Contribution of distinct structural elements to activation of calpain by Ca2+ ions

The effect of Ca2+ in calpain activation is mediated via several binding sites in the enzyme molecule. To test the contribution of structural elements suspected to be part of this Ca2+ relay system, we made a site-directed mutagenesis study on calpains, measuring consequential changes in Ca2+ binding and Ca2+ sensitivity of enzyme activity. Evidence is provided for earlier suggestions that an acidic loop in domain III and the transducer region connecting domains III and IV are part of the Ca2+ relay system. Wild-type Drosophila Calpain B domain III binds two to three Ca2+ ions with a K(d) of 3400 microm. Phospholipids lower this value to 220 microm. Ca2+ binding decreases in parallel with the number of mutated loop residues. Deletion of the entire loop abolishes binding of the ion. The Ca2+ dependence of enzyme activity of various acidic-loop mutants of Calpain B and rat m-calpain suggests the importance of the loop in regulating activity. Most conspicuously, the replacement of two adjacent acidic residues in the N-terminal half of the loop evokes a dramatic decrease in the Ca2+ need of both enzymes, lowering half-maximal Ca2+ concentration from 8.6 to 1.3 mm for Calpain B and from 250 to 7 microm for m-calpain. Transducer-region mutations in m-calpain also facilitate Ca2+ activation with the most profound effect seen upon shortening the region by deletion mutagenesis. All of these data along with structural considerations suggest that the acidic loop and the transducer region form an interconnected, extended structural unit that has the capacity to integrate and transduce Ca2+-evoked conformational changes over a long distance. A schematic model of this "extended transducer" mechanism is presented.     

Ca2+ dependency of calpain 3 (p94) activation

Calpain 3, commonly called p94 in the literature, is the abundant skeletal muscle-specific calpain that is genetically linked to limb girdle muscular dystrophy type 2A. Recently, we showed that p94's insertion sequence 1 (IS1) is a propeptide that must be autoproteolytically cleaved to provide access of substrates and inhibitors to the enzyme's active site. Removal of IS1 from the core of p94 by recombinant methods produced a fully active enzyme. Here we have resolved the discrepancies in the literature about the Ca(2+) requirement of p94 using the protease core. Even at substoichiometric levels of Ca(2+), and in competition with EDTA, autoproteolyzed enzyme slowly accumulated. Because the initial autoproteolytic cleavage is an intramolecular reaction, transient binding of two Ca(2+) ions to the core would be sufficient to promote the reaction that is facilitated by having the scissile peptide lying close to the active site cysteine. The second autolytic cleavage was much slower and required higher Ca(2+) levels, consistent with it being an intermolecular reaction. Other metal ions such as Na(+), K(+), and Mg(2+) cannot substitute for Ca(2+) in catalyzing the intramolecular autoproteolysis of the p94 core or in the subsequent hydrolysis of exogenous substrates. These metal ions increase moderately the activity of this enzyme but only at very high concentrations. Thus, the proteolytic activity of the core of p94 and its deletion mutant lacking NS and IS1 was shown to be strictly Ca(2+)-dependent. We propose a two-stage model of activation of the proteolytic core of p94.     

Activation of erythrocyte membrane Ca2+-ATPase by calpain

Ca2+-ATPase of erythrocyte membranes, prepared from erythrocytes substantially removed of contaminating leukocytes, was found to be activated by calpain isolated from the same source. Saponin or glycodeoxycholate treatment of membranes was essential for elicitation of the calpain response. Unlike the membrane bound ATPase, solubilized ATPase was inactivated by calpain. Digestion of membranes with the protease did not affect the Km (ATP) of Ca2+-ATPase though stimulation of the membrane ATPase by calmodulin could be partially substituted by calpain treatment. As compared with control, Ca2+-ATPase of calpain-digested membranes attained maximal activity at a lower free Ca2+ concentration.     

Bimodal regulation of secretion by cytoplasmic Ca2+ as demonstrated by the parathyroid

Bovine parathyroid cells were used to study parathyroid hormone (PTH) release and the cytoplasmic Ca2+ concentration (Cai2+). When the extracellular Ca2+ concentration was decreased from 3.0 to 0.5 mM, perifused cells reacted with rapid stimulation of PTH release. However, a further reduction of extracellular Ca2+ to less than 10 nM resulted in prompt inhibition. Both effects were readily reversible. Using the intracellular Ca2+ indicator quin-2 also as a buffer for calcium it was possible to control Cai2+ within the 20-600 nM range. PTH release was found to increase with Cai2+ up to 200 nM but was gradually suppressed above this concentration.     

Intracellular alkalinization induces cytosolic Ca2+ increases by inhibiting sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)

Intracellular pH (pHi) and Ca(2+) regulate essentially all aspects of cellular activities. Their inter-relationship has not been mechanistically explored. In this study, we used bases and acetic acid to manipulate the pHi. We found that transient pHi rise induced by both organic and inorganic bases, but not acidification induced by acid, produced elevation of cytosolic Ca(2+). The sources of the Ca(2+) increase are from the endoplasmic reticulum (ER) Ca(2+) pools as well as from Ca(2+) influx. The store-mobilization component of the Ca(2+) increase induced by the pHi rise was not sensitive to antagonists for either IP(3)-receptors or ryanodine receptors, but was due to inhibition of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), leading to depletion of the ER Ca(2+) store. We further showed that the physiological consequence of depletion of the ER Ca(2+) store by pHi rise is the activation of store-operated channels (SOCs) of Orai1 and Stim1, leading to increased Ca(2+) influx. Taken together, our results indicate that intracellular alkalinization inhibits SERCA activity, similar to thapsigargin, thereby resulting in Ca(2+) leak from ER pools followed by Ca(2+) influx via SOCs.     

Purification and properties of carp (Cyprinus carpio) muscle calpain II (high-Ca2+-requiring form of calpain)

Calpain (Ca2+-dependent cysteine proteinase) was purified to apparent homogeneity from carp muscle by the method of DEAE-cellulose, hydroxylapatite and Ultrogel AcA 34 column chromatographies. The purified enzyme is classified as calpain II (high-Ca2+-requiring form of calpain) from the effects of Ca2+ concentration, pH and the antibiotics on the activity. Carp muscle calpain II was inhibited by rat liver calpastatin, the specific inhibitor for calpain. It is probable that the calpain-calpastatin system may play a biologically fundamental and common role in various cells, since the inhibitory effect of calpastatin on calpain from different tissues of different species is well conserved.     

Evidence for a Ca2+-independent association between calpain II and phospholipid vesicles

Possible interactions between calpain II and phospholipids such as phosphatidylinositol, phosphatidylserine and phosphatidylcholine were studied using fluorescence and gel filtration techniques. Changes in fluorescence intensity of purified calpain II show that the enzyme strongly interacts with phosphatidylinositol and phosphatidylserine and to a lesser extent with phosphatidylcholine. These results are corroborated by the gel filtration technique which permits the isolation of the enzyme phospholipid complex. Association between calpain II and various phospholipid vesicles can occur in the absence of calcium. Such binding occurs without any observable change of the molecular mass of the two subunits on SDS-polyacrylamide gel electrophoresis.     

A Ca2+ Current Activated by Release of Intracellular Ca2+ Stores in Rat Basophilic Leukemia Cells (RBL-1)

We have characterized a Ca²⁺ current activated by depletion of intracellular Ca²⁺ stores (capacitative Ca²⁺ entry current) as a first step to investigate the mechanisms underlying communication between the intracellular Ca²⁺ stores and the plasma membrane Ca²⁺ permeability. Whole cell currents in response to voltage ramps from −125 to +60 mV from a holding potential of −40 mV were recorded in rat basophilic leukemia cells (RBL-1 cells) in solutions designed to optimize detection of a Ca²⁺ current. An inwardly rectifying current could be activated upon dialysis of the cell interior with pipette solutions devoid of Ca²⁺ and containing 20 mm BAPTA, a procedure expected to passively deplete intracellular Ca²⁺ stores. The current was maximally activated within 2 min, was sensitive to extracellular Ca²⁺ concentration and was abolished by removal of extracellular Ca²⁺. The current was markedly reduced in the presence of Ni²⁺ or La³⁺. The pathway activated by this protocol was permeant to Ba²⁺, displaying complex permeability characteristics at negative potentials. A small inward Mn²⁺ current consistent with a finite permeability of the pathway to Mn²⁺ was detected. In contrast Ni²⁺ displayed no detectable current carrying ability. Extracellular Na⁺ permeated the pathway in the absence of extracellular Ca²⁺. Under conditions designed to reduce passive depletion of intracellular Ca²⁺ stores, a Ca²⁺ current indistinguishable from that described above was activated by addition of ionomycin. This observation is consistent with the activation of the Ca²⁺ influx pathway occurring as a result of events associated with depletion of intracellular Ca²⁺ stores. Importantly, application of extracellular Ni²⁺ in the presence of ionomycin irreversibly inhibited the current. The presence of an inwardly rectifying K⁺ current in RBL cells could confound studies of the capacitative Ca²⁺ entry current when recorded using pipette solutions devoid of K⁺ since this current would be inward over the voltage range used to investigate the capacitative Ca²⁺ entry current. This study compares an inward rectifying K⁺ current and the capacitative Ca²⁺ entry current in RBL cells and highlights some similarities and differences between the two currents. The results demonstrate that caution should be exercised in interpreting recordings made using extracellular solutions containing even modest amounts of K⁺ when studying the capacitative Ca²⁺ entry current in RBL cells. Received: 12 September 1995/Revised: 18 June 1996     

Intracellular localization of MAP2-related protein (O-map) in prophase I and metaphase II oocytes of Xenopus

An antibody directed against rat brain microtubule-associated protein 2 (MAP2) immunoprecipitated a protein of 240 kDa from a Xenopus oocyte extract. In contrast, in Xenopus brain extract, this antibody recognizes a protein of 280 kDa. The oocyte MAP2-related protein (called O-MAP) is present in both prophase I and metaphase II-blocked oocytes as demonstrated by immunoblotting experiments; it is in vivo phosphorylated. Immunocytochemical studies using the anti-rat brain MAP2 antibody demonstrated that the O-MAP colocalizes within the cortical microtubular array present in both prophase I and metaphase II oocytes. However, O-MAP is not associated with the microtubular structures which are organized during the oocyte prophase-metaphase transition, i.e., a giant cytoplasmic network and both the meiotic spindles. O-MAP therefore appears as a microtubule-associated protein oocyte specific and may play a role in the regulation of microtubule stability and the organization of the oocyte cytoskeleton.     

Association and dissociation of the calcium-binding domains of calpain by Ca2+

The calmodulin-like domain of calpain is important for the association of the calpain large and small subunits. We expressed the calmodulin-like domains of the large subunits of rabbit mu- and m-calpains and their small subunits in E. coli and purified them to homogeneity. Unlike the full-length subunits, the calmodulin-like domains are soluble in buffer containing Ca2+. We performed gel filtration chromatography of the purified proteins and found that all three calmodulin-like domains exist as homodimers in the absence of Ca2+ and dissociate into monomers upon the addition of Ca2+.     

Detection and some properties of calpain II (high-Ca2+-requiring form of calpain) in carp (Cyprinus carpio) erythrocytes

In order to examine the existence of calpain I, a low (micromolar)-Ca2+-requiring form of calpain, in fish tissues, carp erythrocytes were chosen as the experimental material, since only calpain I is known to exist in mammalian erythrocytes. By DEAE-cellulose chromatography, calpain and calpastatin (specific inhibitor for calpain) were separated from carp erythrocyte hemolysate. Carp erythrocyte calpain is classified as calpain II, a high (millimolar)-Ca2+-requiring form of calpain, from the result of Ca2+-requirement for the activity.     

Intracellular localization and physiological function of a rice Ca2+-permeable channel OsTPC1

Two-pore channels (TPCs) are cation channels with a voltage-sensor domain conserved in plants and animals. Rice OsTPC1 is predominantly localized to the plasma membrane (PM), and assumed to play an important role as a Ca²⁺-permeable cation channel in the regulation of cytosolic Ca²⁺ rise and innate immune responses including hypersensitive cell death and phytoalexin biosynthesis in cultured rice cells triggered by a fungal elicitor, xylanase from Trichoderma viride. In contrast, Arabidopsis AtTPC1 is localized to the vacuolar membrane (VM). To gain further insights into the intracellular localization of OsTPC1, we stably expressed OsTPC1-GFP in tobacco BY-2 cells. Confocal imaging and membrane fractionation revealed that, unlike in rice cells, the majority of OsTPC1-GFP fusion protein was targeted to the VM in tobacco BY-2 cells. Intracellular localization and functions of the plant TPC family is discussed.     

Inactivation of calpain I and calpain II by specificity-oriented tripeptidyl chloromethyl ketones

Three new tripeptidyl chloromethyl ketones, Leu-Leu-XCH2Cl, with X representing Phe, Tyr, or Lys, were synthesized and their potencies to inactivate calpains I and II were compared. They were designed to fulfil the specificity requirement of calpains established recently. When compared in terms of the dose for 50% inactivation, Leu-Leu-PheCH2Cl was the strongest inactivator, being 500-600 times more effective than tosyl-PheCH2Cl and 5-14 times more than N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]agmatine (E-64). The potency toward calpain, either I or II, decreased in the order Phe greater than Tyr greater than Lys derivatives greater than E-64, whereas that toward papain was E-64 greater than Lys greater than Phe greater than Tyr derivatives. From the determined kinetic parameters, the Phe derivative was 18.3 and 16.6 times more effective than E-64 on calpains I and II, respectively. Likewise, the rate of the alkylation reaction by these chloromethyl ketones with calpain I was 2-4 times greater than that with calpain II. Leu-Leu-PheCH2Cl and its N-dansylated product should be useful for highly selective affinity labeling of calpains I and II.     

Extracellular nucleotides mediate Ca2+ fluxes in J774 macrophages by two distinct mechanisms

We have studied the effects of extracellular nucleotides on the cytosolic free calcium concentration [( Ca2+]i) in J774 macrophages using quin2 and indo-1 as indicator dyes. Micromolar quantities of ATP induced a biphasic increase in [Ca2+]i: a rapid and transient increase (peak I) which was due to mobilization of Ca2+ from intracellular stores and a second more sustained elevation (peak II) due to influx of extracellular Ca2+. The sustained peak II elevation had two components, a "low threshold" (1 microM ATP) response which saturated at 10-50 microM ATP and a "high threshold" response, apparent at [ATP] greater than 100 microM. The latter component was not seen with nucleotides other than ATP and correlated with an ATP-induced generalized increase in plasma membrane permeability. A variant J774 cell line was isolated which does not demonstrate this ATP-induced increase in plasma membrane permeability; nevertheless, it demonstrated both the release of Ca2+ from intracellular stores and the low threshold component of the Ca2+ influx across the plasma membrane in response to nucleoside di- and triphosphates. Several lines of evidence indicate that the fully ionized (i.e. free acid) forms of nucleoside di- and triphosphates were the ligands that mediated these increases in [Ca2+]i. These data show that extracellular nucleotides mediate Ca2+ fluxes by two distinct mechanisms in J774 cells. In one, the rise in [Ca2+]i is due to release of Ca2+ from intracellular stores and Ca2+ influx across the plasma membrane. This response is elicited preferentially by the free acid forms of purine and pyrimidine nucleoside di- and triphosphates. In the other, the rise in [Ca2+]i reflects a more generalized increase in plasma membrane permeability and is elicited by ATP4- only.     

The cytosol of human erythrocytes contains a highly Ca2+-sensitive thiol protease (calpain I) and its specific inhibitor protein (calpastatin)

The cytosol of human erythrocytes was found to contain a Ca2+-dependent thiol protease (calpain) and its specific inhibitor (calpastatin) by DEAE-cellulose chromatography at pH 8.0, although no proteolytic activity toward casein was detected in the unfractionated hemolysate. The protease required only 40 microM Ca2+ for 50% activation, indicating that it belongs to the highly Ca2+-sensitive type of calpain, namely, calpain I. It was not inactivated by heating at 58 degrees C for 10 min at pH 7.2, the optimal pH for its action on casein. The inhibitor comprised major and minor components, calpastatin H (Mr = 280,000) and caplastatin L (Mr = 48,000). Both were heat-stable proteins which were readily inactivated by tryptic digestion. The inhibition of erythrocyte calpain by erythrocyte calpastatin H or L was not due to sequestering of Ca2+ from the reaction medium by the inhibitor protein. The calpain preparation preferentially digests bands III and IVa of human erythrocyte membrane proteins, with little or no cleavage of the bands corresponding to spectrin.     

Activation of tyrosine hydroxylase by Ca2+-dependent neutral protease, calpain

Two molecular species of Ca2+-dependent neutral protease (calpains I and II) and its endogenous inhibitor (calpastatin) in cytosol fraction of bovine adrenal medulla were separated by hydrophobic interaction chromatography. Both calpains I and II, having low and high Ca2+ requirements for casein hydrolysis, respectively, were found to activate tyrosine hydroxylase(TH) that had been purified from cytosol fraction of bovine adrenal medulla. This activation of TH by calpain was inhibited by leupeptin and the endogenous inhibitor, calpastatin. The activated TH with calpain II, characterized by high-performance gel permeation chromatography, had a reduced Mr of 120,000 from the Mr of 230,000 of native enzyme.     

Intracellular Ca(2+) and Ca(2+)/calmodulin-dependent kinase II mediate acute potentiation of neurotransmitter release by neurotrophin-3

Neurotrophins have been shown to acutely modulate synaptic transmission in a variety of systems, but the underlying signaling mechanisms remain unclear. Here we provide evidence for an unusual mechanism that mediates synaptic potentiation at the neuromuscular junction (NMJ) induced by neurotrophin-3 (NT3), using Xenopus nerve-muscle co-culture. Unlike brain-derived neurotrophic factor (BDNF), which requires Ca(2+) influx for its acute effect, NT3 rapidly enhances spontaneous transmitter release at the developing NMJ even when Ca(2+) influx is completely blocked, suggesting that the NT3 effect is independent of extracellular Ca(2+). Depletion of intracellular Ca(2+) stores, or blockade of inositol 1, 4, 5-trisphosphate (IP3) or ryanodine receptors, prevents the NT3-induced synaptic potentiation. Blockade of IP3 receptors can not prevent BDNF-induced potentiation, suggesting that BDNF and NT3 use different mechanisms to potentiate transmitter release. Inhibition of Ca(2+)/calmodulin-dependent kinase II (CaMKII) completely blocks the acute effect of NT3. Furthermore, the NT3-induced potentiation requires a continuous activation of CaMKII, because application of the CaMKII inhibitor KN62 reverses the previously established NT3 effect. Thus, NT3 potentiates neurotransmitter secretion by stimulating Ca(2+) release from intracellular stores through IP3 and/or ryanodine receptors, leading to an activation of CaMKII.     

Activation of calpain I and calpain II: a comparative study using terbium as a fluorescent probe for calcium-binding sites

The present study demonstrates the activation of calpain I and calpain II by micromolar levels of terbium and has utilized the enhancement in the fluorescence of protein-bound terbium to study and compare the calcium binding sites of the two enzymes. Calpain I and calpain II were isolated from bovine erythrocytes and brain, respectively. While the rates of activation of calpain I by terbium and calcium are comparable, the rate of activation of calpain II was much greater in the presence of terbium than in the presence of calcium. Binding of terbium ions to calpains was monitored by the enhanced terbium fluorescence and by the changes in the intrinsic protein fluorescence of calpains. Stoichiometric titrations indicated that calpain I and calpain II bound four and six molar equivalents of terbium ion, respectively. During the titration, the intrinsic protein fluorescence of calpain II was successively quenched whereas that of calpain I showed an abrupt drop just prior to the saturation. The association constants (Ka) increased from 10(5) to 10(7) M-1 for calpain I and from 10(4) to 10(6) M-1 for calpain II with addition of increasing molar equivalents of terbium. Titration of enzymatic activities with calcium showed that the activation of calpain I required fewer molar equivalents of metal ions than were necessary for the activation of calpain II, in agreement with stoichiometric titration with terbium.