocyte-type pump have been overstated. The fact that the hepatocyte enzyme can be isolated using columns of antibodies directed against the erythrocyte enzyme (133a) will certainly accelerate the analysis of the differences between the two pumps.

III.

ISOLATED

PUMP

A. Isolation, Reconstitution, of PuriJied Punzp

and Properties

As mentioned, purification of the pump to homogeneity in a functionally competent state was made possible by the application of a calmodulin affinity-chromatography column (188). Before this, conventional attempts had yielded preparations that were only partially purified but that had nevertheless yielded information of some importance. Wolf et al. (277) had established that the enzyme could be solubilized from the erythrocyte membrane using Triton X-100 and showed that its inactivation could be prevented by the detergent Tween 20 and phosphatidylcholine. Although the solubilized preparation contained approximately equivalent amounts of three protein components, it is of interest that at least one had a molecular weight in the range that is now known to be that of the pump (145,000, 115,000,105,000). Also of interest is the observation that the heaviest component (but not the other two) could be phosphorylated by ATP. Partial purification of the erythrocyte pump was also reported by Peterson et al. (ZOZ), who dissolved the erythrocyte membranes in Triton X-100 and incorporated the dissolved enzyme in acidic phospholipid liposomes. The reconstituted enzyme was functionally active, and a modest degree of purification was achieved by isoelectric focusing methods. Successful reconstitution of a partially purified preparation of the erythrocyte pump was also reported in 1970 by Haaker and Racker (106). They used asolectin (i.e., cardiolipin containing) liposomes and showed that the ATP:Ca2+ stoichiometry of the transport reaction approached 1. In hindsight, the difficulties encountered by conventional attempts to purify the enzyme were obvious. The pump is present in the erythrocyte membrane in vanishingly small amounts [probably ~0.1% of the total membrane protein (136)]. In addition, it is very unstable and only active in the appropriate phospholipid environment. Calmodulin affinity chromatography circumvented these difficulties and yielded a completely purified and functionally competent enzyme at the end of a relatively rapid procedure. The finding that calmodulin stimulated the erythrocyte pump (98, 126) was the


ERNEST0

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r

25 ‘;2 X

100 pM Ca*+

5 mM

EDTA

IO

5 column

eluate

(

15 (ml)

2. Purification of Ca”’ pump from erythrocyte membrane a calmodulin affinity column. [From Niggli et al. (186).]

FIG.

using

breakthrough that led to the idea of calmodulin affinity chromatography, and the finding that calmodulin activation persisted in solubilized Ca’+-ATPase preparations (162, 189) gave experimental plausibility to the idea. In retrospect, however, if one considers that the erythrocyte membrane contains other calmodulin binding proteins, it was not obvious that the calmodulin affinity column should produce only the Ca2+-ATPase at the end of the EDTA elution step. The fact that it did so was a fortunate case, probably based on the different calmodulin affinities of the various membrane proteins potentially able to bind to the column. The original purification experiment reported by Niggli et al. (188) is shown in Figure 2. Human erythrocyte ghosts were extensively washed with EDTA to remove bound calmodulin and then solubilized with Triton X-100 in the presence of phosphatidylserine to stabilize the solubilized pump. The solubilizate was applied to a sepharose-CNBr-calmodulin column, which was then washed with Ca2’ to remove unbound proteins. This eluted from the column nearly all of the applied protein, including the Mg2+ -dependent ATPase. Replacement of Ca2+ in the elution buffer with EDTA removed from the column the Ca2+ -stimulated ATPase, which was contained in a protein peak corresponding to ~0.1% of the applied protein. The EDTA-eluted peak migrated in sodium dodecyl sulfate-polyacrylamide gels as a protein of approximate molecular weight 130,000 accompanied by minor amounts of a component, the molecular weight of which was approximately double, and was thus suggested to correspond to a dimer of the pump. In the original gel of Niggli et al. (188), traces of lower molecular weight impurities were also visible. Later work (e.g., see Ref. 102) has shown that the time of the Ca2’ washing of the calmodulin column must be increased from the 3 h used by Niggli et al. (188) to 15-18 h to completely free

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the ATPase from contaminants. The purified preparation incorporated radioactivity from [T-~~P]ATP in both the 130,000 and the >ZOO,OOO components, indicating that the heavier component was indeed a dimer of the pump. A s expected, the 32P incorporation was abolished by EDTA and was sensitive to hydroxylamine. The finding that the purified enzyme was no longer stimulated by calmodulin was thus somewhat surprising. This was at variance with the findings of Gietzen et al. (92), who found calmodulin stimulation in a preparation of the purified erythrocyte pump obtained essentially by the method of Niggli et al. (188), except that deoxycholate was used instead of Triton X-100 to solubilize the membranes and phosphatidylcholine was used instead of phosphatidylserine as the stabilizing phospholipid. Whereas the difference in the detergent was most likely irrelevant to calmodulin stimulation, the phospholipid evidently made the difference. As was expected from the previous findings that acidic phospholipids specifically activated the membrane-bound ATPase (219), it was soon found that the preservation of calmodulin activation depended on whether the phospholipid in the environment was acidic or zwitterionic (187, 186). Full calmodulin stimulation was observed in the presence of phospatidylcholine or phosphatidylethanolamine, whereas in the presence of phosphatidylserine, phosphatidylinositol, or cardiolipin the enzyme was already fully stimulated in the absence of calmodulin. The pump was reconstituted in liposomes containing various proportions of acidic and zwitterionic phospholipids (186), and it was found that, depending on the method of reconstitution, 50% activation of the pump (and 50% calmodulin desensitization) were achieved when the liposome membrane contained between ZO-40% acidic phospholipids. This is important information; if one transfers this to the native erythrocyte membrane, which contains -30% phosphatidylserine in the inner monolayer (260), it follows that the pump in vivo is expected to be -50% activated irrespective of the presence of calmodulin. An obvious advantage of calmodulin over phosphatidylserine is that it can be easily modulated up and down in the vicinity of the ATPase, thus providing for a varying degree of activation, whereas modulation of phosphatidylserine in the membrane environment is unlikely. However, the pump is also activated by phosphatidylinositol and its two phosphorylated derivatives (39,47,186, 197), which are present in plasma membranes. Because these phospholipids, at least in some cells types, have rapid turnover rates (19) and because the products of their receptor-stimulated breakdown inositol trisphosphate and diacylglycerol (47; unpublished observations) have no effect on the pump, their modulation of the pump is an attractive possibility (47,197). The fact that optimal activation by phosphatidylserine requires -5,000 mol/mol pump, whereas the optimal molar ratio is much lower in the case of the phosphorylated derivatives of phosphatidylinositol(39), adds interest to the possibility. Another important point in the phospholipid activation experi-


CALCIUM

PUMP

OF

THE

ments was the finding that optimal activation by phosphatidylserine was observed in a Ca2+ concentration range that was well below that required to saturate the phospholipid. This showed that the effect of acidic phospholipids was not related to their ability to bind Ca2+ and to deliver it to the binding site(s) on the pump. If one extends this to the case of calmodulin (a permissible, but not a necessary extrapolation), it can be concluded that the Ca2’ that is eventually transferred across the pump enzyme to the other side of the membrane is not the Ca2+ that was originally bound to calmodulin. A recent extension of the work on the activation of the erythrocyte pump by acidic phospholipids (74) has shown that they activate the pump (i.e., they decrease its K, for Ca2+) to a higher extent than calmodulin does. This (together with additional evidence) has suggested a different mechanism of action of the two compounds, i.e., acidic phospholipids and calmodulin would be targeted to different domains of the pump molecule. This has now been supported by proteolytic and amino acid sequencing work, which is discussed in detail in section IV (286) Three distinct conformational states of the pump have thus been proposed. Conformation A would have low Ca2+ affinity and low Ca2+ pumping rate. Conformations B, and B, would represent the high-activity states of the pump, the Vmax and Ca2+ affinity of which would be regulated i ndependen tly. The form er is the same in the B, and B, states, whereas the latte r is higher in the Bi conformation. The A - B, transition is induced by calmodulin, whereas the A - B, (or B, - B,) transition is induced by the exposure to acidic phospholipids. Support for this suggestion has come from trypsin proteolysis work (see sect. IIIB) that has shown that sequential removal of portions of the pump led first to the loss of calmodulin stimulation and then to the loss of the effects of the acidic phospholipids (74, 285). Loss of calmodulin interaction involves the reduction of the molecular weight of the enzyme to --81,000, whereas the loss of the effects of acidic phospholipids requires further weight reduction below this level. The reconstitution experiments on the ATPase purified from erythrocytes (49,187) but also from heart (44) have used a number of reconstitution techniques. The best results have been obtained using asolectin liposomes (i.e., liposomes containing diphosphatidylglycerol) and the slow cholate dialysis method. Apparently, the reconstituted ATPase yields Ca2+-tight liposomes, since the Ca2+ u ptake reaction slows down to a plateau after some Ca2+ has been accu mulated in par al lel with the decline of the rate of hydrolysis of ATP (49, 187). Addition of Ca2+ionophores at this point discharges the accumulated Ca2’ and stimulates the ATPase reaction severalfold. Evidently, the accumulation process creates a Ca2’ gradient that opposes additional accumulation in the tightly sealed liposomes; essentially no Ca2+cycling occurs under the experimental conditions. Simultaneous measurements of ATP hydrolysis and Ca2+transport during the linear phase after the addition of ATP

PLASMA

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139

produces a Ca2+:ATP stoichiometry approaching 1, offering compelling support for the concept of a I:1 stoichiometry (see sect. IIA). The possibility of balancing charges during the transport of Ca2+ by cotransport of anions (e.g., phosphate) through the band 3 anion channel has been briefly alluded to in section IID (270). These experiments were carried out on erythrocyte ghosts as were later swelling and/or shrinking experiments in the presence of either the penetrant anion Cl- or the impermeant anion gluconate (223) that reached the same conclusion, i.e., the pump operates electrogenically. In other words, the potential generated by the translocation of Ca2+ would be neutralized by the transport of anions through band 3. Experiments on Ca2+-tight reconstituted liposomes, where the purified Ca2+pump evidently was the only protein and where compensatory ion fluxes through other protein components did not occur (191), have shown that the initial rate of ATP-dependent Ca2+ pumping by the ATPase reconstituted in the presence of KC1 is not influenced by the K+-specific ionophore valinomycin, which would have produced a stimulation if the operation of the pump had been obligatorily electrogenie by promoting compensatory efflux of K+. Countertransport of H+ was thus considered a likely possibility and was in fact documented by the direct demonstration of H+ efflux (i.e., of a net H+ efflux after correction for the scalar H+ appearing in the medium as the result of the hydrolysis of ATP) from the reconstituted liposomes into a lightly buffered medium. Calculations by Niggli et al. (191) have suggested an electroneutral 1Ca2+:2H’ countertransport, based, however, on the assumption of a 1:l Ca2+:ATP stoichiometry. Although the concept of an obligatory Ca2+-H+ exchange during the operation of the pump is now generally accepted, recent work has indicated that the pump may be partially electrogenic, e.g., it could exchange I Ca2+for 1 H+ (223). From the discussion so far, it appears that the purified pump is functionally competent. Although most of the work on the purified enzyme has been performed on the erythrocyte pump, the ATPase has also been isolated from a number of other plasma membranes, including heart (41), skeletal muscle (169), and vascular smooth muscle (282). In all cell types, the pump has been found to repeat the essential properties of the erythrocyte enyzme, with the one possible exception being liver. However, the recent work on the hepatocyte enzyme (see sect. IIE) indicates that also in the case of liver the essential properties may be similar to those of the erythrocyte pump. One important aspect of the function of the Ca2+ pump that has been established on the purified erythrocyte enzyme is its reversibility. Preliminary evidence on closed erythrocyte membrane vesicles (79,221,ZSO) had indicated that the Ca2+gradient could be discharged to induce the Ca2+ pump to produce ATP, as had been clearly established by the work of Makinose and Hasselbath (165) for the Ca2+pump of sarcoplasmic reticulum. This was to be expected, in line with the general predic-


140

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tions of Mitchell’s (172) chemiosmotic hypothesis of ATP synthesis by mitochondria and other membrane systems. However, the Ca2+ pump of sarcoplasmic reticulum has been shown to synthesize ATP in the reversal of the ATPase reaction also in the absence of Ca2+ gradients (137), a finding that was at variance with the generally accepted idea that the energy for the synthesis of ATP must come from the discharge of the transmembrane Ca2+ gradient. Extension of the work on the solubilized sarcoplasmic reticulum ATPase by De Meis and co-workers (59-61) has led to the conclusion that the solvation energy of the reactants (Pi, ADP, and Ca”‘) is directly involved in the synthesis of ATP by the enzyme in detergents. The critical point in the experiments by De Meis and co-workers (59-61) was the reduction of the water activity at the active site of the ATPase by inclusion of high concentrations of Me,SO in the reaction medium. Under these conditions the free energy of hydrolysis of the phosphorylated intermediate of the reaction cycle (E2 -P) would be much lower than in water, greatly facilitating its formation. Transformation of the low energy E,- P into a high-energy intermediate capable of phosphorylating ADP, i.e., into the E,-P of the reaction scheme, was achieved by rehydration of the catalytic site in the presence of Ca2+ to occupy low-affinity binding sites. Although the precise role played by Ca2+ in the process is unclear, once Ca2+ and water had gained access to the catalytic site, ATP was indeed formed. Somehow the presence of Ca2+ had favored the transfer of phosphate from E,-P to ADP. Analogous experiments on detergent-solubilized preparations of the purified erythrocyte Ca2’-ATPase (46) have shown that also in this case ATP could be synthesized in the absence of a transmembrane Ca2’ gradient. The experiment was carried out essentially along the lines of those of De Meis and co-workers (59-61) for the sarcoplasmic reticulum pump, although the differences between the two enzymes demanded somewhat particular experimental conditions. Full reversibility of the ATP-linked Ca2+ pumping reaction was demonstrated, i.e., ATP was formed by the plasma membrane Ca2+-ATPase solubilized in Triton X-100 as the level of hydration of the catalytic site previously rendered hydrophobic by the addition of Me,SO was suddenly increased in the presence of Ca2+. B. P,roteol ysis of PurQied

Puwzp

The experiments of controlled proteolysis of the purified (erythrocyte) ATPase have yielded information of great interest on the organization of the functional domains in the molecule. Stieger and Schatzmann (250) have shown that the activation by trypsin corresponds to a decrease of the K, (Ca”‘) of the pump to levels that, under the experimental conditions, were even lower than those obtained with calmodulin. When maximally activated, the pump became fragmented into a number of products, the most prominent having molecular

CARAFOLI

Volume

71

weights of - 100,000 and -40,000. The fragmentation was paralleled by the loss of calmodulin sensitivity, a finding that led Stieger and Schatzmann (250) to suggest that the calmodulin receptor was the fragment of -40,000 that was removed from the main body of the pump, a conclusion that agreed with the work of Enyedi et al. (76) on the enzyme exposed to trypsin in situ. A comprehensive study on the controlled tryptic fragmentation of the purified erythrocyte enzyme has been carried out by Zurini et al. (285). Under the experimental conditions the treatment led to the rapid formation, presumably in sequence, of fragments having approximate sodium dodecyl sulfate-polyacrylamide gel weights of 90,000, 81,000, and 76,000. The first was a transient product, whereas the other two tended to persist for longer times. [1251]iodoazido-calmodulin crosslinked to the 90,000 product but not to the other two products, implying that the calmodulin-interacting domain was contained in a fragment of -9,000 removed from the 90,000 product as proteolysis reduced it to 81,000. The experiments also showed that the treatment almost immediately removed from the enzyme a fragment of molecular weight -33,000, which interacted better than any other of the cleavage products with the hydrophobic probe (m-[1251]iodophenyl)diazirine and thus was suggested to contain particularly hydrophobic intramembrane regions of the pump. The acylphosphate intermediate was not formed by products having molecular weights lower than 76,000, but a product of -48,000, presumably derived from the 76,000 fragment, bound a radioactive ATP analogue and was thus suggested to contain the ATP binding site. The fact that the transient 90,000 product still contained the calmodulin binding domain led to its isolation using a calmodulin-affinity column (285). The fragment could be reconstituted into liposomes and shown to retain ATP-driven Ca2+ transporting ability, but the paucity of the material available precluded a detailed analysis of the properties of the transport process. The experiment, however, is of great significance and deserves repetition and extension. In the cleavage scheme proposed (285) [which has now been supported by recent sequencing work (286; see sect. IV)], the 90,000 fragment was visualized to result from the cleavage of extensive portions of the ATPase molecule at both the NH, and COOH terminus. The portion removed from the NH, terminus was proposed to be the previously mentioned fragment of molecular weight 33,000. Because the cloning work on the ATPase described below has shown that this fragment contains some of the putative transmembrane domains of the molecule, one could clearly rule these domains out from the transmembrane Ca2+ path if proteolytic products that do not contain them indeed transported Ca2+ across reconstituted membranes. Unfortunately the 90,000 product is rapidly degraded and not easy to produce uncontaminated by residual intact ATPase and by other degradation products. In fact, in the original experiment by Zurini et al. (285), traces of a 33,000 contaminant still accompanied the 90,000 frag-


Jtr w i//u r*y 1!I9 I

CALCIUM

PUMP

OF

THE

ment. If the 33,000 and 90,000 fragments only separate after denaturation in the gels, the very low Ca2+ transport activity measured in the experiment could have been due to small amounts of the 33,000 and 90,000 fragments still associated after the trypsin cleavage rather than to the isolated 90,000 fragment that was predominant in the preparation. It will thus be desirable to repeat the work on its transport activity on a completely purified preparation of the 90,000 fragment and to devise methods to isolate the products of molecular weight 81,000 and/or 76,000, which are stable for longer times. A study by Benaim et al. (18) has used trypsin under somewhat different, and milder, conditions and has added significa nt information to th e matter of the organization of the ca lmodulin binding domain in the pump. Under the experimental conditions, initial proteolysis has revealed, in addition to the 90,000 and 81,000 fragments, a product of molecular weight +&OOO that had not been seen under the more drastic conditions used in the study by Zurini et al. (285). This product was greatly favored if the proteolysis was performed in the presence of calmodulin, to the point of becoming, at least for some minutes, the only fragment visible in the high-molecular-weight region. Conversely, proteolysis in the presence of vanadate and Mg2+ accumulated for some minutes the product of molecular weight 81,000. Interestingly, the 85,000 fragment was retained by calmodulin affinity columns, but activity measurements showed it to be less responsive to calmodulin than the product of molecular weight 90,000 (17, 18). The two different proteolysis conditions were tentatively suggested to promote the E, and E, conformations of the pump, respectively; it may be pertinent to mention in this context that spectroscopic work on the purified enzyme has shown independently the existence of two different conformations, presumably corresponding to the E, and E, states (141). The proteolysis scheme derived from these studies is shown in Figure 3. Figure 3 also contains information from later proteolysis work (74), which has shown that the Ca2’ affinity of the 81,000 fragment, which is -0.4-0.5 mm in the absence of calmodulin, can be further increased by acidic phospholipids; EGTA (EDTA) also favors the formation and the accumulation of the product of molecular weight 76,000, which is no longer responsive to acidic phospholipids (74). The scheme for the organization of the calmodulin binding domain shown in Figure 3 is based on these findings and on the following observations (17,18). 1) The products of 90,000 and 85,000 bound calmodulin, whereas the product of 81,000 did not. 2) The first two products had repressed activity in the absence of calmodulin, whereas the latter had high activity even in its absence. The produet of 76,000 had still higher activity in the absence of calmodulin. 3) Whereas the 90,000 fragment responded normally to calmodulin, the 85,000 fragment was less able to do so, despite the fact that it could still bind it. Thus the scheme visualizes the calmodulin binding domain to act as an inhibitory sequence, i.e., it would bind to a domain in the pump that is essential for full activ-

PLASMA

141

MEMBRANE

CaM binding domain

t

PL respons domain

90 KDa

active site Trypsin without effecters + + CaM

Trypsin in the presence of CaM

85 KDa

t

CaM

+ CaM N

-N

Trypsin in EGTA (EDTA)

t

FIG. 3. Scheme for trypsin proteolysis of purified Ca2’ pump in presence of different effecters. Effects of calmodulin (CaM) and acidic phospholipids (PL) on activity of fractions enriched in fragments shown. Scheme is based on work described in Refs. 17,18,74,285,286. Active site is envisaged to be screened by calmodulin binding domain, which would prevent substrate access, thus limiting activity. As domain is removed from site, activity of pump increases. Removal is accomplished in intact pump and in 90,000 fragment by calmodulin interaction or by cleavage and loss of some of its portions (lower molecular mass fragments). Based on recent sequencing work (285), phospholipid-interacting domain has been located away from calmodulin binding domain on its NH,-terminal side. Fully active 76,000 fragment, which has lowest K,(Ca) of all fragments, is visualized to have lost ability to interact with phospholipids due to a structural derangement of this domain.

ity. In principle, the concept is analogous to that proposed for other calmodulin-modulated proteins [e.g., the CAMP-phosphodiesterase (135)] and conveniently explains the phenomenology observed in the trypsin proteolysis work on the isolated pump. As discussed below, the primary structure of the pump has now been elucidated, and some of the functional domains, including the calmodulin binding domain, have been conclusively assigned. Recent work in my laboratory has validated directly the proposal of the calmodulin binding domain as an endogenous inhibitor of the pump (77a). As for the pump domain responsive to acidic phospholipids, recent COOH- and NH,-terminal sequencing work on the major tryptic fragments (286) suggests its location as indi-


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cated in Figure 3 (but see also Fig. 6), i.e., at some distance from the calmodulin binding domain on its NH,-terminal side. Thus the pump would contain two regulatory domains. The main one is COOH terminal and contains the calmodulin responsive region (it also contains the sequence responsive to the CAMP-dependent kinase). The second regulatory domain, located a long distance upstream, contains the region responsive to acidic phospholipids. Future work will decide how closely the proposal illustrated in Figure 3 is related to reality; as emphasized, it will be essential to isolate (and/or to express genetically) the various fragments and to study them separately. With the exception of the experiment described by Zurini et al. (285) on the fragment of 90,000, none of the other fragments has been studied in the purified state [an enriched preparation of the 81,000 component has been found to transport Ca2+ in a liposomal system (17)]. There is no doubt that the domain(s) responsive to calmodulin is in fact separated from the pump bv trypsin digestion (76, 228). However, as pointed out, it is possible-that the trypsin cuts only result in the separation of the NH,-terminal fragment of -33,000 from the molecule under the denaturing conditions of the sodium dodecyl sulfate-polyacrylamide gels. In fact, this seems probable, considering that the separation of the 33,000 fragment would remove from the body of the pump a domain between putative transmembrane helices two and three (see next section) that is preserved in all P-type ion pumps and is thus presumably essential to their function. IV.

PRIMARY OF SOME

STRUCTURE

OF PUMP

OF ITS FUNCTIONAL

AND

IDENTIFICATION

DOMAINS

The elucidation of the primary structure of the plasma membrane Ca2+ pump in 1988 was preceded by partial sequencing work that had established the primary structure of three important functional domains: the fluorescein isothiocyanate (FITC) binding domain (81) that is normally assumed to be part of the binding site for ATP, the domain surrounding the aspartylphosphate (123), and the calmodulin binding domain (1.21). The FITC binding domain has been sequenced after labeling the human erythrocyte ATPase with the reagent, cleaving it with tripsin, and separating the labeled fragment by high-performance liquid chromatography. The domain surrounding the binding site had the sequence F-S-K-G-A-S-E, of which the three central residues are conserved in all P-type ion-motive ATPases. The phosphorylation domain has also been identified in a tryptic fragment of the human erythrocyte pump and has been shown to have the canonical sequence C-S-DK-T-G-T of this domain in all the P-type ATPases (the cysteine and the serine are not conserved in the procaryotic P-type ATPases). The identification of the calmodulin binding domain has been more laborious (121) and has been made possible by the use of a bifunctional, photoactivatable, radioactive, cleavable cross-linker

CARAFOLI

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(62, 120). The reagent was conjugated through an oxysuccinimide moiety situated at one of its ends to lysine residues of calmodulin (probably 75/148). At variance with other calmodulin targets, the integrity of lysine-75 in calmodulin is not required for the interaction with the ATPase. After conjugation to the reagent, Ca2+-saturated calmodulin was covalently attached to its binding site in the human pump through photoactivation of the aryl azide located at the opposite end of the cross-linker. Cleavage of the latter with dithionite at the azo linkage situated between the photoactivable and the oxysuccinimide moieties, followed by removal of calmodulin by dialysis, left the ATPase radioactively labcled in the calmodulin binding domain. Splitting with CNBr followed by separation of the soluble cleavage products yielded a peptide, the sequence of which was NH,-E-L-R-R-G-Q-I-L-W-F-R-G-L-N-R-I-Q-T-Q-I-KV-V-N-A-F-S-S-S-L-H-E-F. The sequence has an obvious predominance of basic amino acids, which is a feature common to the putative calmodulin binding domains of other calmodulin modulated proteins (20, 105, 159). The tryptophan in the NH,-terminal portion of the sequence is also frequently conserved in other putative calmodulin binding domains. Secondary structure predictions show a high probability of helix formation for the 5-6 COOH- and NH,-terminal residues, with the remainder showing strong ,&sheet propensity with a ,& turn in the middle separating the two halves of the domain. In analogy with other Ca2+-dependent calmodulin binding peptides (54), the calmodulin binding domain of the ATPase has strong propensity to form an amphiphilie helix, with the five positive residues on one side and the hydrophobic residues on the other. The location of the domain within the pump molecule was not determined by the labeling and sequencing work. However, chymotryptic cleavage work (121) has shown that the ATPase was completely desensitized to calmodulin, i.e., it had probably lost the calmodulin binding domain, at a time when prominent fragments of high molecular weight were still visible by gel analysis. However, under these conditions 1251-calmodulin only labeled a fragment of molecular weight --X2,000, implying a peripheral location of this domain in the molecule. The carboxy-terminal location was suggested by carboxypeptidase experiments (226) and was definitely established by the cloning work (see below). Whether the pump contains one or two calmodulin binding sites is still an open question. Binding of two molecules has been observed by gel analysis of [‘251]iodoazido-calmodulin cross-linked to the pump (285) and also by stoichometric studies of the binding of iodinated calmodulin to the pump. In the experiments with the cross-linking reagent described (IZI), 50% of the radioactivity remained associated with insoluble peptides after CNB cleavage, suggesting a second calmodulin binding site. If present, however, this second site is most likely not functional, since full activation requires the binding of only one molecule of calmodulin per mole of pump (101). The complete primary structure of the plasma


CALCIUM

PUMP

OF

THE

FIG. 4. Predicted secondary structure of plasma membrane Ca2+ pump. Diagram is based on model of sarcoplasmic reticulum Ca2+-ATPase described in Ref. 164. Secondary structure predictions were performed using University of Wisconsin Genetics Computer Group sequence analysis software package, taking into account alignments of P-type ATPases proposed by Green (103). According to accepted conventions, cu-helices are represented by cylinders and psheets by arrows. 1, putative domain responsive to acidic phospholipids; 2, calmodulin binding domain; 3, domain containing substrate sequence for CAMP-dependent protein kinase. As mentioned in text, this sequence is not present in major isoform of pump expressed in human erythrocytes. 4, flexible hinge, which permits movement of aspartylphosphate and of ATP [fluorescein isothiocyanate (FITC)]-binding lysine to come close in space during reaction cycle (164). M, -Ml,,, putative transmembrane domains.

membrane Ca2’ pump was deduced in 1988 by Shull and Greeb (243) from complementary DNA isolated from rat brain and by Verma et al. (261) from human teratoma libraries. In both cases the approach has been the screening of the library with oligonucleotide probes constructed from suitable tryptic fragments obtained from the human erythrocyte pump (261) or derived from the conserved amino acid sequence of the ATP (FITC) binding site of the P-type ion-motive ATPases (243). The rat brain library has yielded two isoforms of the pump, one having 1,176 amino acids (molecular weight 129,500) and one having 1,198 amino acids (molecular weight 132,605). The isoform from the human teratoma library contains 1,220 amino acids and has a molecular weight of 134,683. This isoform appears to correspond to the first rat brain isoform, with which it shares >99% of the first 1,117 residues, differing significantly only in the COOH-terminal domain. This COOH-terminal difference, however, is due to differential RNA splicing involving a single exon at the COOH-terminal end of the corresponding gene (252). Recent work has led to the identification of two isoforms of the pump expressed in human erythrocytes (251). The most abundant of the two does not contain the CAMP responsive site identified at the COOH-terminus of the teratoma isoform, whereas the minor isoform contains it. The rat brain and human teratoma sequences are

PLASMA

MEMBRANE

143

identical in the catalytic domains [the aspartylphosphate site and the ATP (FITC) binding site]. The socalled “hinge” domain (33) that connects the two previous sites, permitting them to come close in space during the functional cycle, is equally well conserved. The aspartic acid that forms the phosphorylated intermediate is at position 475 in the first rat brain isoform and in the human pump and at position 454 in the second rat brain isoform. The human and rat isoforms also share the essential topological features, i.e., in both species -80% of the total weight of the pump is located outside the membrane domain, protruding into the intracellular space. A secondary structure model of the pump, patterned essentially after that proposed for the sarcoplasmic reticulum Ca2’ pump (164), had assigned 10 putative transmembrane domains (243, 261) using the usual Kyte and Doolittle algorithm (144) (Fig. 4). The transmembrane domains were predicted to be connected by very short loops on the external side of the membrane. Conclusive statements on the topological arrangement of the transmembrane domains would obviously require more than predictions based on the homology with the sarcoplasmic reticulum Ca2’ pump. Advances in this area are now being made by using antibodies raised against synthetic peptides corresponding to selected domains of the pump (F. Hofmann, T. Vorherr, and E. Carafoli, unpublished observations), those forming the putative external loops of the model based on the hydroplots and the analogy with the sarcoplasmic reticulum Ca2’ pump (243,261). As controls, antibodies were raised against the synthetic calmodulin binding domain and an acidic stretch of amino acids immediately NH,-terminal to it (see below). Both domains are necessarily intracellular and would thus un-

FIG. 5. Revised model of membrane architecture of plasma membrane Ca2’ pump based on recent work in my laboratory (F. Hofmann, T. Vorherr, and E. Carafoli, unpublished observations). Details are found in text and in Fig. 4 legend. Numbers in domains spanning membrane refer to putative transmembrane helices shown in model of Fig. 4. Helix 1 and 10 are proposed to be located outside the membrane, in cytosolic space. Second transmembrane helix, spanning residues 270 through 290, had not been predicted in model shown in Fig. 4.


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ambiguously assign the intracellular side of the enzyme. Experiments based on three approaches, i.e., the binding of the antibodies to intact and to permeabilized erythrocytes, to resealed erythrocyte vesicles with opposite membrane polarity, and on competitive enzymelinked immunosorbent (ELISA) assays using antibodies against the complete ATPase molecule, support only in part the provisional model shown in Figure 4. A possible new model is shown in Figure 5. It predicts only light transmembrane domains, assigns a new domain between residues 270 and 290, and places putative transmembrane domains 1, 8, and 10 of the model shown in Figure 4 outside the membrane, on the inner and outer side (domains 8 and lo), respectively. Although this model has some experimental support, it must be tested by additional work. One possible difficulty is the discrepancy with the membrane polarity of some proteolytic cleavage sites in the sarcoplasmic reticulum pump. Whatever the intramembrane architecture of the pump, three main domains protrude into the intracellular ambient. The first protrudes between putative transmembrane helixes two and three and corresponds to the transducing domain of other P-type ion-motive ATPases (164), i.e., to a domain that is postulated to be essential for the coupling of ATP hydrolysis to Ca2+ translocation. It contains both a-helical and antiparallel P-sheet domains, with one of the a-helical domains containing the putative phospholipid-responsive sequence (286). The largest protruding unit (-430 residues) that consists mostly of parallel P-sheets is located between putative transmembrane helixes four and five. It contains the catalytic sites and a domain, called the hinge domain (164), that is postulated to have sufficient flexibility to allow the two catalytic sites to come close in space during the reaction cycle. The fourth unit protrudes from the last putative transmembrane helix with -160 residues and contains the calmodulin binding domain and other domains that identify it as the main regulatory portion of the pump. The calmodulin binding domain is flanked by two acidic stretches, which could in principle represent Ca*+ binding sites. They could regulate the access of Ca*+ to the high-affinity site involved in the catalytic cycle but do not contribute to it, because proteolytic treatments that remove them together with the calmodulin binding domain leave a truncated enzyme that still possesses high Ca*+ affinity (1’7, 18, 285, 286). If these two acidic stretches, particularly the one on the NH,-terminal side of the calmodulin binding domain, do indeed bind Ca*+ in the intact pump, they would thus only function as “Ca*+ filters.” A regulatory function for the acidic sequence NH,-terminal to the calmodulin binding domain in the binding of Ca*+ is indirectly supported by the observation that a synthetic peptide corresponding to it interacts in vitro with the synthetic calmodulin binding domain (266). The location of the high-affinity Ca*+ binding site involved in the catalytic cycle is at the moment unknown, but it need not be located in the vicinity of the calmodulin binding domain; the site is probably formed by conserved resi-

CARAFOLI

Volume 90-85-8

33.5

KDa

1 KDa

35-33.5

71 KDa

(N-terminus)

KDa

(C-terminus)

EAGHGyd

A calcium pump in plasma membrane vesicles from Leishmania braziliensis.

The molecular basis of the modulation of the plasma membrane calcium pump by calmodulin.

Dynamics of the Plasma Membrane Proton Pump.

Evidence for a fourth rat isoform of the plasma membrane calcium pump in the kidney.

Vitamin D and mineral deficiencies increase the plasma membrane calcium pump of chicken intestine.

Binding of calcium by calmodulin: influence of the calmodulin binding domain of the plasma membrane calcium pump.

Hormone-induced phosphorylation of the plasma membrane calcium pump in cultured aortic endothelial cells.

Regulation of the calcium ion pump of sarcoplasmic reticulum: reversible inhibition by phospholamban and by the calmodulin binding domain of the plasma membrane calcium ion pump.

The plasma membrane calcium pump: new ways to look at an old enzyme.

Enhancement of intrarenal plasma membrane calcium pump isoform 1 expression in chronic angiotensin II-infused mice.

Plasma membrane calcium pump expression in human placenta and small intestine.

Microdiversity of human-plasma-membrane calcium-pump isoform 2 generated by alternative RNA splicing in the N-terminal coding region.

The Plasma Membrane Calcium Pump in Pancreatic Cancer Cells Exhibiting the Warburg Effect Relies on Glycolytic ATP.

The calcium pump plasma membrane Ca(2+)-ATPase 2 (PMCA2) regulates breast cancer cell proliferation and sensitivity to doxorubicin.

The calcium pumping ATPase of the plasma membrane.

Relevance of the plasma membrane calcium-ATPase in the homeostasis of calcium in the fetal liver.

Calcium transport across the plasma membrane: stimulation by calmodulin.

Photoaffinity labeling study of the interaction of calmodulin with the plasma membrane Ca2+ pump.

The reversibility of the sarcoplasmic calcium pump.

A circulating inhibitor of the RBC membrane calcium pump in chronic renal failure.

Epitope mapping by deletion mutants reveals the transmembrane topology of the plasma membrane Ca2+ pump.

Evidence for an ATP-driven H(+)-pump in the plasma membrane of the bovine corneal epithelium.

Active transport of calcium in Neurospora plasma membrane vesicles.

Hyperactivation of the human plasma membrane Ca2+ pump PMCA h4xb by mutation of Glu99 to Lys.