Calpain I activates Ca2+ transport by the reconstituted erythrocyte Ca2+ pump

Summary Calpain I purified from human erythrocyte cytosol activates both the ATP hydrolytic activity and the ATP-dependent Ca²⁺ transport function of the Ca²⁺-translocating ATPase solubilized and purified from the plasma membrane of human erythrocytes and reconstituted into phosphatidylcholine vesicles. Following partial proteolysis of the enzyme by calpain I, both the initial rates of calcium ion uptake and ATP hydrolysis were increased to near maximal levels similar to those obtained upon addition of calmodulin. The proteolytic activation resulted in the loss of further stimulation of the rates of Ca²⁺ translocation or ATP hydrolysis by calmodulin as well as an increase of the affinity of the enzyme for calcium ion. However, the mechanistic Ca²⁺/ATP stoichiometric ratio was not affected by the proteolytic treatment of the reconstituted Ca²⁺-translocating ATPase. The proteolytic activation of the ATP hydrolytic activity of the reconstituted enzyme could be largely prevented by calmodulin. Different patterns of proteolysis were obtained in the absence or in the presence of calmodulin during calpain treatment: the 136-kDa enzyme was transformed mainly into a 124-kDa active ATPase fragment in the absence of calmodulin, whereas a 127-kDa active ATPase fragment was formed in the presence of calmodulin. This study shows that calpain I irreversibly activates the Ca²⁺ translocation function of the Ca²⁺-ATPase in reconstituted proteoliposomes by producing a calmodulin-independent active enzyme fragment, while calmodulin antagonizes this activating effect by protecting the calmodulin-binding domain against proteolytic cleavage by calpain.     

Modulation of human erythrocyte Ca2+-ATPase activity by glycerol: The role of calmodulin

The effect of an intracellular cryoprotectant glycerol on human erythrocyte Ca2+-ATPase activity and possible involvement of calmodulin in the regulation of Ca2+-pump under these conditions were investigated. The experiments were carried out using saponin-permeabilized cells and isolated erythrocyte membrane fractions (white ghosts). Addition of rather low concentrations of glycerol to the medium increased Ca2+-ATPase activity in the saponin-permeabilized cells; the maximal effect was observed at 10% glycerol. Subsequent increase in glycerol concentrations above 20% was accompanied by inhibition of Ca2+-ATPase activity. Lack of stimulating effect of glycerol on white ghost Ca2+-ATPase may be attributed to removal of endogenous compounds regulating activity of this ion transport system. Inhibitory analysis using R24571 revealed that activation of Ca2+-ATPase by 10% glycerol was observed only in the case of inhibitor administration after modification of cells with glycerol; in the case of inhibitor addition before erythrocyte contact with glycerol, this phenomenon disappeared. These data suggest the possibility of regulation of human erythrocyte Ca2+-ATPase by glycerol; this regulatory effect may be attributed to both glycerol-induced structural changes in the membrane and also involvement of calmodulin in modulation of catalytic activity of the Ca2+-pump.     

Consequence of aflatoxin B1 ingestion on the membrane-bound liver endoplasmic reticulum Ca2+-ATPase of protein-undernourished Fischer F344 rats

Ingestion of the mycotoxin Aflatoxin B₁ (AFB₁) by protein-undernourished Fischer F344 rats for twelve weeks resulted in significant (p<0.05) increases in the proliferation of liver cells, reduced body weight and increased microsomal Ca²⁺-ATPase activity. Cyt. P₄₅₀ content, -Glutamyl Transferase (GGT) and Glutathione Transferase (GST) activities were unaffected. The ingestion of AFB₁ by normal rats had no effect on all the parameters investigated. It appears that the microsomal Ca²⁺-pumping ATPase of Protein-Undernourished (PU) Fischer F344 rats is more sensitive to the mycotoxin AFB₁ than its counterpart in well-nourished rats. The use of PU rat as animal model for studies on AFB₁ toxicity, particularly the effect of AFB₁ on the regulation of intracellular calcium ion concentration ([Ca²⁺]_i), is suggested.     

Dependence of the resting [Ca2+]cyt of RBL-1 cells on Ca2+ entry

The cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) in resting cells in an equilibrium between several influx and efflux mechanisms. Here we address the question of whether capacitative Ca²⁺ entry to some extent is active at resting conditions and therefore is part of processes that guarantee a constant [Ca²⁺]_cyt. We measured changes of [Ca²⁺]_cyt in RBL-1 cells with fluorometric techniques. An increase of the extracellular [Ca²⁺] from 1.3 mM to 5 mM induced an incrase in [Ca²⁺]_cyt from 105±10 nM to 145±8.5 nM. This increase could be inhibited by 10 μM Gd³⁺, 10 μM La³⁺ or 50 μM 2-aminoethoxydiphenyl borate, blockers of capacitative Ca²⁺ entry. Application of those blockers to a resting cell in a standard extracellular solution (1.3 mM Ca²⁺) resulted in a decrease of [Ca²⁺]_cyt from 105±10 nM to 88.5±10 nM with La³⁺, from 103±12 to 89±12 nM with Gd³⁺ and from 102±12 nM to 89.5±5 nM with 2-aminoethoxydiphenyl borate. From these data, we conclude that capacitative Ca²⁺ entry beside its function in Ca²⁺ signaling contributes to the regulation of resting [Ca²⁺]_cyt.     

Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes

Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca²⁺]_i) in a dose-dependent manner and activated the L-type Ca²⁺ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca²⁺]_i elevation was abolished in the presence of the ET_A receptor blocker BQ123, but was not affected by the ET_B receptor blocker BQ788. ET-1-induced an increase in [Ca²⁺]_i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca²⁺]_i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca²⁺ channel current and an increase of open-state probability (NPo) of an L-type single Ca²⁺ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca²⁺ channel activation and Ca²⁺-induced Ca²⁺ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway. Supported by the National Natural Science Foundation of China (Grant No. 200830870910).     

Renal handling of Ca2+ in diabetes

Ca²⁺ transport in kidney has gained considerable attention in the recent past. Our laboratory has been involved in understanding the regulatory mechanisms underlying Ca²⁺ transport in the kidney across the renal basolateral membrane. We have shown that ANP, a cardiac hormone, mediates its biological functions by acting on its receptors in the kidney basolateral membrane. Furthermore, it has been established that ANP receptors are coupled with Ca²⁺ ATPase, the enzyme that participates in the vectorial translocation of Ca²⁺ from the tubular lumen to the plasma. It is possible that a defect in the ANP-receptor-effector system in diabetes (under certain conditions such as hypertension) may be associated with abnormal Ca²⁺ homeostasis and the development of nephropathy. Accordingly, future studies are needed to establish this hypothesis.     

Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

Ligand binding to transport sites constitutes the initial step in the catalytic cycle of transport ATPases. Here, we consider the well characterized Ca²⁺ ATPase of sarcoplasmic reticulum (SERCA) and describe a series of Ca²⁺ binding isotherms obtained by equilibrium measurements in the presence of various H⁺ and Mg²⁺ concentrations. We subject the isotherms to statistical mechanics analysis, using a model based on a minimal number of mechanistic steps. The analysis allows satisfactory fits and yields information on occupancy of the specific Ca²⁺ sites under various conditions. It also provides a fundamental method for analysis of binding specificity to transport sites under equilibrium conditions that lead to tightly coupled catalytic activation.     

The calmodulin-activated form of the Ca2+-pumping ATPase of the cardiac sarcolemmal membrane produces Ca2+ gradients with a thermodynamic efficiency of 100%

The thermodynamic efficiency of the calmodulin-activated form of the Ca²⁺-pumping ATPase of the bovine cardiac sarcolemma (SL) was evaluated in sealed vesicles under reversible conditions. The free internal Ca²⁺ concentration ([Ca²⁺]_i) established in the SL vesicle lumen by action of the ATPase was determined as a function of the [ATP]/([ADP][P_i]) ratio for the following experimental conditions: 250mM sucrose, 100mM KCI, 0.1mM Mg²⁺, 25mM HEPES, 25mM Tris, pH 7.40, at 37°C, [Ca²⁺]_o=50nM (1mM Ca/EGTA buffer), 0.75mM Mg-ATP, 0.1mM P_i, variable [ADP]. Under these conditions, with the pump working near itsK _m of 64nM, the [Ca²⁺]_i achieved was 18mM, decreasing with increasing [ADP] for [ADP] 0.84mM. A plot of the square of the [Ca²⁺]_i/[Ca²⁺]_o ratio against [ATP]/([ADP][P_i]) gave a straight line with a slope of 1.5×10⁷M. This was in agreement, within the experimental error, with the equilibrium constant for ATP hydrolysis under these conditions (1.09×10⁷M). These results demonstrate (1) tight coupling between Ca²⁺ transport and ATP hydrolysis with a stoichiometry of 2 Ca²⁺ moved per ATP split and (2) a low degree of passive leakage. Analysis at low [ADP] (<0.83mM) showed the unexpected result that ADP increases the rate of theforward reaction of the pump. The maximal effect on the initial rate is a 96±5% increase, with an EC₅₀ of approximately 0.4mM (ADP). Similar but lesser stimulation was observed with CDP. The implications of the above results for the energetics of the pump and for its physiological function in the beating heart are discussed.     

Rapid Ca2+ extrusion via the Na+/Ca2+ exchanger of the human platelet

Summary This communication reports the kinetics of the Na⁺/ Ca²⁺ exchanger and of the plasma membrane (PM) Ca²⁺ pump of the intact human platelet. The kinetic properties of these two systems were deduced by studying the rate of Ca²⁺ extrusion and its Na⁺ dependence for concentrations of cytoplasmic free Ca²⁺ ([Ca²⁺]_cyt) in the 1–10-m range. The PM Ca²⁺ATPase was previously characterized (Johansson, J.S. Haynes, D.H. 1988. J. Membrane Biol. 104:147–163) for [Ca²⁺]_cyt] 1.5 m with the fluorescent Ca²⁺ indicator quin2 (K _d= 115 nm). That study determined that the PM Ca²⁺ pump in the basal state has a V _max = 0.098 mm/min, a K _m= 80 nm and a Hill coefficient = 1.7. The present study extends the measurable range of [Ca²⁺]_cyt with the intracellular Ca²⁺ probe, rhod2 (K _d= 500 nm), which has almost a fivefold lower affinity for Ca²⁺. An Appendix also describes the Mg²⁺ and pH dependence of the K _dand fluorescence characteristics of the commercially available dye, which is a mixture of two molecules. Rates of active Ca²⁺ extrusion were determined by two independent methods which gave good agreement: (i) by measuring Ca²⁺ extrusion into a Ca²⁺-free medium (above citation) or (ii) by the newly developed ionomycin short-circuit method, which determines the ionomycin concentration necessary to short circuit the PM Ca²⁺ extrusion systems. Absolute rates of extrusion were determined by knowledge of how many Ca²⁺ ions are moved by ionomycin per minute. The major findings are as follows: (i) The exchanger is saturable with respect to Ca²⁺ with a K _m= 0.97 ± 0.31 m and V_max = 1.0 ± 0.6 mm/ min. (ii) At high [Ca²⁺]_cyt, the exchanger works at a rate 10 times as large as the basal V _max of the PM Ca²⁺ extrusion pump. (iii) The exchanger can work in reverse after Na⁺ loading of the cytoplasm by monensin. (iv) The PM Ca²⁺ extrusion pump is activated by exposure to [Ca²⁺]_cyt 1.5 m for 20–50 sec. Activation raises the pump V _max to 1.6 ± 0.6 mm/min and the K _mto 0.55 ± 0.24 m. (v) The Ca²⁺ buffering capacity of the cytoplasm is 3.6 mm in the 0.1 to 3 m range of [Ca²⁺]_cyt. In summary, the results show that the human platelet can extrude Ca²⁺ very rapidly at high [Ca²⁺]_cyt. Both the Na⁺/Ca²⁺ exchanger and Ca²⁺ pump activation may prevent inappropriate platelet activation by marginal stimuli.Abbreviations cAMP cyclic adenosine 3,5-monophosphate - cGMP cyclic guanosine 3,5,-monophosphate - Ca-CAM calcium calmodulin; - DT dense tubules - B intrinsic cytoplasmic Ca²⁺ binding sites - R rhod2 or 5-(3,6-bis(dimethylamino)xanth-9-yl)-1-(2-amino-4-hy droxy lphenoxy)-2-(2-amino-5-methylphen- oxy)ethane-N,N,NN-tetraacetic acid - [Ca²⁺]_cyt cytoplasmic Ca²⁺ activity - quin2 2-[[2-bis[(carboxymethyl)amino]-5-methyl-phenoxy]methyl]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline - V or V_extrusion true rate of Ca²⁺ extrusion - fura-2 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,NN-tetraacetic acid - AM acetoxymethyl ester - DMSO dimethylsulfoxide - CTC chlortetracycline - EGTA ethyleneglycol-bis(-aminoethyl ether) N,N,N,N- tetraacetic acid - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - NMDG N-methyl-d-glucamine - PIPES 1,4-piperazine-bis-(ethanesulfonic acid) - HPLC high performance liquid chromatography - _I fraction of high-affinity rhod2 complexed with Ca²⁺ - F the observed fluorescence - F_min the minimal fluorescence observed in the absence of Ca²⁺ - F_max the maximal fluorescence observed when the dye is saturated with Ca²⁺ - X₁ the fraction of high-affinity dye - K _d,1 dissociation constant of high-affinity dye - K _d,2 dissociation constant of the low-affinity dye - -d₁/dt rate of Ca²⁺ removal from the rhod2-Ca complex; - -dF/dt the slope representing the absolute rate of fluorescence decrease in a progress curve - F_max (F_max — F_min)_cyt difference between maximal and minimal fluorescence for cytoplasmic high affinity form of rhod2 - F₅₀ fluorescence of the high-affinity form ofrhod2for[Ca²⁺]_cyt=50 nM - [Ca²⁺]₀ external Ca²⁺concentration - K _p proportionality constant between the total number of Ca²⁺ ions moved and the change in high-affinity rhod2 complexation to Ca² - (d[Ca²⁺]_{cyt, T})/dt rate of Ca²⁺ influx obtained with maximal levels of ionomycin - k_leak rate constant for passive inward Ca²⁺ leakage - k_inno rate constant for ionomycin-mediated Ca²⁺ influx - T total - [rhod2]_cyt,T total intracellular rhod2 concentration - [quin2]_cyt,T total intracellular quin2 concentration - [B]_T total cytoplasmic buffering capacity - A[Ca²⁺]_cyt,T total number of Ca²⁺ ions moved into the cytoplasm - [rhod2-Ca]_{cyt, T} change in concentration of total intracellular high-affinity rhod2 complexed to Ca²⁺ - [B-Ca]_T change in concentration of total cytoplasmic binding sites complexed to Ca²⁺ - [quin2]_{cyt, T} change in concentration of total intracellular quinl complexed to Ca²⁺ - change in the degree of intracellular quin2 saturation - ₁ change in degree of saturation of cytoplasmic high-affinity rhod2 - ₁-/t rate of change in degree of saturation of cytoplasmic high affinityrhod2 - V_obs observed rate of Ca²⁺ removal from the rhod2-Ca complex - V_{8.3 m} the rate of Ca²⁺ removal from the high affinity rhod2-Ca complex at [Ca²⁺]_cyt = 8.3 m - /t rate of change in of the degree of quin2 saturation - [Ca²⁺]_cytT/_t initial linear rate of ionomycin-mediated Ca²⁺ influx - EC₅₀ effective concentration giving a half-maximal effect - [Na⁺]_cyt cytoplasmic Na⁺ activity - CAM calmodulin - ACN acetonitrile - TFA trifuloroacetic acid     

Interaction of Sr2+ with Ca2+-induced Ca2+ release in mitochondria

Respiring rat liver mitochondria are known to spontaneously release the Ca²⁺ taken up when they have accumulated Ca²⁺ over a certain threshold, while Sr²⁺ and Mn²⁺ are well tolerated and retained. We have studied the interaction of Sr²⁺ with Ca²⁺ release. When Sr²⁺ was added to respiring mitochondria simultaneously with or soon after the addition of Ca²⁺, the release was potently inhibited or reversed. On the other hand, when Sr²⁺ was added before Ca²⁺, the release was stimulated. Ca²⁺-induced mitochondrial damage and release of accumulated Ca²⁺ is generally believed to be due to activation of mitochondrial phospholipase A (EC 3.1.1.4.) by Ca²⁺. However, isolated mitochondrial phospholipase A activity was little if at all inhibited by Sr²⁺. The Ca²⁺ -release may thus be triggered by some Ca²⁺ -dependent function other than phospholipase.     

Disulfiram lowers Ca2+, Mg2+-ATPase activity of rat brain synaptosomes

The chronic administration of disulfiram (DS) to rats resulted in significant decrease of synaptosomal Ca²⁺, Mg²⁺-ATPase activity. In vitro studies indicated that DS (ID₅₀=20 M) produced a dose-dependent inhibition of Ca²⁺, Mg²⁺-ATPase. However, diethyldithio-carbamate, a metabolite of DS, failed to modify Ca²⁺, Mg²⁺-ATPase activity, implying that the decrease in ATPase activity in DS administered rats was due to the effect of parent compound. The DS-mediated inhibition (48%) of ATPase activity was comparable with a similar degree of inhibition (49%) achieved by treating the synaptosomal membranes with N-ethylmaleimide (ID₅₀=20 M) in vitro. Furthermore, the inhibition by DS was neither altered by washing the membranes with EGTA nor reversed by treatment with sulfhydryl reagents such as GSH or dithiothreitol. About 74% and 68% decrease of synaptosomal Ca²⁺, Mg²⁺-ATPase specific activity was observed when treated with DS (30 M) and EGTA (100 M) respectively. The remaining 25–30% of total activity is suggested to be of Mg²⁺-dependent ATPase activity. This indicates that both these drugs may act on a common target, calmodulin component that represents 70–75% of total Ca²⁺, Mg²⁺-ATPase activity. Therefore, DS-mediated modulation of synaptosomal Ca²⁺, Mg²⁺-ATPase activity could affect its function of maintaining intracellular Ca²⁺ concentration. This could contribute to the deleterious effects on CNS.     

Opiates inhibit calmodulin activation of a high-affinity Ca2+-stimulated Mg2+-dependent ATPase in synaptic membranes

A high affinity Ca²⁺/Mg²⁺ ATPase has been identified and localized in synaptic membrane subfractions. This enzyme is stimulated by low concentrations of Ca²⁺ (1 M) believed to approximate the range of Ca²⁺ in the synaptosomal cytosol (0.1 to 5.0 M). The opiate agonist levorphanol, in a concentration-dependent fashion, inhibited Ca²⁺-stimulated ATP hydrolysis in lysed synaptic membranes. This inhibition was reversed by naloxone, while dextrorphan, the inactive opiate isomer, was without effect. Inhibition by levorphanol was most pronounced in a subfraction of synaptic membranes (SPM-1). The inhibition of Ca²⁺-stimulated ATP hydrolysis was characterized by a reduction inV _max for Ca²⁺. Levorphanol pretreatment reduced the Hill coefficient (H_N) of 1.5 to 0.7, suggesting cooperative interaction between the opiate receptor and the enzyme protein. Levorphanol, but not dextrorphan, also inhibited (28%) ATP-dependent Ca²⁺ uptake by synaptic membranes. Opiate ligand stereoisomers were tested for their effects on calmodulin stimulating of high affinity Ca²⁺/Mg²⁺ ATPase in synaptic membranes. Levorphanol (10 M), but not the inactive stereoisomer (+)dextrorphan, significantly inhibited (35%) the calmodulin-activated Ca²⁺-dependent ATP hydrolysis activity in a preparation of lysed synaptic membranes. Both Ca²⁺-dependent and calmodulin-dependent stimulation of the enzyme in the presence of optimal concentrations of the other co-substrate were inhibited by levorphanol (35–40%) but not dextrorphan. Inhibition of ATP hydrolysis was characterized by a reduction inV _max for both Ca²⁺ and calmodulin stimulation of the enzyme. Calmodulin stimulation of enzyme activity was most pronounced in SPM-1, the membrane fraction which also exhibits the maximal opiate inhibition (40%) of the Ca²⁺-ATPase. The results demonstrate that opiate receptor activation inhibits a high affinity Ca²⁺/Mg²⁺ ATPase in synaptic plasma membranes in a stereospecific fashion. The inhibition of the enzyme may occur by a mechanism involving both Ca²⁺ and calmodulin. Inhibition of calmodulin activation may contribute to the mechanism by which opiate ligands disrupt synaptosomal Ca²⁺ buffering mechanisms. Changes in the cytosolic distribution of synaptosomal Ca²⁺ following inhibition of Ca²⁺/Mg²⁺ ATPase may underlie some of the pharmacological effects of opiate drugs.     

Ca2+/H+ exchange,lumenal Ca2+ release and Ca2+/ATP coupling ratios in the sarcoplasmic reticulum ATPase

The Ca²⁺ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca²⁺ in intracellular membrane bound compartments, thereby lowering cytosolic Ca²⁺ to induce relaxation. The stored Ca²⁺ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca²⁺, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca²⁺. We review here biochemical and biophysical evidence demonstrating that release of bound Ca²⁺ into the lumen of SR requires Ca²⁺/H⁺ exchange at the low affinity Ca²⁺ sites. Rise of lumenal Ca²⁺ above its dissociation constant from low affinity sites, or reduction of the H⁺ concentration by high pH, prevent Ca²⁺/H⁺ exchange. Under these conditions Ca²⁺ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca²⁺pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.     

Monoclonal antibodies to the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum identify polymorphic forms of the enzyme and indicate the presence in the enzyme of a classical high-affinity Ca2+ binding site

In order to determine whether polymorphic forms of the Ca²⁺ + Mg²⁺-dependent ATPase exist, we have examined the cross-reactivity of five monoclonal antibodies prepared against the rabbit skeletal muscle sarcoplasmic reticulum enzyme with proteins from microsomal fractions isolated from a variety of muscle and nonmuscle tissues. All of the monoclonal antibodies cross-reacted in immunoblots against rat skeletal muscle Ca²⁺ + Mg²⁺-dependent ATPase but they cross-reacted differentially with the enzyme from chicken skeletal muscle. No cross-reactivity was observed with the Ca²⁺ + Mg²⁺-dependent ATPase of lobster skeletal muscle. The pattern of antibody cross-reactivity with a 100,000 dalton protein from sarcoplasmic reticulum and microsomes isolated from various muscle and nonmuscle tissues of rabbit demonstrated the presence of common epitopes in multiple polymorphic forms of the Ca²⁺ + Mg²⁺-dependent ATPase. One of the monoclonal antibodies prepared against the purified Ca²⁺ + Mg²⁺-dependent ATPase of rabbit skeletal muscle sarcoplasmic reticulum was found to cross-react with calsequestrin and with a series of other Ca²⁺-binding proteins and their proteolytic fragments. Its cross-reactivity was enhanced in the presence of EGTA and diminished in the presence of Ca²⁺. Its lack of cross-reactivity with proteins that do not bind Ca²⁺ suggests that it has specificity for antigenic determinants that make up the Ca²⁺-binding sites in several Ca²⁺-binding proteins including the Ca²⁺ + Mg²⁺-dependent ATPase.This paper is dedicated to the memory of Dr. David E. Green.     

Characterization of two different Ca2+ uptake and IP3-sensitive Ca2+ release mechanisms in microsomal Ca2+ pools of rat pancreatic acinar cells

We have examined the effect of the Ca²⁺ (Mg²⁺)-ATPase inhibitors thapsigargin (TG) and vanadate on ATP-dependent ⁴⁵Ca²⁺ uptake into IP₃-sensitive Ca²⁺ pools in isolated microsomes from rat pancreatic acinar cells. The inhibitory effect of TG was biphasic. About 40–50% of total Ca²⁺ uptake was inhibited by TG up to 10 nm (apparent K_i4.2 nm, Ca²⁺ pool I). An additional increase of inhibition up to 85–90% of total Ca²⁺ uptake could be achieved at 15 to 20 nm of TG (apparent K_i12.1 nm, Ca²⁺ pool II). The rest was due to TG-insensitive contaminating plasma membranes and could be inhibited by vanadate (apparent K_i10 m). In the absence of TG, increasing concentrations of vanadate also showed two phases of inhibition of microsomal Ca²⁺ uptake. About 30–40% of total Ca²⁺ uptake was inhibited by 100 m of vanadate (apparent K_i18 m, Ca²⁺ pool II). The remaining 60–70% could be inhibited either by vanadate at concentrations up to 1 mm (apparent K_i300 m) or by TG up to 10 nm (Ca²⁺ pool I). The amount of IP₃-induced Ca²⁺ release was constant at 25% over a wide range of Ca²⁺ filling. About 10–20% remained unreleasable by IP₃. Reduction of IP₃ releasable Ca²⁺ in the presence of inhibitors showed similar dose-response curves as Ca²⁺ uptake (apparent K_i 3.0 nm for IP₃-induced Ca²⁺ release as compared to 4.2 nm for Ca²⁺ uptake at TG up to 10 nm) indicating that the highly TG-sensitive Ca²⁺ pump fills the IP₃-sensitive Ca²⁺ pool I. At TG concentrations >10 nm which blocked Ca²⁺ pool II the apparent K_i values were 11.3 and 12.1 nm, respectively. For inhibition by vanadate up to 100 m the apparent K_i values were 18 m for Ca²⁺ uptake and 7 m for Ca²⁺ release (Ca²⁺ pool II). At vanadate concentrations up to 1 mm the apparent K_i values were 300 and 200 m, respectively (Ca²⁺ pool I). Both Ca²⁺ pools I and II also showed different sensitivities to IP₃. Dose-response curves for IP₃ in the absence of inhibitors (control) showed an apparent K_m value for IP₃ at 0.6 m. In the presence of TG (inhibition of Ca²⁺ pool I) the curve was shifted to the left with an apparent K_m for IP₃ at 0.08 m. In the presence of vanadate (inhibition of Ca²⁺ pool II), the apparent K_m for IP₃ was 2.1 m. These data allow the conclusion that there are at least three different Ca²⁺ uptake mechanisms present in pancreatic acinar cells: TG- and IP₃ insensitive but highly vanadate-sensitive Ca²⁺ uptake occurs into membrane vesicles derived from plasma membranes. Two Ca²⁺ pools with different TG-, vanadate- and IP₃-sensitivities are most likely located in the endoplasmic reticulum at different cell sites, which could have functional implications for hormonal stimulation of pancreatic acinar cells.This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 246. The authors wish to thank Dr. KlausDieter Preuß for valuable discussions and Mrs. Gabriele Mörschbächer for excellent secretarial help.     

Mapping the BKCa channel's "Ca2+ bowl": side-chains essential for Ca2+ sensing

There is controversy over whether Ca(2+) binds to the BK(Ca) channel's intracellular domain or its integral-membrane domain and over whether or not mutations that reduce the channel's Ca(2+) sensitivity act at the point of Ca(2+) coordination. One region in the intracellular domain that has been implicated in Ca(2+) sensing is the "Ca(2+) bowl". This region contains many acidic residues, and large Ca(2+)-bowl mutations eliminate Ca(2+) sensing through what appears to be one type of high-affinity Ca(2+)-binding site. Here, through site-directed mutagenesis we have mapped the residues in the Ca(2+) bowl that are most important for Ca(2+) sensing. We find acidic residues, D898 and D900, to be essential, and we find them essential as well for Ca(2+) binding to a fusion protein that contains a portion of the BK(Ca) channel's intracellular domain. Thus, much of our data supports the conclusion that Ca(2+) binds to the BK(Ca) channel's intracellular domain, and they define the Ca(2+) bowl's essential Ca(2+)-sensing motif. Overall, however, we have found that the relationship between mutations that disrupt Ca(2+) sensing and those that disrupt Ca(2+) binding is not as strong as we had expected, a result that raises the possibility that, when examined by gel-overlay, the Ca(2+) bowl may be in a nonnative conformation.     

Effect of lead on the erythrocyte (Ca2+,Mg2+)-ATPase activity Calmodulin involvement

Summary I have investigated the effect of lead on the erythrocyte ghosts (Ca²⁺,Mg²⁺)-ATPase, with special attention to the role of calmodulin in this phenomena. Under regular incubation conditions, lead inhibits the enzyme with an IC₅₀ of 6.0 µM. The presence of exogenously added calmodulin apparently does not change this inhibitory value. DTT added during the incubation period does not affect the inhibitory action of lead. However, when the membranes are preincubated with DTT, an important IC₅₀ displacement is observed, either with or without added calmodulin. Since [¹²⁵I]calmodulin binding to the membranes is enhanced when lead is used, the possibility of a lead/calmodulin complex that optimally stimulates the enzyme using lead concentrations between 1.0 and 10.0 µM, is suggested. Based on the experimental data, I propose two well defined actions of lead; first, an inhibitory action upon the ATPase above 1.0 µM lead, most probably related to essential sullphydryl groups in the enzyme; and second, a direct action of lead upon calmodulin at lead concentrations below 1.0 µM.A preliminary report has been presented at the 5^th European Bioenergetics Conference. Aberystwyth, Wales. 20–26 July 1988. EBEC Reports. vol 5; p294 (1988).     

The SERCA3-type of organellar Ca2+pumps

Of all the SERCA pumps, SERCA3 was the latest to be described and the least well known. Its primary structure deviates more than usual from the other members of the SERCA family. It is not known whether its remarkably low affinity for Ca²⁺ (K_0.5 > 1M) observed upon expression in the COS cell system occurs also in its normal cellular context. SERCA3 is particularly expressed at high levels in different types of blood cells and related cells like platelets, lymphocytes, mast cells and arterial endothelial cells. It is also found in cerebellar Purkinje neurons. The physiological significance of this expression pattern remains unknown.     

Sarcolemmal Na+-Ca2+ exchange and Ca2+-pump activities in cardiomyopathies due to intracellular Ca2+-overload

In order to identify defects in Na⁺-Ca²⁺ exchange and Ca²⁺-pump systems in cardiomyopathic hearts, the activities of sarcolemmal Na⁺-dependent Ca²⁺ uptake, Na⁺-induced Ca²⁺ release, ATP-dependent Ca²⁺ uptake and Ca²⁺-stimulated ATPase were examined by employing cardiomyopathic hamsters (UM-X7.1) and catecholamine-induced cardiomyopathy produced by injecting isoproterenol into rats. The rates of Na⁺-dependent Ca²⁺ uptake, ATP-dependent Ca²⁺ uptake and Ca²⁺-stimulated ATPase activities of sarcolemmal vesicles from genetically-linked cardiomyopathic as well as catecholamine-induced cardiomyopathic hearts were decreased without any changes in Na⁺-induced Ca²⁺-release. Similar results were obtained in Ca²⁺-paradox when isolated rat hearts were perfused for 5 min with a medium containing 1.25 mM Ca²⁺ following a 5 min perfusion with Ca²⁺-free medium. Although a 2 min reperfusion of the Ca²⁺-free perfused hearts depressed sarcolemmal Ca²⁺-pump activities without any changes in Na⁺-induced Ca²⁺-release, Na⁺-dependent Ca²⁺ uptake was increased. These results indicate that alterations in the sarcolemmal Ca²⁺-efflux mechanisms may play an important role in cardiomyopathies associated with the development of intracellular Ca²⁺ overload.