Activation of phospholipase C delta1 through C2 domain by a Ca(2+)-enzyme-phosphatidylserine ternary complex.

The concentration of free Ca(2+) and the composition of nonsubstrate phospholipids profoundly affect the activity of phospholipase C delta1 (PLCdelta1). The rate of PLCdelta1 hydrolysis of phosphatidylinositol 4,5-bisphosphate was stimulated 20-fold by phosphatidylserine (PS), 4-fold by phosphatidic acid (PA), and not at all by phosphatidylethanolamine or phosphatidylcholine (PC). PS reduced the Ca(2+) concentration required for half-maximal activation of PLCdelta1 from 5.4 to 0.5 microM. In the presence of Ca(2+), PLCdelta1 specifically bound to PS/PC but not to PA/PC vesicles in a dose-dependent and saturable manner. Ca(2+) also bound to PLCdelta1 and required the presence of PS/PC vesicles but not PA/PC vesicles. The free Ca(2+) concentration required for half-maximal Ca(2+) binding was estimated to be 8 microM. Surface dilution kinetic analysis revealed that the K(m) was reduced 20-fold by the presence of 25 mol % PS, whereas V(max) and K(d) were unaffected. Deletion of amino acid residues 646-654 from the C2 domain of PLCdelta1 impaired Ca(2+) binding and reduced its stimulation and binding by PS. Taken together, the results suggest that the formation of an enzyme-Ca(2+)-PS ternary complex through the C2 domain increases the affinity for substrate and consequently leads to enzyme activation.     

Conformational changes of (Ca2+-Mg2+)-ATPase of erythrocyte plasma membrane caused by calmodulin and phosphatidylserine as revealed by circular dichroism and fluorescence studies

Two spectroscopic techniques, circular dichroism and steady-state fluorescence, were employed in order to study conformational changes of the purified, detergent-solubilized (Ca2+-Mg2+)-ATPase of porcine erythrocyte ghost membranes. Circular dichroism (CD) spectra in the peptide region were obtained from the purified (Ca2+-Mg2+)-ATPase of porcine erythrocyte ghost membranes with the aim to investigate the secondary structure of the enzyme in the presence of calmodulin (CaM) or phosphatidylserine (PS), as well as in the E1 and E2 states. The E1 conformation was stabilized by 10 microM free Ca2+, while the E2 conformation was stabilized by 0.1 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). It was found that the E1 and E2 states of the enzyme strikingly differed in their secondary structure (66% and 46% of calculated alpha-helix content, respectively). In the presence of Ca2+, PS decreased the helical content of the ATPase to 61%, while CaM to 55%. Quenching of intrinsic fluorescence of (Ca2+-Mg2+)-ATPase by acrylamide, performed in the presence of Ca2+, gave evidence for a single class of tryptophan residues with Stern-Volmer constant (KSV) of 10 M-1. Accessibility of tryptophan residues varied depending on the conformational status of the enzyme. Addition of PS and CaM decreased the KSV value to 7.6 M-1 and 8.5 M-1, respectively. In the absence of Ca2+, KSV was 7.0 M-1. KI and CsCl were less effective as quenchers. The fluorescence energy transfer between (Ca2+-Mg2+)-ATPase tryptophan residues and dansyl derivative of covalently labeled CaM occurred in the presence of EGTA, but was further promoted by Ca2+. It is concluded that the interaction of CaM and PS with (Ca2+-Mg2+)-ATPase results in different conformational states of the enzyme. CD and fluorescence spectroscopy allowed to distinguish these states from the E1 and E2 conformational forms of the ATPase.     

Determination of the intracellular dissociation constant, K(D), of the fluo-3. Ca(2+) complex in mouse sperm for use in estimating intracellular Ca(2+) concentrations.

In order to calculate the actual, rather than the relative, intracellular Ca(2+) concentration (Ca(2+))(i) in mammalian sperm cells, using fluorescent probes whose fluorescence emission differs between the probe. Ca(2+) complex and free probe, the value of the dissociation constant for the probe. Ca(2+) complex, K(D), is required. Interaction of the probe with cellular components may change the intracellular value of K(D) from that determined in buffered solution. We had previously shown that fluo-3, whose Ca(2+) complex is highly fluorescent whereas free fluo-3 is not, could be used to monitor changes of (Ca(2+))(i) in mouse sperm. In this report, we describe a method for determining K(D) for the fluo-3. Ca(2+) complex in mouse sperm suspended in medium MJB, a medium in which the sperm remain viable, but which contains high Ca(2+). The method involved treating the sperm with ionomycin to provide a plasma membrane Ca(2+) carrier, with nigericin to eliminate pH gradient, and with gramicidin D to eliminate membrane potential, such that (Ca(2+))(i) equilibrates with medium Ca(2+) concentration (Ca(2+))(e), then titrating (Ca(2+))(e) with EGTA in added aliquots to near nil concentration. At EGTA concentrations in excess of total medium Ca(2+), an approximation algorithm was used to calculate (Ca(2+))(e), based on the known K(D) for the EGTA. Ca(2+) complex. The fluorescence of the intracellular fluo-3. Ca(2+) complex, F, decreased with increasing additions of EGTA; (Ca(2+))(i) = (Ca(2+))(e) was plotted as a linear function of F/[F(max) - F]; the slope gives K(D). At 37 degrees C, intracellular K(D) was calculated to be 0.636 +/- 0.018 microM (+/-SEM, n = 8). At 37 degrees C and 20 degrees C, K(D) values in MJB were calculated to be 0.502 +/- 0.022 and 0.578 +/- 0.029 (+/-SEM, n =8 and n = 6), respectively. The higher intracellular K(D) value implies probe interaction with cytosol components, primarily those in the head, as this compartment is the major contributor to sperm fluorescence. Changes in (Ca(2+))(i), monitored with fluo-3 fluorescence, that occur on interaction of capacitated mouse sperm with the zona pellucida and may now be quantified, using 0.636 microM for K(D) of the intracellular fluo-3. Ca(2+) complex.     

(Ca2+ + Mg2+)-ATPase in enriched sarcolemma from dog heart

An enriched fraction of plasma membranes was prepared from canine ventricle by a process which involved thorough disruption of membranes by vigorous homogenization in dilute suspension, sedimentation of contractile proteins and mitochondria at 3000 X g followed by sedimentation of a microsomal fraction at 200 000 X g. The microsomal suspension was then fractionated on a discontinuous sucrose gradient. Particles migrating in the density range 1.0591--1.1083 were characterized by (Na+ + K+)-ATPase activity and [3H]ouabain binding as being enriched in sarcolemma and were comprised of nonaggregated vesicles of diameter approx. 0.1 micron. These fractions contained (Ca2+ + Mg2+)-ATPase which appreared endogenous to the sarcolemma. The enzyme was solubilized using Triton X-100 and 1 M KCl and partially purified. Optimal Ca2+ concentration for enzyme activity was 5--10 microM. Both Na+ and K+ stimulated enzyme activity. It is suggested that the enzyme may be involved in the outward pumping of Ca2+ from the cardiac cell.     

Inhibition of Acanthamoeba myosin I heavy chain kinase by Ca(2+)-calmodulin.

The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosins I depends on phosphorylation of their single heavy chains by myosin I heavy chain kinase. Kinase activity is enhanced > 50-fold by autophosphorylation at multiple sites. The rate of kinase autophosphorylation is increased approximately 20-fold by acidic phospholipids independent of the presence of Ca2+ and diglycerides. We show in this paper that Ca(2+)-calmodulin inhibits phospholipid-stimulated autophosphorylation of myosin I heavy chain kinase and hence also inhibits the catalytic activity of unphosphorylated kinase in the presence of phospholipid. Ca(2+)-calmodulin does not inhibit kinase activity in the absence of phospholipid. Micromolar Ca(2+)-calmodulin also inhibits binding of myosin I heavy chain kinase to phospholipid vesicles and purified plasma membranes. Proteolytic removal of a 7-kDa NH2-terminal segment from the 97-kDa kinase prevents binding of both calmodulin and phospholipid; therefore, we propose that they bind to the same or overlapping sites. These data provide a mechanism by which Ca2+ could inhibit the actin-activated Mg(2+)-ATPase activity of the myosin I isozymes in vivo and thus regulate myosin I-dependent motile activities.     

Ca(2+)-binding site in Rhodobacter sphaeroides cytochrome C oxidase

Cytochrome c oxidase (COX) from R. sphaeroides contains one Ca(2+) ion per enzyme that is not removed by dialysis versus EGTA. This is similar to COX from Paracoccus denitrificans [Pfitzner, U., Kirichenko, A., Konstantinov, A. A., Mertens, M., Wittershagen, A., Kolbesen, B. O., Steffens, G. C. M., Harrenga, A., Michel, H., and Ludwig, B. (1999) FEBS Lett. 456, 365-369] and is in contrast to the bovine oxidase, which binds Ca(2+) reversibly. A series of R. sphaeroides mutants with replacements of the E54, Q61, and D485 residues, which form the Ca(2+) coordination sphere in subunit I, has been generated. The substitutions for the E54 residue do not assemble normally. Mutants with the Q61 replacements are active and retain the tightly bound Ca(2+); their spectra are not perturbed by added Ca(2+) or EGTA. The D485A mutant is active, binds to Ca(2+) reversibly, like the mitochondrial oxidase, and exhibits the red shift in the heme a absorption spectrum upon Ca(2+) binding for both reduced and oxidized states of heme a. The K(d) value of 6 nM determined by equilibrium titrations is much lower than that reported for the homologous D477A mutant of Paracoccus denitrificans or for bovine COX (K(d) = 1-3 microM). The rate of Ca(2+) binding with the D485A oxidase (k(on) = 5 x 10(3) M(-1) s(-1)) is comparable to that observed earlier for bovine COX, but the off-rate is extremely slow (approximately 10(-3) s(-1)) and highly temperature-dependent. The k(off) /k(on) ratio (190 nM) is about 30-fold higher than the equilibrium K(d) of 6 nM, indicating that formation of the Ca(2+)-adduct may involve more than one step. Sodium ions reverse the Ca(2+)-induced red shift of heme a and dramatically decrease the rate of Ca(2+) binding to the D485A mutant COX. With the D485A mutant, 1 Ca(2+) competes with 1 Na(+) for the binding site, whereas 2 Na(+) compete with 1 Ca(2+) for binding to the bovine oxidase. This finding indicates that the aspartic residue D442 (a homologue of R. sphaeroides D485) may be the second Na(+) binding site in bovine COX. No effect of Ca(2+) binding to the D485A mutant is evident on either the steady-state enzymatic activity or several time-resolved partial steps of the catalytic cycle. It is proposed that the tightly bound Ca(2+) plays a structural role in the bacterial oxidases while the reversible binding with the mammalian enzyme may be involved in the regulation of mitochondrial function.     

Voltage-independent changes in L-type Ca(2+) current uncoupled from SR Ca(2+) release in cardiac myocytes

To determine the effect of voltage-independent alterations of L-type Ca(2+) current (I(Ca)) on the sarcoplasmic reticular (SR) Ca(2+) release in cardiac myocytes, we measured I(Ca) and cytosolic Ca(2+) transients (Ca(i)(2+); intracellular Ca(2+) concentration) in voltage-clamped rat ventricular myocytes during 1) an abrupt increase of extracellular [Ca(2+)] (Ca(o)(2+)) or 2) application of 1 microM FPL-64176, a Ca(2+) channel agonist, to selectively alter I(Ca) in the absence of changes in SR Ca(2+) loading. On the first depolarization in higher Ca(o)(2+), peak I(Ca) was increased by 46 +/- 6% (P < 0.001), but the increases in the maximal rate of rise of Ca(i)(2+) (dCa(i)(2+)/dt(max), where t is time; an index of SR Ca(2+) release flux) and the Ca(i)(2+) transient amplitude were not significant. Rapid exposure to FPL-64176 greatly slowed inactivation of I(Ca), increasing its time integral by 117 +/- 8% (P < 0.001) without significantly increasing peak I(Ca), dCa(i)(2+)/dt(max), or amplitude of the corresponding Ca(i)(2+) transient. Prolongation of exposure to higher Ca(o)(2+) or FPL-64176 did not further increase peak I(Ca) but greatly increased dCa(i)(2+)/dt(max), Ca(i)(2+) transient amplitude, and the gain of Ca(2+) release (dCa(i)(2+)/dt(max)/I(Ca)), evidently due to augmentation of the SR Ca(2+) loading. Also, the time to peak dCa(i)(2+)/dt(max) was significantly increased in the continuous presence of higher Ca(o)(2+) (by 37 +/- 5%, P < 0.001) or FPL-64176 (by 63 +/- 5%, P < 0.002). Our experiments provide the first evidence of a marked disparity between an increased peak I(Ca) and the corresponding SR Ca(2+) release. We attribute this to saturation of the SR Ca(2+) release flux as predicted by local control theory. Prolongation of the SR Ca(2+) release flux, caused by combined actions of a larger I(Ca) and maximally augmented SR Ca(2+) loading, might reflect additional Ca(2+) release from corbular SR.     

Stimulatory effect of regucalcin on ATP-dependent Ca(2+) uptake activity in rat liver mitochondria

The effect of Ca(2+)-binding protein regucalcin on Ca(2+)-ATPase activity in isolated rat liver mitochondria was investigated. The presence of regucalcin (0.1, 0.25, and 0.5 microM) in the enzyme reaction mixture led to a significant increase in Ca(2+)-ATPase activity. Regucalcin significantly stimulated ATP-dependent (45)Ca(2+) uptake by the mitochondria. Ruthenium red (10(-5) M) or lanthanum chloride (10(-4) M), an inhibitor of mitochondrial Ca(2+) uptake, completely inhibited regucalcin (0.25 microM)-increased mitochondrial Ca(2+)-ATPase activity and (45)Ca(2+) uptake. The effect of regucalcin (0.25 microM) in increasing Ca(2+)-ATPase activity was completely inhibited by the presence of digitonin (10(-2)%), a solubilizing reagent of membranous lipids, or vanadate (10(-5) M), an inhibitor of phosphorylation of ATPase. The activatory effect of regucalcin (0.25 microM) on Ca(2+)-ATPase activity was not further enhanced in the presence of dithiothreitol (2.5 mM), a protecting reagent of the sulfhydryl (SH) group of the enzyme, or calmodulin (0.60 microM), a modulator protein of Ca(2+) action that could increase mitochondrial Ca(2+)-ATPase activity. The present study demonstrates that regucalcin can stimulate Ca(2+) pump activity in rat liver mitochondria, and that the protein may act on an active site (SH group)-related to phosphorylation of mitochondrial Ca(2+)-ATPase.     

Activating and deactivating roles of lipid bilayers on the Ca(2+)-ATPase/phospholamban complex

The physicochemical properties of the lipid bilayer shape the structure and topology of membrane proteins and regulate their biological function. Here, we investigated the functional effects of various lipid bilayer compositions on the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) in the presence and absence of its endogenous regulator, phospholamban (PLN). In the cardiac muscle, SERCA hydrolyzes one ATP molecule to translocate two Ca(2+) ions into the SR membrane per enzymatic cycle. Unphosphorylated PLN reduces SERCA's affinity for Ca(2+) and affects the enzymatic turnover. We varied bilayer thickness, headgroup, and fluidity and found that both the maximal velocity (V(max)) of the enzyme and its apparent affinity for Ca(2+) (K(Ca)) are strongly affected. Our results show that (a) SERCA's V(max) has a biphasic dependence on bilayer thickness, reaching maximum activity with 22-carbon lipid chain length, (b) phosphatidylethanolamine (PE) and phosphatidylserine (PS) increase Ca(2+) affinity, and (c) monounsaturated lipids afford higher SERCA V(max) and Ca(2+) affinity than diunsaturated lipids. The presence of PLN removes the activating effect of PE and shifts SERCA's activity profile, with a maximal activity reached in bilayers with 20-carbon lipid chain length. Our results in synthetic lipid systems compare well with those carried out in native SR lipids. Importantly, we found that specific membrane compositions closely reproduce PLN effects (V(max) and K(Ca)) found in living cells, reconciling an ongoing controversy regarding the regulatory role of PLN on SERCA function. Taken with the physiological changes occurring in the SR membrane composition, these studies underscore a possible allosteric role of the lipid bilayers on the SERCA/PLN complex.     

Role of regucalcin as an activator of Ca(2+)-ATPase activity in rat liver microsomes.

The effect of Ca(2+)-binding protein regucalcin on Ca(2+)-ATPase activity in isolated rat liver microsomes was investigated. The presence of regucalcin (0.1-1.0 microM) in the enzyme reaction mixture led to a significant increase in Ca(2+)-ATPase activity. Regucalcin significantly stimulated ATP-dependent (45)Ca(2+) uptake by the microsomes. Thapsigargin (10(-6) M), a specific inhibitor of microsomal Ca(2+) pump enzyme (Ca(2+)-ATPase), clearly inhibited regucalcin (0.5 microM)-increased microsomal Ca(2+)-ATPase activity. Liver microsomal Ca(2+)-ATPase activity was markedly decreased by N-ethylmaleimide (NEM; 2.5 mM), while the activity was clearly elevated by dithiothreitol (DTT; 2.5 mM), indicating that the sulfhydryl (SH) group of the enzyme is an active site. The effect of regucalcin (0.5 microM) in increasing Ca(2+)-ATPase activity was completely inhibited by the presence of NEM (2.5 mM) or digitonin (10(-2) %), a solubilizing reagent of membranous lipids. Moreover, the effect of regucalcin on enzyme activity was seen in the presence of Ca(2+) ionophore (A23187; 10(-7) M). The present study demonstrates that regucalcin can stimulate Ca(2+) pump activity in rat liver microsomes, and that the protein may act the SH groups of microsomal Ca(2+)-ATPase.     

Distinction between Ca(2+) pump and Ca(2+)/H(+) antiport activities in synaptic vesicles of sheep brain cortex

Synaptic vesicles, isolated from a sheep brain cortex, accumulate Ca(2+) in a manner that depends on the pH and pCa values. In the presence of 100 microM CaCl(2), most of the Ca(2+) taken up by the vesicles was vanadate-inhibited (86%) at pH 7.4, whereas at pH 8.5, part of the Ca(2+) accumulated (36%) was DeltapH-dependent (bafilomycin and CCCP inhibited) and part was insensitive to those drugs (31%). We also observed that both vanadate-sensitive and bafilomycin-sensitive Ca(2+) accumulations were completely released by the Ca(2+) ionophore, ionomycin, and that these processes work with high (K(0.5)=0.6 microM) and low (K(0.5)=217 microM) affinity for Ca(2+), respectively. The DeltapH-dependent Ca(2+) transport appears to be largely operative at Ca(2+) concentrations (>100 microM) which completely inhibited the vanadate-sensitive Ca(2+) uptake. These Ca(2+) effects on the Ca(2+) accumulation were well correlated with those observed on the vanadate-inhibited Ca(2+)-ATPase and bafilomycin-inhibited H(+)-ATPase, respectively. The Ca(2+)-ATPase activity reached a maximum at about 25 microM (pH 7.4) and sharply declined at higher Ca(2+) concentrations. In contrast, Ca(2+) had a significant stimulatory effect on the H(+)-ATPase between 250 and 500 microM Ca(2+) concentration. Furthermore, we found that DeltapH-sensitive Ca(2+) transport was associated with proton release from the vesicles. About 21% of the maximal proton gradient was dissipated by addition of 607.7 microM CaCl(2) to the reaction medium and, if CaCl(2) was present before the proton accumulation, lower pH gradients were reached. Both vanadate-inhibited and bafilomycin-inhibited systems transported Ca(2+) into the same vesicle pool of our preparation, suggesting that they belong to the same cellular compartment. These results indicate that synaptic vesicles of the sheep brain cortex contain two distinct mechanisms of Ca(2+) transport: a high Ca(2+) affinity, proton gradient-independent Ca(2+) pump that has an optimal activity at pH 7.4, and a low Ca(2+) affinity, proton gradient-dependent Ca(2+)/H(+) antiport that works maximally at pH 8.5.     

Ca(2+)-calmodulin-dependent protein kinases Ia and Ib from rat brain I. Identification, purification, and structural comparisons.

Two Ca(2+)-calmodulin (CaM)-dependent protein kinases were purified from rat brain using as substrate a synthetic peptide based on site 1 (site 1 peptide) of the synaptic vesicle-associated protein, synapsin I. One of the purified enzymes was an approximately 89% pure protein of M(r) = 43,000 which bound CaM in a Ca(2+)-dependent fashion. The other purified enzyme was an apparently homogenous protein of M(r) = 39,000 accompanied by a small amount of a M(r) = 37,000 form which may represent a proteolytic product of the 39-kDa enzyme. The 39-kDa protein bound CaM in a Ca(2+)-dependent fashion. Gel filtration analysis indicated that both enzymes are monomers. The 43- and 39-kDa enzymes are named Ca(2+)-CaM-dependent protein kinases Ia and Ib (CaM kinases Ia, Ib), respectively. The specific activities of CaM kinases Ia and Ib were similar (5-8 mumol/min/mg protein). CaM kinase Ia (but not CaM kinase Ib) activity was enhanced by addition of a CaM-Sepharose column wash (non-binding) fraction suggesting the existence of an "activator" of CaM kinase Ia. Both kinases phosphorylated exogenous substrates (site 1 peptide and synapsin I) in a Ca(2+)-CaM-dependent fashion and both kinases underwent autophosphorylation. CaM kinase Ia autophosphorylation was Ca(2+)-CaM-dependent and occurred exclusively on threonine while CaM kinase Ib autophosphorylation showed Ca(2+)-CaM independence and occurred on both serine and threonine. Proteolytic digestion of autophosphorylated CaM kinases Ia and Ib yielded phosphopeptides of differing M(r). These characteristics, as well as enzymatic and regulatory properties (DeRemer, M. F., Saeli, R. J. Brautigen, D. L., and Edelman, A. M. (1992) J. Biol. Chem. 267, 13466-13471), indicate that CaM kinases Ia and Ib are distinct and possibly previously unrecognized enzymes.     

Characterization of a Ca(2+)-binding site in human annexin II by site-directed mutagenesis.

Annexin II, a major cytoplasmic substrate of the src tyrosine kinase, is a member of the annexin family of Ca2+/phospholipid-binding proteins. It is composed of a short N-terminal tail (30 residues) followed by four so-called annexin repeats (each 70-80 residues in length) which share sequence homologies and are thought to form (a) new type(s) of Ca(2+)-binding site(s). We have produced wild-type and site specifically mutated annexin II molecules to compare their structure and biochemistry. The recombinant wild-type annexin II displays biochemical and spectroscopical properties resembling those of the authentic protein purified from mammalian cells. In particular, it shows the Ca(2+)-induced blue shift in fluorescence emission which is typical for this annexin. Replacement of the single tryptophan in annexin II (Trp-212) by a phenylalanine abolishes the fluorescence signal and allows the unambiguous assignment of the Ca(2+)-sensitive spectroscopic properties to Trp-212. This residue is located in the third annexin repeat in a highly conserved stretch of 17 amino acids which are also found in the other repeats and known as the endonexin fold. To study the precise architecture of the Ca2+ site which must reside in close proximity to Trp-212, we changed several residues of the endonexin fold in repeat 3 by site-directed mutagenesis. An analysis of these mutants by fluorescence spectroscopy and Ca(2+)-dependent phospholipid binding reveals that Gly-206 and Thr-207 seem indispensible for a correct folding of this Ca(2+)-binding site.     

Conformational changes in sarcoplasmic reticulum Ca(2+)-ATPase mutants: effect of mutations either at Ca(2+)-binding site II or at tryptophan 552 in the cytosolic domain

By analyzing, after expression in yeast and purification, the intrinsic fluorescence properties of point mutants of rabbit Ca(2+)-ATPase (SERCA1a) with alterations to amino acid residues in Ca(2+)-binding site I (E(771)), site II (E(309)), in both sites (D(800)), or in the nucleotide-binding domain (W(552)), we were able to follow the conformational changes associated with various steps in the ATPase catalytic cycle. Whereas Ca(2+) binding to purified wild-type (WT) ATPase in the absence of ATP leads to the rise in Trp fluorescence expected for the so-called E2 --> E1Ca(2) transition, the Ca(2+)-induced fluorescence rise is dramatically reduced for the E(309)Q mutant. As this purified E(309)Q mutant retains the ability to bind Ca(2+) at site I (but not at site II), we tentatively conclude that the protein reorganization induced by Ca(2+) binding at site II makes the major contribution to the overall Trp fluorescence changes observed upon Ca(2+) binding to both sites. Judging from the fluorescence response of W(552)F, similar to that of WT, these changes appear to be primarily due to membranous tryptophans, not to W(552). The same holds for the fluorescence rise observed upon phosphorylation from P(i) (the so-called E2 --> E2P transition). As for WT ATPase, Mg(2+) binding in the absence of Ca(2+) affects the fluorescence of the E(309)Q mutant, suggesting that this Mg(2+)-dependent fluorescence rise does not reflect binding of Mg(2+) to Ca(2+) sites; instead, Mg(2+) probably binds close to the catalytic site, or perhaps near transmembrane span M3, at a location recently revealed by Fe(2+)-catalyzed oxidative cleavage. Mutation of W(552) hardly affects ATP-induced fluorescence changes in the absence of Ca(2+), which are therefore mostly due to membranous Trp residues, demonstrating long-range communication between the nucleotide-binding domain and the membranous domain.     

A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein

Recently, several groups have developed green fluorescent protein (GFP)-based Ca(2+) probes. When applied in cells, however, these probes are difficult to use because of a low signal-to-noise ratio. Here we report the development of a high-affinity Ca(2+) probe composed of a single GFP (named G-CaMP). G-CaMP showed an apparent K(d) for Ca(2+) of 235 nM. Association kinetics of Ca(2+) binding were faster at higher Ca(2+) concentrations, with time constants decreasing from 230 ms at 0.2 microM Ca(2+) to 2.5 ms at 1 microM Ca(2+). Dissociation kinetics (tau approximately 200 ms) are independent of Ca(2+) concentrations. In HEK-293 cells and mouse myotubes expressing G-CaMP, large fluorescent changes were observed in response to application of drugs or electrical stimulations. G-CaMP will be a useful tool for visualizing intracellular Ca2+ in living cells. Mutational analysis, together with previous structural information, suggests the residues that may alter the fluorescence of GFP.     

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.     

Ca(2+) influx and opening of Ca(2+)-activated K(+) channels in muscle fibers from control and mdx mice

Using the patch-clamp technique, we demonstrate that, in depolarized cell-attached patches from mouse skeletal muscle fibers, a short hyperpolarization to resting value is followed by a transient activation of Ca(2+)-activated K(+) channels (K(Ca)) upon return to depolarized levels. These results indicate that sparse sites of passive Ca(2+) influx at resting potentials are responsible for a subsarcolemmal Ca(2+) load high enough to induce K(Ca) channel activation upon muscle activation. We then investigate this phenomenon in mdx dystrophin-deficient muscle fibers, in which an elevated Ca(2+) influx and a subsequent subsarcolemmal Ca(2+) overload are suspected. The number of Ca(2+) entry sites detected with K(Ca) was found to be greater in mdx muscle. K(Ca) activity reflecting subsarcolemmal Ca(2+) load was also found to be independent of the activity of leak channels carrying inward currents at negative potentials in mdx muscle. These results indicate that the sites of passive Ca(2+) influx newly described in this study could represent the Ca(2+) influx pathways responsible for the subsarcolemmal Ca(2+) overload in mdx muscle fibers.     

Physiological characterization of the Na(+)/Ca(2+) exchanger (NCX) in hepatopancreatic and antennal gland basolateral membrane vesicles isolated from the freshwater crayfish Procambarus clarkii

The purpose of this study was to physiologically characterize the basolateral Na(+)/Ca(2+) exchanger (NCX) in basolateral membrane vesicles (BLMVs) of hepatopancreas and antennal gland of intermolt crayfish. Conditions were optimized to measure Na(+)-dependent Ca(2+) uptake and retention in the BLMV including use of intravesicular (IV) oxalate and measuring initial uptake rates at 20 s. Na(+)-dependent Ca(2+) uptake rate into BLMV was temperature insensitive. Na(+)-dependent Ca(2+) uptake rate was dependent upon free Ca(2+) with saturable Michaelis-Menten kinetics determined as follows: hepatopancreas, maximal uptake rate (J(max))=2.45 nmol/mg per min, concentration at which carrier operates at half-maximal uptake rate (K(m))=0.69 microM Ca(2+); antennal gland, J(max)=13.2 nmol/mg per min, K(m)=0.59 microM Ca(2+). The two vesicle populations exhibited different sensitivity to putative NCX inhibitors. Benzamil had no effect on Na(+)-dependent Ca(2+) uptake rate in hepatopancreas; in antennal gland it was inhibitory at concentrations up to 30 microM and was stimulatory at higher concentrations. Conversely the inhibitor quinacrine was inhibitory at 10 microM in hepatopancreas and was stimulatory at 1000 microM; meanwhile it was ineffective in antennal gland BLMV. Short circuiting the BLMV had no effect on Na(+)-dependent Ca(2+) uptake rate suggesting that the process may be electroneutral. Compared with another prominent basolateral transporter in hepatopancreas the plasma membrane Ca(2+) ATPase (PMCA), the NCX has 70-fold greater J(max) (at comparable temperature) and a lower affinity. In antennal gland the NCX has 40-fold greater J(max) and a lower affinity. In hepatopancreas and antennal gland BLMV NCX appears to determine the rate of basolateral Ca(2+) efflux in intermolt.     

A thapsigargin-sensitive Ca(2+) pump is present in the pea Golgi apparatus membrane

The Golgi apparatus behaves as a bona fide Ca(2+) store in animal cells and yeast (Saccharomyces cerevisiae); however, it is not known whether this organelle plays a similar role in plant cells. In this work, we investigated the presence of an active Ca(2+) accumulation mechanism in the plant cell Golgi apparatus. Toward this end, we measured Ca(2+) uptake in subcellular fractions isolated from the elongating zone of etiolated pea (Pisum sativum) epicotyls. Separation of organelles using sucrose gradients showed a strong correlation between the distribution of an ATP-dependent Ca(2+) uptake activity and the Golgi apparatus marker enzyme, xyloglucan-fucosyltransferase. The kinetic parameters obtained for this activity were: the rate of maximum Ca(2+) uptake of 2.5 nmol mg min(-1) and an apparent K(m) for Ca(2+) of 209 nM. The ATP-dependent Ca(2+) uptake was strongly inhibited by vanadate (inhibitor concentration causing 50% inhibition [I(50)] = 126 microM) and cyclopiazonic acid (I(50) = 0.36 nmol mg protein(-1)) and was not stimulated by calmodulin (1 microM). Addition of Cd(2+) and Cu(2+) at nanomolar concentration inhibited the Ca(2+) uptake, whereas Mn(2+), Fe(2+), and Co(2+) had no significant effect. Interestingly, the active calcium uptake was inhibited by thapsigargin (apparent I(50) = 88 nM), a well-known inhibitor of the endoplasmic reticulum and Golgi sarco-endoplasmic reticulum Ca(2+) ATPase from mammalian cells. A thapsigargin-sensitive Ca(2+) uptake activity was also detected in a cauliflower (Brassica oleracea) Golgi-enriched fraction, suggesting that other plants may also possess thapsigargin-sensitive Golgi Ca(2+) pumps. To our knowledge, this is the first report of a plant Ca(2+) pump activity that shows sensitivity to low concentrations of thapsigargin.     

Ca2+-stimulated, Mg2+-dependent ATPase in bovine thyroid plasma membranes

An isolated plasma membrane fraction from bovine thyroid glands contained a Ca2+-stimulated, Mg2+-dependent adenosine triphosphatase ((Ca2+ + Mg2+)-ATPase) activity which was purified in parallel to (Na+ + K+)-ATPase and adenylate cyclase. The (Ca2+ + Mg2+)-ATPase activity was maximally stimulated by approx. 200 microM added calcium in the presence of approx. 200 microM EGTA (69.7 +/- 5.2 nmol/mg protein per min). In EGTA-washed membranes, the enzyme was stimulated by calmodulin and inhibited by trifluoperazine.