Differential regulation of Ca2+/calmodulin-dependent enzymes by plant calmodulin isoforms and free Ca2+ concentration.

Multiple calmodulin (CaM) isoforms are expressed in plants, but their biochemical characteristics are not well resolved. Here we show the differential regulation exhibited by two soya bean CaM isoforms (SCaM-1 and SCaM-4) for the activation of five CaM-dependent enzymes, and the Ca(2+) dependence of their target enzyme activation. SCaM-1 activated myosin light-chain kinase as effectively as brain CaM (K(act) 1.8 and 1.7 nM respectively), but SCaM-4 produced no activation of this enzyme. Both CaM isoforms supported near maximal activation of CaM-dependent protein kinase II (CaM KII), but SCaM-4 exhibited approx.12-fold higher K(act) than SCaM-1 for CaM KII phosphorylation of caldesmon. The SCaM isoforms showed differential activation of plant and animal Ca(2+)-ATPases. The plant Ca(2+)-ATPase was activated maximally by both isoforms, while the erythrocyte Ca(2+)-ATPase was activated only by SCaM-1. Plant glutamate decarboxylase was activated fully by SCaM-1, but SCaM-4 exhibited an approx. 4-fold increase in K(act) and an approx. 25% reduction in V(max). Importantly, SCaM isoforms showed a distinct Ca(2+) concentration requirement for target enzyme activation. SCaM-4 required 4-fold higher [Ca(2+)] for half-maximal activation of CaM KII, and 1.5-fold higher [Ca(2+)] for activation of cyclic nucleotide phosphodiesterase than SCaM-1. Thus these plant CaM isoforms provide a mechanism by which a different subset of target enzymes could be activated or inhibited by the differential expression of these CaM isoforms or by differences in Ca(2+) transients.

Effects of volatile anesthetic on the Ca2+-ATPase activation by dimerization. Distance-dependent quenching analysis and fluorescence energy transfer studies.

The phenomenological distance-dependent quenching (DDQ) model was employed to investigate the character of the interaction between volatile anesthetics (VAs) and the plasma membrane Ca2+-ATPase (PMCA). The simultaneous analysis of the frequency-domain and steady-state data of tryptophan (Trp) fluorescence quenching by a VA points to a specific character of the apparent quenching effect of the VA, possibly arising from a significant contribution of static quenching. The apparent contributions of both static and dynamic quenching may be due to VA binding in the PMCA, which results in the modification of the conformational substates of the enzyme. To characterize further the molecular consequences of VA binding, we investigated its effects on the process of PMCA activation by self-association. VA shifted the equilibrium from enzyme dimers to monomers, as monitored by the loss of fluorescence energy transfer. The shift was apparently due to the VA-induced decrease in the affinity of PMCA molecules for self-association. Addition of a large molecular mass dextran to increase the proximity between enzyme monomers induced re-association of the VA-impaired PMCA, while the Ca2+-ATPase activity was not recovered. The results are congruent with a dual VA effect on PMCA, a shift in the monomer/dimer equilibrium, and an inactivation of both monomers and dimers.

Estrogen increases cGMP in selected brain regions and in cerebral microvessels.

We have previously reported that exogenous and endogenous estrogen can amplify residual cerebral blood flow during experimental cerebral ischemia. Because estrogen has been linked to nitric oxide and cyclic guanosine monophosphate (cGMP) signaling in noncerebral tissue, we tested the hypothesis that long-term 17beta-estradiol treatment increases basal cGMP in brain homogenates and cerebral microvessels in female rabbits. We also determined whether there are important baseline gender-specific differences in regional cGMP. Adult female rabbits were implanted with 17beta-estradiol pellets, 10 mg (F10, n = 10) or 50 mg (F50, n = 13), and compared with untreated females (F, n = 19) and males with negligible estrogen (M, n = 19) (plasma 17beta-estradiol levels of 4+/-4 pg/mL in M, 7+/-5 pg/mL in F, 141+/-74 pg/mL in F10, and 289 +/-10 pg/mL in F50). Cyclic GMP was determined by radioimmunoassay in cerebellum, hypothalamus, caudate nucleus, hippocampus, and cortex. Cerebral microvessels were harvested from additional cohorts of untreated males and females or estradiol-implanted females (n = 6 per group). Basal cGMP was higher in F versus M only in cerebellum. Estrogen-induced increases in regional cGMP were prominent in hippocampus at all doses (M = 43+/-26, F = 43+/-21, F10 = 84+/-24, F50 = 117+/-55 fmol/mg protein) and in cortex at the high dose (M = 78+/-55, F = 88+/-51, F10 = 69+/-34, F50 = 143+/-52 fmol/mg protein). Similarly, microvascular cGMP increased only in females treated with the 50 mg dose (M = 77+/-13, F = 86+/-25, F10O = 106+/-35, F50 = 192+/-88 fmol/mg protein). Therefore, 17beta-estradiol increases cGMP content in parenchymal regions that are known physiologic targets for reproductive steroids but are also areas of selective vulnerability to ischemic insult. Further, high doses of estrogenic steroids could amplify cGMP signaling within the cerebral microvasculature.

Spectroscopic analysis of halothane binding to the plasma membrane Ca2+-ATPase.

The intrinsic tryptophan (Trp) fluorescence of the plasma membrane Ca2+-ATPase (PMCA) is significantly quenched by halothane, a volatile anesthetic common in clinical practice. It has been proposed that halothane inhibition of the Ca2+-ATPase activity results from conformational changes following anesthetic binding in the enzyme. We have investigated whether the observed quenching reflects halothane binding to PMCA. We have shown that the quenching is dose dependent and saturable and can be fitted to a binding curve with an equilibrium constant K(Hal) = 2.1 mM, a concentration at which the anesthetic approximately half-maximally inhibits the Ca2+-ATPase activity. The relatively low sensitivity of halothane quenching of Trp fluorescence to the concentration of phosphatidylcholine and detergent in the PMCA preparation concurs with the quenching resulting from anesthetic binding in the PMCA molecule. Analysis of the Trp fluorescence quenching by acrylamide indicates that the Trp residues are not considerably exposed to the solvent (Stern-Volmer quenching constant of 2.9 M(-1)) and do not differ significantly in their accessibility to halothane. Other volatile anesthetics, diethyl ether and diisopropyl ether, reduce the quenching caused by halothane in a dose-dependent manner, suggesting halothane displacement from its binding site(s). These observations indicate that halothane quenching of intrinsic Trp fluorescence of PMCA results from anesthetic binding to the protein. The analysis, used as a complementary approach, provides new information to the still rudimentary understanding of the process of anesthetic interaction with membrane proteins.

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 rat cerebellum by calmodulin affinity chromatography. The enzyme was identified as plasma membrane Ca2+-ATPase by its interaction with calmodulin and monoclonal antibodies produced against red blood cell (RBC) Ca2+-ATPase, and by thapsigargin insensitivity. The purpose of the study was to establish whether two regulators of the RBC Ca2+-ATPase, calmodulin and protein kinase C (PKC), affect the Ca2+-ATPase isolated from excitable cells and whether their effects are comparable to those on the RBC Ca2+-ATPase. We found that calmodulin and PKC activated both enzymes. There were significant quantitative differences in the phosphorylation and activation of the SM versus RBC Ca2+-ATPase. The steady-state Ca2+-ATPase activity of SM Ca2+-ATPase was approximately 3 fold lower and significantly less stimulated by calmodulin. The initial rate of PKC catalyzed phosphorylation (in the presence of 12-myristate 13-acetate phorbol) was approximately two times slower for SM enzyme. While phosphorylation of RBC Ca2+-ATPase approached maximum level at around 5 min, comparable level of phosphorylation of SM Ca2+-ATPase was observed only after 30 min. The PKC-catalyzed phosphorylation resulted in a statistically significant increase in Ca2+-ATPase activity of up to 20-40%, higher in the SM Ca2+-ATPase. The differences may be associated with diversities in Ca2+-ATPase function in erythrocytes and neuronal cells and different isoforms composition.

Novel components and enzymatic activities of the human erythrocyte plasma membrane calcium pump.

The plasma membrane Ca2+ pump is essential for the maintenance of cystolic calcium ion concentration levels in eukaryotes. Here we show that the Ca2+-ATPase, purified from human erythrocytes, contains two homopolymers, poly(3-hydroxybutyrate) (PHB) and inorganic polyphosphate (polyP), which form voltage-activated calcium channels in the plasma membranes of Escherichia coli and other bacteria. Furthermore, we demonstrate that the plasma membrane Ca2+-ATPase may function as a polyphosphate kinase, i.e. it exhibits ATP-polyphosphate transferase and polyphosphate-ADP transferase activities. These findings suggest a novel supramolecular structure for the functional Ca2+-ATPase, and a new mechanism of uphill Ca2+ extrusion coupled to ATP hydrolysis.

Entropy-driven interactions of anesthetics with membrane proteins.

Thermodynamic analysis of anesthetic effects on Ca2+-ATPase activity was performed to evaluate the feasibility of anesthetic binding and gain insight into the molecular events underlying the anesthetic-enzyme interactions. The Ca2+-ATPases, integral membrane proteins vital in cellular Ca2+ regulation, are suitable models for investigation of the mechanism of anesthetic action on membrane proteins that are targeted by the anesthetics. Ca2+-ATPase of plasma membrane, PMCA, and SERCA1 in the intracellular sarcoplasmic reticulum membrane were used to study two general anesthetics: halothane, a halogenated two-carbon alkane; and propofol, an intravenous, strongly lipophilic-substituted phenol. Interactions of both anesthetics result in a negative Gibbs free energy change, which in both enzymes is more favorable for the more lipophilic propofol than halothane. Temperature dependence (more negative change in Gibbs free energy at increased temperature) is in agreement with predominantly nonpolar interactions. The interactions are entropy-driven, characterized by positive enthalpy which is overcompensated by positive entropy changes. This is in contrast to the reported in literature enthalpy-driven anesthetic binding to soluble proteins. The possible contributions to the observed positive entropy change are discussed including displacement of ordered water molecules by anesthetic binding in nonpolar cavities in the membrane proteins and subtle structural rearrangements.

Heterogeneous halothane binding in the SR Ca2+-ATPase.

The activity of various Ca2+-ATPases is affected by volatile anesthetics, such as halothane, commonly used in clinical practice. The effect on the enzyme in skeletal muscle sarcoplasmic reticulum (SR) is biphasic, including stimulation at clinical anesthetic concentrations and subsequent inhibition at higher concentrations. We have previously proposed that the action of a volatile anesthetic on Ca2+-ATPases results from its binding in the interior of the enzyme molecule [Lopez, M.M. and Kosk-Kosicka, D. (1995) J. Biol. Chem. 270, 28239-28245]. Presently, we investigated whether the anesthetic interacts directly with the skeletal muscle SR Ca2+-ATPase (SERCA1) as evidenced by binding. Photoaffinity labeling with [14C]halothane demonstrated that the anesthetic binds saturably to SR membranes, and that approximately 80% of the binding is specific, with a KI of 0.6 mM. The KI value agrees well with the concentration at which halothane half-maximally activates SERCA1. SDS gel electrophoresis of labeled membranes indicates that 38-56% of [14C]halothane incorporates into SERCA1, and 38-53% in lipids. Distribution of label among the three fragments produced by controlled tryptic digestion of SERCA1 suggests heterogeneous halothane binding presumably in discrete sites in the enzyme. The results provide the first direct evidence that halothane binds to SERCA1. Potentially this binding could be related to anesthetic effect on enzyme's function.

Analysis of phosphorylation and mutation of tyrosine residues of calmodulin on its activation of the erythrocyte Ca(2+)-transporting ATPase.

The role played by the phosphorylation sites of calmodulin on its ability to activate the human erythrocyte Ca(2+)-transporting ATPase (Ca(2+)-ATPase) was evaluated. Phosphorylation of mammalian calmodulin on serine/threonine residues by casein kinase II decreased its affinity for Ca(2+)-ATPase by twofold. In contrast, tyrosine phosphorylation of mammalian calmodulin by the insulin-receptor kinase did not significantly alter calmodulin-stimulated Ca(2+)-ATPase activity. Two variant calmodulins, each containing only one tyrosine residue (the second Tyr is replaced by Phe) were also examined: [F138]calmodulin, a mutant containing tyrosine at position 99, and wheat germ calmodulin which has tyrosine at position 139. The concentrations of [F138]calmodulin and wheat germ calmodulin required for half-maximal activation of Ca(2+)-ATPase were tenfold and fourfold higher, respectively, than mammalian calmodulin. Phosphorylation at Tyr99 of [F138]calmodulin shifted its affinity for Ca(2+)-ATPase towards that of mammalian calmodulin. However, phosphorylation at Tyr139 of wheat germ calmodulin had essentially no effect on its interaction with Ca(2+)-ATPase. Thus, all of the observed effects of both phosphorylation and substitution of residues of calmodulin are on its affinity for Ca(2+)-ATPase, not on Vmax. The effects are dependent on the site of phosphate incorporation. Replacement of tyrosine with phenylalanine has a larger effect than phosphorylation of tyrosine, suggesting that the observed functional alterations reflect a secondary conformational change in the C-terminal half of calmodulin, the region that is important in its activation of Ca(2+)-ATPase.

Volatile anesthetics selectively inhibit the Ca(2+)-transporting ATPase in neuronal and erythrocyte plasma membranes.

BACKGROUND: The activity of the plasma membrane Ca(2+)-transporting adenosine triphosphatase (PMCA) is inhibited by volatile anesthetics at clinical concentrations. The goal of the current study was to determine whether the inhibition is selective as compared to other adenosine triphosphatases (ATPases) and another group of general anesthetics, barbiturates. In addition, the authors determined whether the response to anesthetics of the enzymes in neuronal membranes is similar to that in erythrocyte membranes. METHODS: The effects of halothane, isoflurane, and sodium pentobarbital on four different ATPase activities were studied at 37 degrees C in two distinct plasma membrane preparations, human red blood cells and synaptosomal membranes from rat cerebellum. RESULTS: Inhibition patterns of the PMCA by halothane and isoflurane at anesthetic concentrations were vary similar in red blood cells and synaptosomal membranes. The half-maximal inhibition (I50) occurred at 0.25-0.30 mM halothane and 0.30-0.32 mM isoflurane. The PMCA in both membranes was significantly more sensitive to the inhibitory action of volatile anesthetics (I50 = 0.75-1.15 minimum alveolar concentration) than were other ATPases, such as the Na+,K+-ATPase (I50 approximately 3 minimum alveolar concentration) or Mg(2+)-ATPase (I50 > or = 5 minimum alveolar concentration). In contrast, sodium pentobarbital inhibited the PMCA in both membranes only at approximately 100-200-fold above its anesthetic concentrations. The other ATPases were inhibited at similar pentobarbital concentrations (I50 = 11-22 mM). CONCLUSIONS: The findings demonstrate analogous response of the PMCA of neuronal and erythrocyte cells to two groups of general anesthetics. The PMCA activity is selectively inhibited by volatile anesthetics at their clinical concentrations. The enzyme in vivo may then be a pharmacologic target for volatile anesthetics but not for barbiturates.

The active species of plasma membrane Ca2+-ATPase are a dimer and a monomer-calmodulin complex.

The purified plasma membrane Ca2+-ATPase is fully activated through the enzyme concentration-dependent self-association at physiologically relevant Ca2+ concentrations (Kosk-Kosicka, D., and Bzdega, T. (1988) J. Biol. Chem. 263, 18184-18189; Kosk-Kosicka, D., Bzdega, T., and Wawrzynow, A. (1989) J. Biol. Chem. 264, 19495-19499). We have previously shown that the Ca2+-ATPase activity of the oligomeric enzyme is independent of calmodulin, in contrast to another active enzyme species, a presumable monomer, that is activated by calmodulin binding. Presently, we have succeeded in determining the molecular mass of the two active enzyme species by equilibrium ultracentrifugation. For the calmodulin-dependent species, the molecular mass is 170 +/- 30 kDa, which is consistent with predominantly monomeric Ca2+-ATPase with bound calmodulin. The molecular mass of calmodulin-independent oligomers is 260 +/- 34 kDa, indicating that they are dimers. Results of experiments performed under different calcium and potassium concentrations and in the presence of dextran that causes molecular crowding verify a strict Ca2+ requirement of the dimerization process. We conclude that the active species of the Ca2+-ATPase are a monomer-calmodulin complex and a dimer.

Mechanism of inhibition of the plasma membrane Ca(2+)-ATPase by barbiturates.

We have demonstrated that sodium pentobarbital inhibited the activation of the human red blood cell plasma membrane Ca(2+)-ATPase produced by dimerization of enzyme monomers or by calmodulin binding to enzyme monomers. The effects of the barbiturate were dose-dependent. Both Vmax and Ca2+ affinity were reduced. The Ca(2+)-ATPase activity of the dimeric enzyme was distinctly less sensitive with respect to the effective inhibitory concentrations of pentobarbital and to the rate of onset of inhibition than was the calmodulin-dependent activation of enzyme monomers. Temperature dependence of the inhibition was in agreement with direct, nonpolar interactions of pentobarbital with a water-exposed nonpolar patch on the surface of this transmembrane protein. The barbiturate prevented the increase of intrinsic tryptophan fluorescence associated with substrate Ca2+ binding to the enzyme dimer. On the basis of the barbiturate effects we propose a model for the action of detergent-like compounds on the enzyme. They inhibit Ca(2+)-ATPase activity by binding to a nonpolar patch on the water-exposed dimerization surface of the enzyme monomer, part of which is also the binding site for calmodulin. The model assumes that their binding to the nonpolar patch on the monomer interferes with dimerization and weakens but does not prohibit calmodulin binding, whose activation of the enzyme is then submaximal. The model should be applicable to other proteins as the two activation pathways studied have been demonstrated for various enzymes.

How do volatile anesthetics inhibit Ca(2+)-ATPases?

Volatile anesthetics at concentrations that are used in clinical practice to induce anesthesia selectively inhibit activity of the plasma membrane Ca(2+)-transport ATPase (Kosk-Kosicka, D., and Roszczynska, G. (1993) Anesthesiology 79, 774-780). We have investigated the mechanism of the inhibitory action of several anesthetics on the purified erythrocyte Ca(2+)-ATPase by employing fluorescence spectroscopy measurements that report changes in the environment of intrinsic tryptophans and of an extrinsic probe attached in the active site of the enzyme. We have shown that the observed inhibition of the Ca(2+)-dependent activation of the enzyme correlates well with the elimination of the Ca(2+)-induced conformation change that is important for the proper function of the enzyme. Analysis of the anesthetics effects on the total tryptophan fluorescence indicates a significant effect on enzyme conformation. Similar changes have been observed in the sarcoplasmic reticulum Ca(2+)-ATPase. We propose that volatile anesthetics inhibit Ca(2+)-ATPase by interacting with nonpolar sites in protein interior, in analogy to the binding demonstrated for myoglobin, hemoglobin, and adenylate kinase (Schoenborn, B. P., and Featherstone, R. M. (1967) Adv. Pharmacol. 5, 1-17; Tilton, R. F., Kuntz, I. D., and Petsko, G. A. (1984) Biochemistry 23, 2849-2857). Such binding is expected to modify conformational substate(s) of the enzyme and perturb its function. We view this process as an example of a general phenomena of interaction of small molecules with internal sites in proteins.

Self-association of plasma membrane Ca(2+)-ATPase by volume exclusion.

At enzyme concentrations above 40 nM the configuration of the purified plasma membrane Ca(2+)-ATPase is that of calmodulin-insensitive dimers. Dilution of the enzyme generates progressively higher proportions of calmodulin-sensitive monomers with lower Vmax and Ca2+ sensitivity than the dimeric enzyme. Dimerization from monomeric state had not been documented before. We investigated whether concentration by volume exclusion, obtained by addition of a large molecular weight dextran to a monomeric Ca(2+)-ATPase would elicit dimer-like behavior. Dextran induced self-association of monomers, as monitored by fluorescence energy transfer, but the Ca2+ sensitivity of the re-associated monomers was lower than that of the native dimers. These results suggest that the self-association reaction is structurally but not functionally reversible, and also document the existence of a hitherto unknown kinetic state of the oligomerized Ca(2+)-ATPase, with high Vmax but low Ca(2+)-sensitivity.

Neutral organic solute effects on the activity of the plasma membrane Ca(2+)-ATPase.

We have compared effects of dimethylsulfoxide (Me2SO) and two polyols on the Ca(2+)-ATPase purified from human erythrocytes. As studied under steady-state conditions over a broad solute concentration range and temperature, Me2SO, glycerol, and xylitol do not inhibit the Ca(2+)-ATPase activity; this is in contrast to numerous other organic solutes that we have investigated. Under specific experimental conditions, Me2SO (but not glycerol) substantially increases Ca(2+)-ATPase activity, suggesting a possible facilitation of enzyme oligomerization. The activation is more pronounced at low Ca2+ concentrations. In contrast to glycerol, Me2SO shows no protective effect on enzyme structure as assessed by determining residual Ca(2+)-ATPase activity after exposing the enzyme to thermal denaturation at 45 degrees C. Under these conditions several other organic solutes strongly enhance the denaturating effect of temperature. Because of the temperature dependence of its effect on the Ca(2+)-ATPase activity we believe that Me2SO activates the Ca(2+)-ATPase by indirect water-mediated interactions.

Stabilizing and destabilizing effects on plasma membrane Ca(2+)-ATPase activity.

We have examined the temperature-dependent effects of several organic compounds on the activity of the purified Ca(2+)-ATPase of erythrocytes. The monomeric enzyme was activated either by interaction with calmodulin or by oligomerization in the absence of calmodulin. Of the four homologous solute series studied including polyols, alkanols, aprotic solvents, and N-methyl derivatives of formamide and acetamide only polyols stabilized the enzyme over a broad range of concentration and temperature. Similarity of Ca(2+)-ATPase activity patterns at 25 and 37 degrees C and in the presence of glycerol is in agreement with indirect, stabilizing interactions. Glycerol also protected the Ca(2+)-ATPase from thermal denaturation at 45 degrees C. Within each homologous series, inhibitory effects increased with increasing solute concentration and with increasing structural similarity to detergents, indicating that direct destabilizing interactions are responsible for the observed inhibition. These were comparable to the destabilizing effect of urea. Oligomers were more resistant to all inhibitory solutes as compared to calmodulin-activated monomers suggesting that the nonpolar patches of the oligomerized enzyme are less accessible to solutes.

Inhibition of plasma membrane Ca(2+)-ATPase activity by volatile anesthetics.

BACKGROUND: The precise sites and mechanisms of action of volatile anesthetics remain unknown. Recently, several integral membrane proteins have been suggested as potential targets to which anesthetics can bind at hydrophobic regions. Impairment of cell Ca2+ homeostasis has been postulated as one of the possible mechanisms of anesthetic action. To test these hypotheses, the authors selected the human erythrocyte Ca(2+)-ATPase as a model membrane protein. This enzyme is an integral membrane protein that is instrumental in maintaining Ca2+ homeostasis in the cell in which it is the sole Ca(2+)-transporting system. Thus, any functional alteration of the Ca(2+)-ATPase by anesthetics may lead to serious perturbations in Ca(2+)-regulated processes in the cell. METHODS: The Ca(2+)-ATPase activity was measured as a function of increased concentration of four volatile anesthetics: halothane, isoflurane, enflurane, and desflurane. RESULTS: All four anesthetics significantly inhibited the Ca(2+)-ATPase activity in a dose-dependent manner. The half-maximal inhibition occurred at anesthetic concentrations from 0.3 to 0.7 vol% at 37 degrees C, which, except for desflurane, is a clinically relevant concentration range. The greater the clinical potency of the volatile anesthetics studied, the less was the concentration required to inhibit the Ca(2+)-ATPase activity. The inhibition was less at 25 degrees C than at 37 degrees C, which is consistent with direct interactions of the nonpolar interfaces of the enzyme with the nonpolar of the portions of the anesthetics. CONCLUSIONS: The authors' findings indicate that the Ca(2+)-ATPase is a suitable model for investigating the mechanism of action of volatile anesthetics on the integral membrane protein, and that this inhibition may be specific.