Resistance to thapsigargin-induced intracellular calcium mobilization in a multidrug resistant tumour cell line

A multidrug resistant (MDR) cell line, derived from the human leukaemic cell K562 and selected for its resistance to Vincristine, was shown to be resistant to Thapsigargin (TG). A concentration of 50 nM TG was toxic to K562 cells whereas the MDR cell line, known as Lucena I cells, survived unaffected for up to seven days in culture. Similarly, no intracellular Ca²⁺ mobilization was observed in the MDR cell line treated with TG. This effect was not a result of TG extrusion by P glycoprotein (Pgp), as no mobilization was observed even in the presence of the Pgp inhibitors Verapamil (5 M) and Cyclosporin A (0.16 M). In the present study, both cell lines expressed comparable levels of Bcl-2 making it unlikely that Bcl-2 was involved in this process. Similarly, no overexpression of the endoplasmic reticulum Ca²⁺ ATPase (SERCA) could be detected in the MDR cell line and Ca²⁺ uptake by vesicles of the two cell types were equally sensitive to TG. These results confirm that MDR cells do not mobilize Ca²⁺ in the presence of TG but go against the possibility that this might be due to TG extrusion or to the overexpression of a resistant SERCA isoform.     

The role of ion antiporters in the maintenance of intracellular pH in rat vascular smooth muscle cells

Vascular smooth muscle intracellular pH is maintained by the Na⁺/H⁺ and Cl^–/HCO ₃ ^– antiporters. The Na⁺/H⁺ exchanger is a major route of H⁺ extrusion in most eukaryotic cells and is present in vascular smooth muscle cells in a similar capacity. It extrudes H^– into the extracellular space in exchange for Na⁺. The Cl^–/HCO ₃ ^– exchanger plays an analogous role to lower the pH of vascular smooth muscle cells when increases in intracellular pH occur. Its activity has also been demonstrated in A7r5 and A10 vascular smooth muscle cells. The Na⁺/H⁺ exchanger is regulated by a number of agents which act through inositol trisphosphate/diacylglycerol, to stimulate the antiporter. Calcium-calmodulin dependent protein kinase may also activate the antiporter in vivo. Phosphorylation of the Cl^–/HCO ₃ ^– exchanger has also been observed but its physiological role is not known. Both these antiporters exist in the plasma membrane as integral proteins with free acidic cytoplasmic termini. These regions may be important in sensing changes in intracellular pH, to which these antiporters respond.Abbreviations CaM Calmodulin - DCCD Dicylohexyl-Carbodiimide - DG Diacylglycerol - DIDS-4 4-Diisthiocyanostilbene-2,2-Disulfonic Acid - IP₃ Inositol Trisphosphate - PKC protein Kinase C - SITS-4 4-Acetamido-4-Isothiocyanstilbene-2,2-Disulfonate - VSMC Vascular Smooth Muscle Cell     

Comparison of Ni-sensitive and Ni-resistant strains of Nostoc muscorum

A wild-type Ni-sensitive (Ni^s) strain of Nostoc muscorum ISU spontaneously yielded mutants resistant to inhibition by 40 M Ni with a frequency of about 10^-7. A Ni-resistant (Ni^r) mutant was deficient in the activities of urease and uptake hydrogenase. Cellular Ni uptake in the Ni^s strain was dependent on concentration (40 to 120 M) and time (0 to 30 min) (V_max=0.51 nmol/g protein.min; K_m=92 M). The Ni bioconcentration factor for such cells ranged between 0.95×10³ and 1.89×10³. Ni uptake in spheroplast preparations from Ni^s cells followed almost the same trend as intact cells except that the bioconcentration factor was slightly less [(0.82 to 1.39)×10³]. In contrast, Ni uptake in the Ni^r intact cells was not concentration dependent and also the uptake was saturated, even at 40 M, within 10 min. Spheroplasts from the Ni^r strain showed a Ni bioconcentration factor of 1.19×10³ compared with 4.41×10³ for intact cells. The invariably lower Ni uptake by spheroplasts was attributed to altered membrane transport properties.R.K. Asthana, A.L. Singh and S.P. Singh are with the Algal Research Laboratory, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi-221 005, India.     

Role of SH Groups in the Functioning of Ca2+-Transporting ATPases Regulating Ca2+ Homeostasis and Exocytosis

Using a ^Ca2+-sensitive fluorescent indicator, Fura-2/AM, and a metallochromic dye, arsenazo, we measured the intracellular concentration of Ca²⁺ ([Ca²⁺] _i ) and the content of total calcium in isolated acinar cells of the rat submandibular salivary gland. It was shown that the influence of a mercaptide-forming compound, sodium p-chloromercuribenzoate (pChMB), increased both the [Ca²⁺] _i and content of total calcium but did not change the intensity of exocytosis. Such a situation is probably related to the fact that pChMB inhibits plasmalemmal Ca²⁺-ATPase (PMCA). The absence of changes in the exocytotic activity can be explained as follows: the influence of a pChMB-induced significant increase in the [Ca²⁺] _i is neutralized due to the functioning of Ca²⁺-ATPases of the endoplasmic reticulum (SERCA), which pump Ca²⁺ into the store. Incubation of a microsomal fraction with pChMB resulted in suppression of the specific PMCA and SERCA activities with apparent constants of inhibition (I ₅₀) 245 and 52 M, respectively. Dithiothreitol (DTT, 0.1 mM) increased the PMCA and SERCA activities (probably facilitating the access of substrate to the active centers of ATPases at the expense of a decrease in the number of disulfide bonds, which is followed by changes in the conformation of intracellular hydrophilic loops of their molecules). Dithiothreitol also recovered the suppression of PMCA and SERCA activities induced by pre-incubation with pChMB (by 45 and 32%, respectively); these activities did not, however, reach the initial levels. A probable interpretation of this fact is that DTT shields from the action of pChMB only superficial but not sterically less accessible SH groups. Limited proteolysis of the microsomes by -chymotrypsin decreased the specific PMCA and SERCA activities by 16 and 60%, respectively. Incubation of the microsomes in an -chymotrypsin-containing medium (15 sec) with subsequent addition of 150 M pChMB exerted almost no influence on the PMCA activity, whereas the SERCA activity dramatically increased (by 146%). This fact allows us to suggest that -chymotrypsin is capable of eliminating the inhibitory effect of pChMB on the SERCA activity; the mechanism of this effect remains unknown. Therefore, functionally important SH groups are present in the catalytic and active centers of both PMCA and SERCA; superficial SH groups dominate in the PMCA molecules, whereas SERCA is controlled by more deeply localized SH groups.     

BH3-mimetic gossypol-induced autophagic cell death in mutant BRAF melanoma cells with high expression of p21

Aims

The aim of the present study was to identify the potential therapeutic effects of BH3-mimetic gossypol on melanoma cells with acquired resistance to BRAF inhibitors.

Main methods

The IC₅₀ values of gossypol were determined using MTT assays in three melanoma cell lines with different resistances to BRAF inhibitor. The effects of gossypol on three melanoma cell lines were further examined by immunoblotting analysis, cell cycle analysis, flow cytometric apoptotic assay and autophagy assay. The functional role of autophagy in gossypol-induced growth inhibition was investigated using siRNA-mediated knockdown of Beclin-1.

Key findings

Gossypol retained its efficacy in BRAF-V600E melanoma clones with acquired resistance to BRAF inhibitors through a mechanism independent of MEK–ERK inhibition. Gossypol caused G₂/M arrest in both BRAF mutant A375P and A375P/Mdr cells with high expression of p21^Cip1, regardless of their drug resistance. Interestingly, we determined that the lack of gossypol-induced mitotic arrest in BRAF-WT-harboring SK-MEL-2 cells was associated with a low level of p21^Cip1 expression. In addition, gossypol preferentially induced autophagy and apoptosis in the gossypol-sensitive cells and not in the gossypol-resistant SK-MEL-2 cells. In particular, alleviation of autophagy by knockdown of Beclin-1 partially caused a resistance to gossypol-induced cell cycle arrest at G₂/M in BRAF-V600E cells with a concomitant decreased induction of apoptosis.

Significance

Taken together, these results suggest that gossypol may exhibit potential for the treatment of BRAF inhibitor-resistant tumors, but a functional p21^Cip1 is a prerequisite for a positive response to its clinical application.     

Characterization of inositol 1,4,5-trisphosphate-sensitive (IsCaP) and-insensitive (IisCaP) nonmitochondrial Ca2+ pools in rat pancreatic acinar cells

Summary We have measured Ca²⁺ uptake and Ca²⁺ release in isolated permeabilized pancreatic acinar cells and in isolated membrane vesicles of endoplasmic reticulum prepared from these cells. Ca²⁺ uptake into cells was monitored with a Ca²⁺ electrode, whereas Ca²⁺ uptake into membrane vesicles was measured with⁴⁵Ca²⁺. Using inhibitors of known action, such as the H⁺ ATPase inhibitors NBD-Cl and NEM, the Ca²⁺ ATPase inhibitor vanadate as well as the second messenger inositol 1,4,5-trisphosphate (IP₃) and its analog inositol 1,4,5-trisphosphorothioate (IPS₃), we could functionally differentiate two non-mitochondrial Ca²⁺ pools. Ca²⁺ uptake into the IP₃-sensitive Ca²⁺ pool (IsCaP) occurs by a MgATP-dependent Ca²⁺ uptake mechanism that exchanges Ca²⁺ for H⁺ ions. In the absence of ATP Ca²⁺ uptake can occur to some extent at the expense of an H⁺ gradient that is established by a vacuolar-type MgATP-dependent H⁺ pump present in the same organelle. The other Ca²⁺ pool takes up Ca²⁺ by a vanadate-sensitive Ca²⁺ ATPase and is insensitive to IP₃ (IisCaP). The IsCaP is filled at higher Ca²⁺ concentrations (10^–6 mol/liter) which may occur during stimulation. The low steady-state [Ca²⁺] of 10^–7 mol/liter is adjusted by the IisCaP.It is speculated that both Ca²⁺ pools can communicate with each other, the possible mechanism of which, however, is at present unknown.     

Mechanistic origin of the kinetic cooperativity for the ATPase activity of sarcoplasmic reticulum

The (Ca²⁺-Mg²⁺)-ATPase from sarcoplasmic reticulum presents negative cooperativity for the hydrolysis of Mg²⁺-ATP at different concentration ranges of this substrate. A kinetic model is proposed according to which Mg²⁺-ATP may bind to three different enzymatic species present during the catalytic cycle, E (K ₁=1 µM), EP.Ca₂ (K ₉=500 µM) and *EP (K ₇=20 µM), accelerating the release of P_i. The fact that each of these species has a different affinity for Mg²⁺-ATP allows a significant enhancement of the rate of P_i release to the medium at the different ranges of Mg²⁺-ATP concentration where the enzyme shows a kinetic cooperativity. The kinetic analysis of this mechanism yields an equation which is a ratio of two cubic polynomials (3:3 rate equations) with respect to Mg²⁺-ATP and which may explain the negative cooperativity of the enzyme at different concentration ranges of Mg²⁺-ATP.Abbreviations: EGTA, ethylene glycol bis(-aminoethylether)-N,N,N,N-tetraacetic acid; I.U., international units; piruvate kinase (EC 2.7.1.40); lactate dehydrogenase (EC 1.1.1.27); ATP phosphohydrolase (EC 3.8.1.3).     

NADP+-dependent acetaldehyde dehydrogenase from Zymomonas mobilis

An NADP⁺-linked acetaldehyde dehydrogenase (EC 1.2.1.4) from the ethanol producing bacterium Zymomonas mobilis was purified 180-fold to homogeneity. The enzyme is a cytosolic protein with an isoelectric point of 8.0 and has an apparent molecular weight of 210000. It showed a single band in sodium dodecylsulfate gel electrophoresis with a molecular weight of 55000, which indicates that it consists of four probably identical subunits. The apparent K _m values for the substrate acetaldehyde were 57 M and for the cosubstrate NADP⁺ 579 M. The enzyme was almost inactive with NAD⁺ as cofactor. Several other aldehydes besides acetaldehyde were accepted as a substrate but not formaldehyde or trichloroacetaldehyde. In anaerobically grown cells of Zymomonas mobilis the enzyme showed a specific activity of 0.035 U/mg protein but its specific activity could be increased up to 0.132 U/mg protein by adding acetaldehyde to the medium during the exponential growth phase or up to 0.284 U/mg protein when cells were grown under aeration. The physiological role of the enzyme is discussed.Abbreviations ALD-DH acetaldehyde dehydrogenases from Z. mobilis - DTT dithiothreitol - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - SDS sodium dodecylsulfate Dedicated to Prof. Dr. H.-G. Schlegel, Universität Göttingen, on the occasion of his 65th birthday     

Influence of Ca2+ channel modulators on [3H]purine release from rat cultured glial cells

[³H]Purine release from rat striatum astrocyte cultures was studied at 14 days in vitro (DIV). Superfusion of cultures with a Ca²⁺-free medium +0.5 mM ethylene glycol-bis(-aminoethylether)N,N,N,N-tetracetic acid (EGTA) reduced the electrically evoked [³H]purine release. Nimodipine only at the concentration of 10 M modified [³H]purine outflow whereas 0.1 M -conotoxin and 0.03–0.1 M nitrendipine reduced the evoked one. Superfusion of cultures with 0.1 M -conotoxin +0.1 M nitrendipine antagonized the evoked [³H]purine release similarly to each drug given alone. Neither nitrendipine nor -conotoxin influenced the uptake of⁴⁵Ca²⁺ by the cultures. The treatment of cells with the Ca²⁺ agonist Bay K 8644 did not affect [³H]purine release or the⁴⁵Ca²⁺ uptake. The drug did not either alter [Ca²⁺]_i, evaluated by loading the cells with 3 M Fura-2/AM. 10–30 M 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), a blocker of intracellular Ca²⁺ discharge, significantly reduced the evoked [³H]purine release. On the other hand, 2 M thapsigargin, an inhibitor of the ion store Ca²⁺ ATPase, was able to increase either the culture [³H]purine release or the [Ca²⁺]_i. Together, the findings indicate that voltage-sensitive calcium channels (VSCC_s) of the neuronal N and L-types are not involved in the modulation of [³H]purine release from rat cultured astrocytes whereas Ca²⁺ coming from intracytoplasmic stores seems to play a prevailing role. Moreover, agents which block VSCC_s seem to be able to affect [³H]purine outflow with mechanisms other than VSCC gating.     

Na+-transport modulation induces isoform-specific expression of Na+,K+-ATPase α-subunit isoforms in C2C12 skeletal muscle cell

Changes in demands for Na⁺ transport alter expression of the Na⁺,K⁺-ATPase subunit isoforms. In skeletal muscle, the effects of these changes on expression the 2 isoform, the major isoform expressed in differentiated muscle cell, is not known. Therefore, this study examines regulation of the -subunit isoforms by Na⁺ in the C₂C₁₂ skeletal muscle cell that expresses the 1 and 2 isoforms. Western blot analysis showed that in differentiating C₂C₁₂ muscle cell, but not in undifferentiated myoblast, veratridine, a Na⁺ channel activator, greatly increased expression of the 2 isoform; expression of 1 was unaltered. Because the level of -actinin was unaltered, the data suggest that veratridine treatment did not significantly alter the progression of cell differentiation. Furthermore, a reduction in Na⁺ transport by tetrodotoxin again failed to alter expression of a1. Thus, in C₂C₁₂ skeletal muscle cell, changes in Na⁺ transport alters expression of the 2, but not the 1 isoform. These results differ from those observed previously in muscle cells that express only the 1 isoform. Because mammalian skeletal muscle expresses both the 1- and 2-subunit isoforms, the differential regulation that was observed may be physiologically relevant in these muscle cells in vivo.     

The Nootropic Drug Vinpocetine Inhibits Veratridine-Induced [Ca2+]i Increase in Rat Hippocampal CA1 Pyramidal Cells

The alkaloid derivative vinpocetine (14-ethoxycarbonyl-(3,16-ethyl)-14,15-eburnamine; Cavinton) has a well known beneficial effect on brain function in hypoxic and ischemic conditions. While it increases CNS blood flow and improves cellular metabolism, relatively little is known about vinpocetine's underlying molecular mechanisms on the single cell level. Since apoptotic and necrotic cell damage is always preceded by an increase in [Ca²⁺]_i, this study investigated the effect of vinpocetine on [Ca²⁺]_i increases in acute brain slices. Sodium influx is an early event in the biochemical cascade that takes place during ischemia. The alkaloid veratridine can activate this Na⁺ influx, causing depolarization and increasing [Ca²⁺]_i in the cells. Therefore, it can be used to simulate an ischemic attack in brain cells. Using a cooled CCD camera-based ratio imaging system and cell loading with fura 2/AM, the effect of vinpocetine on [Ca²⁺]_i changes in single pyramidal neurons in the vulnerable CA1 region of rat hippocampal slices was investigated. Preperfusion and continuous administration of vinpocetine (10 M) significantly inhibited the elevation in [Ca²⁺]_i induced by veratridine (10 M). When the drug was administered after veratridine, it could accelerate the recovery of cellular calcium levels. Piracetam, another nootropic used in clinical practice, could attenuate the elevation of [Ca²⁺]_i only at a high, 1 mM, concentration. We have concluded that vinpocetine, at a pharmacologically relevant concentration, can decrease pathologically high [Ca²⁺]_i levels in individual rat hippocampal CA1 pyramidal neurons; this effect might contribute to the neuroprotective property of the drug.     

Rat osseous plate alkaline phosphatase: Effect of neutral protease digestion on the hydrolysis of pyrophosphate and nitrohenylphospate

Collagenase treatment, commonly used to prepare alkaline phosphatase-rich matrix vesicles from epiphyseal cartilage growth plates, seems to affect the integrity of this membrane-bound enzyme. Alkaline phosphatase-rich rat osseous plates were incubated with 1000 U/mL collagenase for 3 h, at 37°C and after purification on Sepharose 4B, kinetic studies were performed using nitrophenylphosphate and pyrophosphate as substrates.The optimum apparent pH for the hydrolysis of p-nitrophenylphosphate and pyrophosphate increased from 9.4 to 10.25 and from 8.0 to 9.0, respectively, as a consequence of collagenase treatment. In the absence of Mg²⁺ ions, the enzyme hydrolyzed PNPP with K_M = 322.5 ± 15.3 M and V = 965.2 ± 45.8 U/mg, while in the presence of 2 mM Mg²⁺ ions, V increased 66%. Cobalt (K_0.5 = 5.3 ± 0.3 M) and manganese (K_0.5 = 0.72 ± 0.03 M) ions stimulated the PNPPase activity of the collagenase-treated enzyme, but with a lower apparent affinity when compared with that of not-treated enzyme. In the absence of Mg²⁺ ions pyrophosphate was hydrolyzed according to Michaelis-Menten kinetics (K_M = 105.1 ± 6.3 M and V = 64.9 ± 3.9 U/mg), but site-site interactions (n_H = 1.2) were observed in the presence of 2 mM Mg²⁺ ions (V = 110.8 ± 5.5 U/mg; K_0.5 = 42.7 ± 2.0 M).To our knowledge this is the first report showing significant alterations on phosphohydrolytic activity and metal binding properties of bone alkaline phosphatase due to associated neutral proteases in collagenase preparations often used for the isolation of matrix vesicles.     

Importance of Ca2+ influx by Na+/Ca2+ exchange under normal and sodium-loaded conditions in mammalian ventricles

Na⁺/Ca²⁺ exchange (NCX) is a major Ca²⁺ extrusion system in cardiac myocytes, but can also mediate Ca²⁺ influx and trigger sarcoplasmic reticulum Ca²⁺ release. Under conditions such as digitalis toxicity or ischemia/reperfusion, increased [Na⁺]_i may lead to a rise in [Ca²⁺]_i through NCX, causing Ca²⁺ overload and triggered arrhythmias. Here we used an agent which selectively blocks Ca²⁺ influx by NCX, KB-R7943 (KBR), and assessed twitch contractions and Ca²⁺ transients in rat and guinea pig ventricular myocytes loaded with indo-1. KBR (5 M) did not alter control steady-state twitch contractions or Ca²⁺ transients at 0.5 Hz in rat, but significantly decreased them in guinea pig myocytes. When cells were Na⁺-loaded by perfusion of strophanthidin (50 M), the addition of KBR reduced diastolic [Ca²⁺]_i and abolished spontaneous Ca²⁺ oscillations. In guinea pig papillary muscles exposed to substrate-free hypoxic medium for 60 min, KBR (10 M applied 10 min before and during reoxygenation) reduced both the incidence and duration of reoxygenation-induced arrhythmias. KBR also enhanced the recovery of developed tension after reoxygenation. It is concluded that (1) the importance of Ca²⁺ influx via NCX for normal excitation-contraction coupling is species-dependent, and (2) Ca²⁺ influx via NCX may be critical in causing myocardial Ca²⁺ overload and triggered activities induced by cardiac glycoside or reoxygenation.     

Processes Maintaining Calcium Homeostasis in Acinar Cells of the Rat Submandibular Salivary Gland

Using a Ca²⁺-sensitive fluorescent indicator, fura-2/AM, we recorded calcium transients in secretory cells of isolated acini of the rat submandibular salivary gland; these transients were induced by hyperpotassium-induced depolarization (after an increase in [K⁺] _e up to 50 mM) of the plasma membrane of the above cells. Calcium transients were significantly suppressed by 50 M nifedipine. Addition of 10 M carbonyl cyanide m-chlorophenylhydrazone to the normal extracellular solution was accompanied by a rise in [Ca²⁺] _i , whereas when hyperpotassium solution is used the effect was less expressed. Blockers of CA²⁺-ATPase in the cellular membrane and in the endoplasmic reticulum, eosin Y (5 M) and cyclopiazonic acid (CPA, 5 M), respectively, evoked a significant increase in [Ca²⁺] _i and a decrease in the K⁺-depolarization-induced calcium transient. Extracellular application of caffeine (2, 10, or 30 mM) was accompanied by a concentration-dependent rise in [Ca²⁺] _i . Therefore, potassium depolarization of the plasma membrane of acinar cells of the rat submandibular salivary gland activates both the voltage-dependent Ca²⁺ influx and Ca²⁺-induced Ca²⁺ release from the endoplasmic reticulum; the initial level of [Ca²⁺] _i was restored at the joint involvement of Ca²⁺-ATPases in the plasma membrane and the membranes of the endoplasmic reticulum and mitochondria.     

Is the Ca2+-ATPase from sarcoplasmic reticulum also a heat pump?

We calculate, using the first law of thermodynamics, the membrane heat fluxes during active transport of Ca²⁺ in the Ca²⁺-ATPase in leaky and intact vesicles, during ATP hydrolysis or synthesis conditions. The results show that the vesicle interior may cool down during hydrolysis and Ca²⁺-uptake, and heat up during ATP synthesis and Ca²⁺-efflux. The heat flux varies with the SERCA isoform. Electroneutral processes and rapid equilibration of water were assumed. The results are consistent with the second law of thermodynamics for the overall processes. The expression for the heat flux and experimental data, show that important contributions come from the enthalpy of hydrolysis for the medium in question, and from proton transport between the vesicle interior and exterior. The analysis give quantitative support to earlier proposals that certain, but not all, Ca²⁺-ATPases, not only act as Ca²⁺-pumps, but also as heat pumps. It can thus help explain why SERCA 1 type enzymes dominate in tissues where thermal regulation is important, while SERCA 2 type enzymes, with their lower activity and better ability to use the energy from the reaction to pump ions, dominate in tissues where this is not an issue.

Neuronal Control of Exocytosis and Calcium Homeostasis

We showed that 5 M acetylcholine (ACh) and 100 M norepinephrine (NE) cause increases in the total Ca²⁺ content in acinar cells by 30 and 87% and in the exocytosis intensity by 15 and 20%, respectively. Application of 5 M ACh and 100 M NE increased the free cytosolic Ca²⁺ concentration ([Ca²⁺] _i ) by 87 ± 2 and 140 ± 7 nM, respectively. Application of ACh and NE in a Ca²⁺-free external solution caused a [Ca²⁺] _i increase that was 40 and 67% lower than in physiological solution. We postulate that the exocytosis developing upon neural stimulation of the gland results from generation of Ca²⁺ transients that are spreading from the basal to the apical region of the exocrine cell, where secretory granules are concentrated.     

Cyclopiazonic Acid Is Complexed to a Divalent Metal Ion When Bound to the Sarcoplasmic Reticulum Ca2+-ATPase

We have determined the structure of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) in an E2·P_i-like form stabilized as a complex with , an ATP analog, adenosine 5′-(β,γ-methylene)triphosphate (AMPPCP), and cyclopiazonic acid (CPA). The structure determined at 2.5Å resolution leads to a significantly revised model of CPA binding when compared with earlier reports. It shows that a divalent metal ion is required for CPA binding through coordination of the tetramic acid moiety at a characteristic kink of the M1 helix found in all P-type ATPase structures, which is expected to be part of the cytoplasmic cation access pathway. Our model is consistent with the biochemical data on CPA function and provides new measures in structure-based drug design targeting Ca²⁺-ATPases, e.g. from pathogens. We also present an extended structural basis of ATP modulation pinpointing key residues at or near the ATP binding site. A structural comparison to the Na⁺,K⁺-ATPase reveals that the Phe⁹³ side chain occupies the equivalent binding pocket of the CPA site in SERCA, suggesting an important role of this residue in stabilization of the potassium-occluded E2 state of Na⁺,K⁺-ATPase.The Ca²⁺-ATPase from sarco(endo)plasmic reticulum of rabbit skeletal muscle (SERCA,⁵ isoform 1a) is a thoroughly studied member of the P-type ATPase family (1). SERCA possesses 10 transmembrane helices (M1 through M10) with both the N terminus and the C terminus facing the cytoplasmic side and three cytoplasmic domains, inserted in loops between M2 and M3 (A-domain) and between M4 and M5 (P- and N-domain) (2). The enzyme mediates the uptake of Ca²⁺ ions into the lumen of the sarcoplasmic reticulum (SR) after their release into the cytoplasm through calcium release channels during muscle contraction (3). SERCA, plasma membrane Ca²⁺-ATPase, and a third, Golgi-located secretory pathway Ca²⁺-ATPase are important factors in calcium and manganese homeostasis, transport, signaling, and regulation (4, 5).Crystal structures of all major states in the reaction cycle of SERCA have been determined. These include the Ca₂E₁·ATP state (6, 7) with high affinity Ca²⁺ binding sites accessible from the cytoplasmic side of the SR membrane, the calcium-occluded transition state (6), the open E2P state with luminal facing ion binding sites that have low affinity for Ca²⁺ and high affinity for protons (8) and the proton-occluded H_2–3E2[ATP] state with a bound modulatory ATP (9). This considerable amount of structural information has turned the Ca²⁺-ATPase into a valuable model system for studies on structural rearrangements that take place during the catalytic cycle of P-type ATPases. SERCA is considered a promising drug target in medical research, with a particular focus on prostate cancer and infectious diseases. Several compounds have already been shown to bind and inhibit SERCA by stabilizing the enzyme in a particular conformational state. Thapsigargin (TG), cyclopiazonic acid (CPA), and 2,5-di-(tert-butyl) hydroquinone (BHQ) stabilize an E2-like state, and 1,3-dibromo-2,4,6-tri (methylisothiouronium)benzene stabilizes an E1-P-like conformation (10–13). CPA is a toxic indole tetramic acid first isolated from Penicillium cyclopium (14) and later found to be produced by Aspergillus versicolor and Aspergillus flavus. Like TG, CPA specifically binds to and inhibits SERCA with nanomolar affinity (15). Indeed, CPA is widely used in biochemical and physiological studies on Ca²⁺ signaling and muscle function, where it causes Ca²⁺ store depletion due to specific inhibition of Ca²⁺ reuptake by SERCA. CPA and TG were originally proposed to bind to similar sites on SERCA (16), but recent crystal structures have shown a distinct site of interaction (17, 18). Despite these structural insights, a previously demonstrated magnesium dependence of CPA binding (19) remained unexplained, and opposing CPA binding modes were observed (see below).Tetramic acids are synthesized naturally, and more than 150 natural derivatives have been isolated from bacterial and fungal species (reviewed in Ref. 20). Tetramic acids possessing a 3-acyl group have the ability to chelate divalent metal ions. For instance, tenuazonic acid from the fungus Phoma sorghina has been shown to form complexes with Ca²⁺ and Mg²⁺ (21), as well as heavier metals such as Cu(II), Ni(II), and Fe(III) (22).Previously published crystallographic structures of the SERCA·CPA complex (PDB ID 2O9J and 2EAS) demonstrated that CPA binds within the proposed calcium access channel of SERCA. However, the structures did not reveal a role for magnesium, and the orientation of CPA within this binding site differed in the two studies (17, 18). To address these ambiguities, we have determined the crystal structure of SERCA in complex with , AMPPCP (an ATP analog), and Mn²⁺·CPA. The structure reveals novel insight into CPA binding, which we find to be mediated by a divalent cation, as demonstrated by means of the anomalous scattering properties of Mn²⁺. Further and improved refinement using previously deposited data (PDB ID 2O9J and 2OA0), in light of our new findings, also revealed a strong plausibility for a magnesium ion bound at this site. Furthermore, we find a new configuration of the bound AMPPCP nucleotide, addressing the modulatory role of ATP binding to the E2·P_i occluded conformation of SERCA.     

Identification and characterization of high-affinity Ca2+-ATPase associated with axonal plasma membranes of dog mesenteric nerves

The microsomal fraction isolated from dog mesenteric nerve fibres was found to contain ATPase activity stimulated by micromolar concentrations of Ca ions. Such a high-affinity Ca²⁺-ATPase (hereafter referred to as HA Ca-ATPase) followed a Michaelis-Menten kinetics with K_m for Ca ions of 0.4 M and V_max=12.5±2.4 mol P_i.mg^–1h^–1. The examination of the subcellular origin of HA Ca-ATPase revealed that this enzyme is associated with axonal plasma membranes as documented by its co-purification with several plasma membrane marker enzymes and with tetrodotoxin-sensitive³H-saxitoxin binding. The addition of exogenous magnesium ions (Mg) resulted in a non-competitive inhibition of HA Ca-ATPase with K_i=0.5 mM. The reaction velocity of HA Ca-ATPase was also inhibited by other divalent ions with the order of potency Mg>Mn >ZnCo>Ni. In contrast to low affinity (high K_m) Mg- and Ca-ATPase, the HA Ca-ATPase was insensitive to the inhibition by sodium azide (10 mM) and sodium fluoride (10 mM). Similarly, the specific activity of HA Ca-ATPase was unaffected by vanadate (100 M) and N-ethylmaleinimide (100 M). It is concluded that axonal plasma membranes of dog mesenteric nerves contain HA Ca-ATPase which seems to be unrelated to calcium-transporting Mg-dependent, Ca-stimulated ATPase.Abbreviations used BSA bovine serum albumin - HA Ca-ATPase high-affinity Ca²⁺-ATPase - K-pNPPase onabain-sensitive, K⁺-stimulated p-nitrophenyl phosphatase - NEM N-ethylmaleinimide - SIM 250 mM sucrose, 10 mM imidazole-HCl pH 7.4 - TRIS tris (hydroxymethyl) aminomethane