Allosteric modulation of Leishmania donovani plasma membrane Ca(2+)-ATPase by endogenous calmodulin.

The plasma membrane of the human pathogen Leishmania donovani possesses a high-affinity transmembrane Ca(2+)-ATPase that has its catalytic site oriented toward the cytoplasmic milieu (Ghosh, J., Ray, M., Sarkar, S., and Bhaduri, A. (1990) J. Biol. Chem. 265, 11345-11351). When the enzyme is studied in its more authentic, physiologically relevant, membrane-associated form, it exhibits pronounced sigmoidal kinetics with Ca2+ (K0.5 approximately 700 nM) in a trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid buffering system that effectively complexes all available Mg2+. Addition of exogenous Mg2+ (60 microM) completely abolishes sigmoidicity and establishes strictly hyperbolic kinetics, and the Km for Ca2+ reduces to 100 nM. Mg2+ can be replaced by heterologous calmodulin. The exclusive dependence of the enzyme on only Ca2+ for its activity and its positive allosteric modulation by Mg2+ distinguish this enzyme from other well-characterized plasma membrane Ca(2+)-ATPases. Employing this Ca(2+)-ATPase as the assay system, a soluble endogenous activating protein factor was purified that, by several criteria, corresponds to authentic calmodulin. The parasite calmodulin shifts the kinetics to hyperbolic kinetics, increases the Vmax 2-fold, and most important lowers the Km (approximately 100 nM) to a physiological level. The interaction with endogenous calmodulin thus converts the enzyme from a totally inactive to a fully active state.     

Mechanism of modulation of the plasma membrane Ca(2+)-ATPase by arachidonic acid

The intracellular level of long chain fatty acids controls the Ca(2+) concentration in the cytoplasm. The molecular mechanisms underlying this Ca(2+) mobilization are not fully understood. We show here that the addition of low micromolar concentrations of fatty acids directly to the purified plasma membrane Ca(2+)-ATPase enhance ATP hydrolysis, while higher concentration decrease activity, exerting a dual effect on the enzyme. The effect of arachidonic acid is similar in the presence or absence of calmodulin, acidic phospholipids or ATP at the regulatory site, thereby precluding these sites as probable acid binding sites. At low arachidonic acid concentrations, neither the affinity for calcium nor the phosphoenzyme levels are significantly modified, while at higher concentrations both are decreased. The action of arachidonic acid is isoenzyme specific. The increase on ATP hydrolysis, however, is uncoupled from calcium transport, because arachidonic acid increases the permeability of erythrocyte membranes to calcium. Oleic acid has no effect on membrane permeability while linoleic acid shows an effect similar to that of arachidonic acid. Such effects might contribute to the entry of extracellular Ca(2+) following to fatty acid release.     

Novel effects of calmodulin and calmodulin antagonists on the plasma membrane (Ca2+ + Mg2+)-ATPase from rabbit kidney proximal tubules.

In this work we report an unusual pattern of activation by calmodulin on the (Ca2+ + Mg2+)-ATPase from basolateral membranes of kidney proximal tubule cells. The activity of the ATPase depleted of calmodulin is characterized by a high Ca2+ affinity (Km = 2.2-3.4 microM) and a biphasic dependence on ATP concentration. The preparation responded to the addition of calmodulin by giving rise to a new Ca2+ site of very high affinity (Km less than 0.05 microM). Calmodulin antagonists had diverse effects on ATPase activity. Compound 48/80 inhibited calmodulin-stimulated activity by 70%, whereas calmidazolium did not modify this component. In the absence of calmodulin, 48/80 still acted as an antagonist, increasing the Km for Ca2+ to 5.7 microM and reducing enzyme turnover by competing with ATP at the low affinity regulatory site. Calmidazolium did not affect Ca2+ affinity, but it did displace ATP from the regulatory site. At fixed Ca2+ (30 microM) and ATP (5 mM) concentrations, Pi protected against 48/80 and potentiated inhibition by calmidazolium. At 25 microM ATP, Pi protected against calmidazolium inhibition. We propose that the effects of ATP and Pi arise because binding of the drugs to the ATPase occurs mainly on the E2 forms.     

Caloxin 1b3: a novel plasma membrane Ca(2+)-pump isoform 1 selective inhibitor that increases cytosolic Ca(2+) in endothelial cells

The purpose of this study was to invent an extracellular inhibitor selective for the plasma membrane Ca(2+) pump(s) (PMCA) isoform 1. PMCA extrude Ca(2+) from cells during signalling and homeostasis. PMCA isoforms are encoded by 4 genes (PMCA1-4). Pig coronary artery endothelium and smooth muscle express the genes PMCA1 and 4. We showed that the endothelial cells contained mostly PMCA1 protein while smooth muscle cells had mostly PMCA4. A random peptide phage display library was screened for binding to synthetic extracellular domain 1 of PMCA1. The selected phage population was screened further by affinity chromatography using PMCA from rabbit duodenal mucosa which expressed mostly PMCA1. The peptide displayed by the selected phage was termed caloxin 1b3. Caloxin 1b3 inhibited PMCA Ca(2+)-Mg(2+)-ATPase in the rabbit duodenal mucosa (PMCA1) with a greater affinity (inhibition constant=17±2 μM) than the PMCA in the human erythrocyte ghosts (PMCA4, inhibition constant=45±4 μM). The affinity of caloxin 1b3 was also higher for PMCA1 than for PMCA2 and 3 indicating its selectivity for PMCA1. Consistent with an inhibition of PMCA1, caloxin 1b3 addition to the medium increased cytosolic Ca(2+) concentration in endothelial cells. Caloxin 1b3 is the first known PMCA1 selective inhibitor. We anticipate caloxin 1b3 to aid in understanding PMCA physiology in endothelium and other tissues.     

Structure of the rat plasma membrane Ca(2+)-ATPase isoform 3 gene and characterization of alternative splicing and transcription products. Skeletal muscle-specific splicing results in a plasma membrane Ca(2+)-ATPase with a novel calmodulin-binding domain.

We have isolated the rat gene encoding isoform 3 of the plasma membrane Ca(2+)-ATPase (PMCA3) and have determined its exon/intron organization. The PMCA3 gene contains 24 exons and spans approximately 70 kilobases. In addition, we have analyzed the splicing and polyadenylation patterns leading to the production of an alternative 4.5-kilobase (PMCA3) skeletal muscle mRNA that differs from the previously characterized 7.5-kilobase brain mRNA (Greeb, J., and Shull, G. E. (1989) J. Biol. Chem. 264, 18569-18576). cDNA cloning, Northern blot hybridization, and polymerase chain reaction analyses of the 4.5-kilobase mRNA demonstrate (i) the inclusion of a novel 68-nucleotide exon (exon 22) that is specific for skeletal muscle and significantly alters the calmodulin-binding domain and (ii) the utilization of an alternative polyadenylation site following exon 23 which eliminates the last coding exon (exon 24) and 3'-untranslated sequence of the 7.5-kilobase mRNA. We have also identified a 42-nucleotide exon (exon 8) that is included in the skeletal muscle PMCA3 mRNAs, but may be either included or excluded in the brain mRNAs. Exon 8 is inserted immediately before the sequence encoding a putative phospholipid binding domain and thus may alter regulatory interactions of the enzyme with acidic phospholipids.     

Single-molecule dynamics reveal an altered conformation for the autoinhibitory domain of plasma membrane Ca(2+)-ATPase bound to oxidatively modified calmodulin

We used single-molecule polarization modulation methods to investigate the activation of the plasma membrane Ca(2+)-ATPase (PMCA) by oxidized calmodulin (CaM). Oxidative modification of methionine residues of CaM to their corresponding sulfoxides is known to inhibit the ability of CaM to activate PMCA. Single-molecule polarization methods were used to measure the orientational mobility of fluorescently labeled oxidized CaM bound to PMCA. We previously identified two distinct populations of PMCA-CaM complexes characterized by high and low orientational mobilities, with the low-mobility population appearing at a subsaturating Ca(2+) concentration [Osborn, K. D., et al. (2004) Biophys. J. 87, 1892-1899]. We proposed that the high-mobility population corresponds to PMCA-CaM complexes with a dissociated (and mobile) autoinhibitory domain, whereas the low-mobility population corresponds to PMCA-CaM complexes where the autoinhibitory domain is not dissociated and therefore the enzyme is not active. In the present experiments, performed with PMCA complexed with oxidatively modified CaM at a saturating Ca(2+) concentration, we found a large population of molecules with an orientationally immobile autoinhibitory domain. In contrast, native CaM bound to PMCA was characterized almost entirely by the more orientationally mobile population at a similar Ca(2+) concentration. The addition of 1 mM ATP to complexes of oxidized CaM with PMCA reduced but did not abolish the low-mobility population. These results indicate that the decline in the ability of oxidized CaM to activate PMCA results at least in part from its reduced ability to induce conformational changes in PMCA that result in dissociation of the autoinhibitory domain after CaM binding.     

Activation of an islet cell plasma membrane (Ca2+ + Mg2+)-ATPase by calmodulin and Ca-EGTA

Islet cell plasma membranes contain a calcium-stimulated and magnesium-dependent ATPase (Ca2+ + Mg2+)-ATPase) which requires calmodulin for maximum enzyme activity (Kotagal, N., Patke, C., Landt, M., McDonald, J., Colca, J., Lacy, P., and McDaniel, M. (1982) FEBS Lett. 137, 249-252). Investigations indicated that exogenously added calmodulin increases the velocity and decreases the Km for Ca2+ of the high affinity (Ca2+ + Mg2+)-ATPase. These studies routinely employed the chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to maintain Ca2+ concentrations in the submicromolar range. During the course of these investigations, it was found unexpectedly that increasing the concentrations of EGTA (0.1-4 mM) and total calcium in the media, while maintaining constant free Ca2+ levels, increased the velocity of the high affinity (Ca2+ + Mg2+)-ATPase. The free calcium concentrations under these conditions were verified by a calcium-sensitive electrode. The (Ca2+ + Mg2+)-ATPase maximally activated by 2-4 mM EGTA was not further stimulated by calmodulin, whereas camodulin stimulation increased as the concentration of EGTA in the media was decreased. A similar enhancement by Ca-EGTA was observed on active calcium transport by the plasma membrane-enriched fraction. Moreover, Ca-EGTA had a negligible effect on both active calcium transport as well as Ca2+-stimulated ATPase activity by the islet cell endoplasmic reticulum, processes which are not stimulated by calmodulin. The results indicate that stimulation by Ca-EGTA may be used to differentiate calcium transport systems by these subcellular organelles. Furthermore, the concentration of EGTA routinely employed to maintain free Ca2+ levels may itself obscure effects of calmodulin and other physiological agents on calcium-dependent activities.     

The plasma membrane Ca2+ pump isoform 4a differs from isoform 4b in the mechanism of calmodulin binding and activation kinetics: implications for Ca2+ signaling

The inhibition by the regulatory domain and the interaction with calmodulin (CaM) vary among plasma membrane calcium pump (PMCA) isoforms. To explore these differences, the kinetics of CaM effects on PMCA4a were investigated and compared with those of PMCA4b. The maximal apparent rate constant for CaM activation of PMCA4a was almost twice that for PMCA4b, whereas the rates of activation for both isoforms showed similar dependence on Ca2+. The inactivation of PMCA4a by CaM removal was also faster than for PMCA4b, and Ca2+ showed a much smaller effect (2- versus 30-fold modification). The rate constants of the individual steps that determine the overall rates were obtained from stopped-flow experiments in which binding of TA-CaM was observed by changes in its fluorescence. TA-CaM binds to two conformations of PMCA4a, an "open" conformation with high activity, and a "closed" one with lower activity. Compared with PMCA4b (Penheiter, A. R., Bajzer, Z., Filoteo, A. G., Thorogate, R., T?r?k, K., and Caride, A. J. (2003) Biochemistry 41, 12115-12124), the model for PMCA4a predicts less inhibition in the closed form and a much faster equilibrium between the open and closed forms. Based on the available kinetic parameters, we determined the constants to fit the shape of a Ca2+ signal in PMCA4b-overexpressing Chinese hamster ovary cells. Using the constants for PMCA4a, and allowing small variations in parameters of other systems contributing to a Ca2+ signal, we then simulated the effect of PMCA4a on the shape of a Ca2+ signal in Chinese hamster ovary cells. The results reproduce the published data (Brini, M., Coletto, L., Pierobon, N., Kraev, N., Guerini, D., and Carafoli, E. (2003) J. Biol. Chem. 278, 24500-24508), and thereby demonstrate the importance of altered regulatory kinetics for the different functional properties of PMCA isoforms.     

Plasma membrane Ca(2+) pump isoform 3f is weakly stimulated by calmodulin

Isoform 3f of the plasma membrane Ca(2+) pump is a major isoform of this pump in rat skeletal muscle. It has an unusual structure, with a short carboxyl-terminal regulatory region of only 33 residues when compared with the 77 to 124 residues found in the other isoforms. Also, whereas the regulatory regions of the other isoforms, downstream of the alternative splice, consist of two homologous groups, the sequence of 3f is not related to either group. A synthetic peptide representing the calmodulin binding domain of isoform 3f had a much lower calmodulin affinity (with a K(d) of 15 nM) than the corresponding peptide of isoform 2b (K(d) value was 0.2 nM). The characteristics of this domain were further studied by making chimeras of the 3f regulatory region with the catalytic core of isoform 4 and by making the full-length isoform 3f. Both constructs bound to calmodulin-Sepharose. The chimera was fully active without calmodulin, showing no stimulation of activity when calmodulin was added. The full-length isoform 3f was slightly activated by calmodulin. These data show that the regulatory region of isoform 3f is only a weak autoinhibitor of the enzyme, in contrast to the properties of all the other isoforms studied so far. Rather, this isoform is a special-purpose, constitutively active form of the enzyme, expressed primarily in skeletal muscle and as a minor isoform in brain.     

The C-terminal 165 amino acids of the plasma membrane Ca(2+)-ATPase confer Ca2+/calmodulin sensitivity on the Na+,K(+)-ATPase alpha-subunit.

The C-terminal 165 amino acids of the rat brain plasma membrane (PM) Ca(2+)-ATPase II containing the calmodulin binding auto-inhibitory domain was connected to the C-terminus of the ouabain sensitive chicken Na+,K(+)-ATPase alpha 1 subunit. Expression of this chimeric molecule in ouabain resistant mouse L cells was assured by the high-affinity binding of [3H]ouabain. In the presence of Ca2+/calmodulin, this chimeric molecule exhibited ouabain inhibitable Na+,K(+)-ATPase activity; the putative chimeric ATPase activity was absent in the absence of Ca2+/calmodulin and activated by Ca2+/calmodulin in a dose-dependent manner. Furthermore, this chimeric molecule could bind monoclonal IgG 5 specific to the chicken Na+,K(+)-ATPase alpha 1 subunit only in the presence of Ca2+/calmodulin, suggesting that the epitope for IgG 5 in this chimera is masked in the absence of Ca2+/calmodulin and uncovered in their presence. These results propose a direct interaction between the calmodulin binding auto-inhibitory domain of the PM Ca(2+)-ATPase and the specific regions of the Na+,K(+)-ATPase alpha 1 subunit that are structurally homologous to the PM Ca(2+)-ATPase. A comparison of the deduced amino acid sequences revealed several possible regions within the Na+,K(+)-ATPase that might interact with the auto-inhibitory domain of the PM Ca(2+)-ATPase.     

Mediating molecular recognition by methionine oxidation: conformational switching by oxidation of methionine in the carboxyl-terminal domain of calmodulin

The C-terminus of calmodulin (CaM) functions as a sensor of oxidative stress, with oxidation of methionine 144 and 145 inducing a nonproductive association of the oxidized CaM with the plasma membrane Ca(2+)-ATPase (PMCA) and other target proteins to downregulate cellular metabolism. To better understand the structural underpinnings and mechanism of this switch, we have engineered a CaM mutant (CaM-L7) that permits the site-specific oxidation of M144 and M145, and we have used NMR spectroscopy to identify structural changes in CaM and CaM-L7 and changes in the interactions between CaM-L7 and the CaM-binding sequence of the PMCA (C28W) due to methionine oxidation. In CaM and CaM-L7, methionine oxidation results in nominal secondary structural changes, but chemical shift changes and line broadening in NMR spectra indicate significant tertiary structural changes. For CaM-L7 bound to C28W, main chain and side chain chemical shift perturbations indicate that oxidation of M144 and M145 leads to large tertiary structural changes in the C-terminal hydrophobic pocket involving residues that comprise the interface with C28W. Smaller changes in the N-terminal domain also involving residues that interact with C28W are observed, as are changes in the central linker region. At the C-terminal helix, (1)H(alpha), (13)C(alpha), and (13)CO chemical shift changes indicate decreased helical character, with a complete loss of helicity for M144 and M145. Using (13)C-filtered, (13)C-edited NMR experiments, dramatic changes in intermolecular contacts between residues in the C-terminal domain of CaM-L7 and C28W accompany oxidation of M144 and M145, with an essentially complete loss of contacts between C28W and M144 and M145. We propose that the inability of CaM to fully activate the PMCA after methionine oxidation originates in a reduced helical propensity for M144 and M145, and results primarily from a global rearrangement of the tertiary structure of the C-terminal globular domain that substantially alters the interaction of this domain with the PMCA.     

Localization and regulation of plasma membrane Ca(2+)-ATPase in bovine spermatozoa

Calcium (Ca(2+)) signals, produced by the opening of plasma membrane entry channels, regulate a number of functions in spermatozoa such as capacitation and motility. The mechanisms of Ca(2+) removal from the sperm, required to restore resting [Ca(2+)](i), include plasma membrane Ca(2+)-dependent ATPase (PMCA) isoenzymes as well as a plasma membrane Na(+)-Ca(2+) exchanger. We have recently shown that bovine sperm PMCA is stimulated by PDC-109, a secretory protein of bovine seminal vesicles. To demonstrate the subcellular localization and regulation of bovine sperm PMCA, we have performed cell fractionation, enzyme activity determination and Western blotting studies of PMCA in spermatozoa removed from the cauda epididymidis of bull. Fractionation of sperm heads and tails resulted in a distinct association of ATPase activity with the tail membrane fraction. In vitro stimulation studies with PDC-109 using intact and fractionated sperm showed an increase in enzyme activity up to 105% in sperm tail membranes. Furthermore, thapsigargin inhibition did not alter the stimulatory effect of PDC-109 on ATPase activity, indicating that no sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), but only PMCA isoenzymes are involved in this effect. Western blotting studies using a polyvalent PMCA antibody showed the exclusive presence of a 135 kDa band in the tail plasma membrane fraction. To elucidate whether or not the stimulatory effect was a direct one or indirectly mediated through PKA and PKC activation, PKA and PKC inhibitors, respectively, were used in the Ca(2+)-ATPase activity assays, which was followed by PDC-109 stimulation. The stimulatory effect of PDC-109 on PMCA was still observed under these conditions, while no phosphotyrosine proteins could be detected by Western blotting in sperm extracts following PDC-109 treatment. Co-immunoprecipitation studies, PDC-109 affinity chromatography as well as overlay blots failed to show a strong association of both PMCA and PDC-109, pointing to an indirect, perhaps phospholipid-mediated effect.     

Trypanosoma brucei plasma membrane-type Ca(2+)-ATPase 1 (TbPMC1) and 2 (TbPMC2) genes encode functional Ca(2+)-ATPases localized to the acidocalcisomes and plasma membrane, and essential for Ca(2+) homeostasis and growth

Trypanosoma brucei adaptation and survival in its host involve integrated regulation of Ca(2+) pumps (Ca(2+)-ATPases), which are essential in calcium ion homeostasis. Here we report the cloning and sequencing of two genes (TbPMC1 and TbPMC2) encoding plasma membrane-type Ca(2+)-ATPases (PMCAs) of T. brucei, an agent of African trypanosomiasis. Indirect immunofluorescence analysis using antibodies against the proteins and against epitope tags introduced into each protein showed that TbPMC1 co-localized with the vacuolar H(+)-pyrophosphatase to the acidocalcisomes while TbPMC2 localized to the plasma membrane. Northern and Western blot analyses revealed that TbPMC1 and TbPMC2 are up-regulated during blood stages. TbPMC1 and TbPMC2 suppressed the Ca(2+) hypersensitivity of a mutant of S. cerevisiae that has a defect in vacuolar Ca(2+) accumulation. T. brucei Ca(2+)-ATPase genes were functionally characterized by using double-stranded RNA interference (RNAi) methodology to produce inducible Ca(2+)-ATPase-deficient procyclic forms. Similar results were obtained with bloodstream form trypomastigotes, except that the RNAi system was leaky and mRNA and protein levels recovered with time. The induction of dsRNA (RNAi) caused gross morphological alterations, and growth inhibition of procyclic forms. Induction of RNAi against TbPMC1 but not against TbPMC2 caused elevated levels of cytosolic Ca(2+) and decreased mobilization of Ca(2+) from intracellular stores following ionophore addition. These results establish that T. brucei PMCA-Ca(2+)-ATPases are essential for parasite viability and validate them as targets for drug development.     

Inhibition of plasma membrane Ca(2+)-ATPase by CrATP. LaATP but not CrATP stabilizes the Ca(2+)-occluded state

The bidentate complex of ATP with Cr(3+), CrATP, is a nucleotide analog that is known to inhibit the sarcoplasmic reticulum Ca(2+)-ATPase and the Na(+),K(+)-ATPase, so that these enzymes accumulate in a conformation with the transported ion (Ca(2+) and Na(+), respectively) occluded from the medium. Here, it is shown that CrATP is also an effective and irreversible inhibitor of the plasma membrane Ca(2+)-ATPase. The complex inhibited with similar efficiency the Ca(2+)-dependent ATPase and the phosphatase activities as well as the enzyme phosphorylation by ATP. The inhibition proceeded slowly (T(1/2)=30 min at 37 degrees C) with a K(i)=28+/-9 microM. The inclusion of ATP, ADP or AMPPNP in the inhibition medium effectively protected the enzyme against the inhibition, whereas ITP, which is not a PMCA substrate, did not. The rate of inhibition was strongly dependent on the presence of Mg(2+) but unaltered when Ca(2+) was replaced by EGTA. In spite of the similarities with the inhibition of other P-ATPases, no apparent Ca(2+) occlusion was detected concurrent with the inhibition by CrATP. In contrast, inhibition by the complex of La(3+) with ATP, LaATP, induced the accumulation of phosphoenzyme with a simultaneous occlusion of Ca(2+) at a ratio close to 1.5 mol/mol of phosphoenzyme. The results suggest that the transport of Ca(2+) promoted by the plasma membrane Ca(2+)-ATPase goes through an enzymatic phospho-intermediate that maintains Ca(2+) ions occluded from the media. This intermediate is stabilized by LaATP but not by CrATP.     

The calmodulin binding domain of the plasma membrane Ca2+ pump interacts both with calmodulin and with another part of the pump

Synthetic peptides corresponding to the calmodulin-binding domain of the human erythrocyte Ca2+ pump were prepared representing residues 2-29 (C28W), 2-21 (C20W), 2-16 (C15W), and 16-29 (C14) of the sequence (James, P., Maeda, M., Fisher, R., Verma, A. K., Krebs, J., Penniston, J. T., and Carafoli, E. (1988) J. Biol. Chem. 263, 2905-2910). Peptides C28W, C20W, and C15W bound to calmodulin with an apparent 1:1 stoichiometry in the presence of Ca2+ and inhibited the activation of the Ca2+ pump by calmodulin, while C14 was ineffective. Substituting tyrosine (C28Y) or alanine (C28A) for the tryptophan residue lowered the affinity for calmodulin. The estimated Kd values for the calmodulin-peptide complexes were 0.1 nM for C28W, 5-15 nM for C20W, C28Y, and C28A, and 700-1700 nM for C15W. The Ca2+ pump in inside-out erythrocyte membrane vesicles was activated by proteolytic removal of the endogenous calmodulin-binding domain. Addition of C20W or C28W then inhibited calmodulin-independent Ca2+ transport, while a calmodulin-binding peptide from another enzyme had no effect. The inhibition of the pump by C20W was purely competitive with Ca2+, while C28W decreased the Vmax and increased the K1/2 for Ca2+, restoring the pump activity nearly to its low basal level. The results suggest that a calmodulin-binding peptide from any enzyme has two kinds of specificity: it shares with peptides from other enzymes the ability to bind to calmodulin, but only it has the specificity to interact with its own (proteolytically activated) enzyme.     

Protein kinase C and calmodulin effects on the plasma membrane Ca2+-ATPase from excitable and nonexcitable cells

We have purified Ca2+-ATPase from synaptosomal membranes (SM)1 from ratcerebellum by calmodulin affinity chromatography. The enzyme was identifiedas plasma membrane Ca2+-ATPase by its interaction with calmodulin andmonoclonal antibodies produced against red blood cell (RBC) Ca2+-ATPase, andby thapsigargin insensitivity. The purpose of the study was to establishwhether two regulators of the RBC Ca2+-ATPase, calmodulin and protein kinaseC (PKC), affect the Ca2+-ATPase isolated from excitable cells and whethertheir effects are comparable to those on the RBC Ca2+-ATPase. We found thatcalmodulin and PKC activated both enzymes. There were significantquantitative differences in the phosphorylation and activation of the SMversus RBC Ca2+-ATPase. The steady-state Ca2+-ATPase activity of SMCa2+-ATPase was approximately 3 fold lower and significantly less stimulatedby calmodulin. The initial rate of PKC catalyzed phosphorylation (in thepresence of 12-myristate 13-acetate phorbol) was approximately two timesslower for SM enzyme. While phosphorylation of RBC Ca2+-ATPase approachedmaximum level at around 5 min, comparable level of phosphorylation of SMCa2+-ATPase was observed only after 30 min. The PKC-catalyzedphosphorylation resulted in a statistically significant increase inCa2+-ATPase activity of up to 20-40%, higher in the SM Ca2+-ATPase.The differences may be associated with diversities in Ca2+-ATPase functionin erythrocytes and neuronal cells and different isoforms composition.     

Study of calmodulin binding to the alternatively spliced C-terminal domain of the plasma membrane Ca2+ pump.

The C-terminal regions of the four human plasma membrane Ca2+ pump isoforms 1a-d generated from alternatively spliced RNA have been expressed in Escherichia coli, and the recombinant proteins have been purified to a very high degree. The C-termini of isoforms 1a, 1c, and 1d contain an insert encoded by an alternatively spliced exon which is homologous to the calmodulin binding domain of isoform 1b. In isoforms 1c and 1d (29 and 38 amino acid insertions, respectively), subdomain A of the original calmodulin binding site of isoform 1b is followed by the spliced-in domain, which is then followed by subdomain B of the original calmodulin binding site. The positive charges of histidine residues at positions 27, 28, and 38 of the alternatively spliced sequence are likely to be responsible for the observed pH-dependent calmodulin binding to the novel "duplicated" binding site. The affinity of calmodulin for the C-terminal domains of isoforms 1a, 1c, and 1d, which contain the histidine-rich inserts, is much higher at pH 5.9 than at pH 7.2. A synthetic peptide (I31) containing 31 amino acids of the alternatively spliced sequence (from residue 9 to 40) also binds calmodulin with strong pH dependency. Alternative splicing in the C-terminal domain is proposed to confer pH dependence to the regulation of the activity of Ca2+ pump isoforms.     

In calmodulin-IQ domain complexes, the Ca(2+)-free and Ca(2+)-bound forms of the calmodulin C-lobe direct the N-lobe to different binding sites

We have investigated the roles played by the calmodulin (CaM) N- and C-lobes in establishing the conformations of CaM-IQ domain complexes in different Ca(2+)-free and Ca(2+)-bound states. Our results indicate a dominant role for the C-lobe in these complexes. When the C-lobe is Ca(2+)-free, it directs the N-lobe to a binding site within the IQ domain consensus sequence. It appears that the N-lobe must be Ca(2+)-free to interact productively with this site. When the C-lobe is Ca(2+)-bound, it directs the N-lobe to a site upstream of the consensus sequence, and it appears that the N-lobe must be Ca(2+)-bound to interact productively with this site. A model for switching in CaM-IQ domain complexes is presented in which the N-lobe adopts bound and extended positions that depend on the status of the Ca(2+)-binding sites in each CaM lobe and the compositions of the two N-lobe binding sites. Ca(2+)-dependent changes in the conformation of the bound C-lobe that appear to be responsible for directed N-lobe binding are also identified. Changes in the equilibria between extended and bound N-lobe positions may control bridging interactions in which the extended N-lobe is bound to another CaM-binding domain. Ca(2+)-dependent control of bridging interactions with CaM has been implicated in the regulation of ion channel and unconventional myosin activities.