Unravelling the Interaction of Thapsigargin with the Conformational States of Ca2+-ATPase from Skeletal Sarcoplasmic Reticulum

Fortea, Maria-Isabel; Soler, Fernando; Fernández-Belda, Francisco

doi:10.1074/jbc.m103949200

Cited by 13 publications

(12 citation statements)

References 26 publications

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“…The final equilibrium of Ca 2ϩ and Mic binding to the enzyme is independent of the order of addition, although the slow Ca 2ϩ binding transition in the presence of Mic is responsible for the distinct behavior of the Ca 2ϩ -bound and Ca 2ϩ -free enzymatic forms that we observe. Stabilization of the Ca 2ϩ -free enzyme is the main effect of clotrimazole (35) and other high-affinity inhibitors of the enzyme (14,31,37).…”

Section: Discussionmentioning

confidence: 99%

Inhibition of sarcoplasmic reticulum Ca²⁺-ATPase by miconazole

Lax

Soler

Fernández-Belda

2002

American Journal of Physiology-Cell Physiology

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The inhibition of sarcoplasmic reticulum Ca2+-ATPase activity by miconazole was dependent on the concentration of ATP and membrane protein. Half-maximal inhibition was observed at 12 microM miconazole when the ATP concentration was 50 microM and the membrane protein was 0.05 mg/ml. When ATP was 1 mM, a low micromolar concentration of miconazole activated the enzyme, whereas higher concentrations inhibited it. A qualitatively similar response was observed when Ca2+ transport was measured. Likewise, the half-maximal inhibition value was higher when the membrane concentration was raised. Phosphorylation studies carried out after sample preequilibration in different experimental settings shed light on key partial reactions such as Ca2+ binding and ATP phosphorylation. The miconazole effect on Ca2+-ATPase activity can be attributed to stabilization of the Ca2+-free enzyme conformation giving rise to a decrease in the rate of the Ca2+ binding transition. The phosphoryl transfer reaction was not affected by miconazole.

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Section: Discussionmentioning

confidence: 99%

Inhibition of sarcoplasmic reticulum Ca²⁺-ATPase by miconazole

Lax

Soler

Fernández-Belda

2002

American Journal of Physiology-Cell Physiology

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“…Samples of SR vesicles as prepared (native vesicles) or in the presence of Ca 2+ ionophore A23187 (leaky vesicles) were used. SERCA1a is by far the most abundant protein in the microsomal preparation from rabbit skeletal muscle [26] and the only one bearing high affinity Ca 2+ sites. The experimental protocols described in this study, i.e., rates of active Ca 2+ transport and Ca 2+ -dependent ATP hydrolysis, Ca 2+ binding to the high affinity transport sites and phosphoenzyme (EP) formed from ATP in the presence of Ca 2+ have been successfully applied by others for the functional characterization of SERCA using the abovementioned microsomal fraction [27].…”

Section: Methodsmentioning

confidence: 99%

Inhibition Mechanism of the Intracellular Transporter Ca2+-Pump from Sarco-Endoplasmic Reticulum by the Antitumor Agent Dimethyl-Celecoxib

et al. 2014

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Dimethyl-celecoxib is a celecoxib analog that lacks the capacity as cyclo-oxygenase-2 inhibitor and therefore the life-threatening effects but retains the antineoplastic properties. The action mechanism at the molecular level is unclear. Our in vitro assays using a sarcoplasmic reticulum preparation from rabbit skeletal muscle demonstrate that dimethyl-celecoxib inhibits Ca2+-ATPase activity and ATP-dependent Ca2+ transport in a concentration-dependent manner. Celecoxib was a more potent inhibitor of Ca2+-ATPase activity than dimethyl-celecoxib, as deduced from the half-maximum effect but dimethyl-celecoxib exhibited higher inhibition potency when Ca2+ transport was evaluated. Since Ca2+ transport was more sensitive to inhibition than Ca2+-ATPase activity the drugs under study caused Ca2+/Pi uncoupling. Dimethyl-celecoxib provoked greater uncoupling and the effect was dependent on drug concentration but independent of Ca2+-pump functioning. Dimethyl-celecoxib prevented Ca2+ binding by stabilizing the inactive Ca2+-free conformation of the pump. The effect on the kinetics of phosphoenzyme accumulation and the dependence of the phosphoenzyme level on dimethyl-celecoxib concentration were independent of whether or not the Ca2+–pump was exposed to the drug in the presence of Ca2+ before phosphorylation. This provided evidence of non-preferential interaction with the Ca2+-free conformation. Likewise, the decreased phosphoenzyme level in the presence of dimethyl-celecoxib that was partially relieved by increasing Ca2+ was consistent with the mentioned effect on Ca2+ binding. The kinetics of phosphoenzyme decomposition under turnover conditions was not altered by dimethyl-celecoxib. The dual effect of the drug involves Ca2+-pump inhibition and membrane permeabilization activity. The reported data can explain the cytotoxic and anti-proliferative effects that have been attributed to the celecoxib analog. Ligand docking simulation predicts interaction of celecoxib and dimethyl-celecoxib with the intracellular Ca2+ transporter at the inhibition site of hydroquinones.

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“…In this regard, RR was as effective as thapsigargin, a drug often used to evoke Ca 2+ translocation [18]. (B) RR, perhaps because of the multiple oxidation states of ruthenium, was a potent ROS scavenger, and could protect cells from adverse effects of PDT.…”

Section: Apoptotic Responses To Pdt: the Role Of Bcl-2mentioning

confidence: 99%

Death pathways associated with photodynamic therapy

Kessel

2006

Medical Laser Application

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When the mitochondria and/or the endoplasmic reticulum were targeted by photodynamic therapy, photodamage to the anti-apoptotic protein Bcl-2 was observed. This led to an apoptotic outcome if that death pathway was available. Lysosomal photodamage ultimately resulted in activation of the pro-apoptotic protein Bid, also leading to apoptosis. Photodamage to the plasma membrane was associated with migration of sensitizers to the cytosol and procaspase photodamage, with apoptosis impaired. Where apoptosis was unavailable because of lack of necessary components of the program, an autophagic outcome has been observed. It is also clear that autophagy can occur along with apoptosis as a PDT response, and may play a role in immunologic responses to photodamaged tumor cells.

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Unravelling the Interaction of Thapsigargin with the Conformational States of Ca2+-ATPase from Skeletal Sarcoplasmic Reticulum

Cited by 13 publications

References 26 publications

Inhibition of sarcoplasmic reticulum Ca²⁺-ATPase by miconazole

Inhibition of sarcoplasmic reticulum Ca²⁺-ATPase by miconazole

Inhibition Mechanism of the Intracellular Transporter Ca2+-Pump from Sarco-Endoplasmic Reticulum by the Antitumor Agent Dimethyl-Celecoxib

Death pathways associated with photodynamic therapy

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Unravelling the Interaction of Thapsigargin with the Conformational States of Ca2+-ATPase from Skeletal Sarcoplasmic Reticulum

Cited by 13 publications

References 26 publications

Inhibition of sarcoplasmic reticulum Ca2+-ATPase by miconazole

Inhibition of sarcoplasmic reticulum Ca2+-ATPase by miconazole

Inhibition Mechanism of the Intracellular Transporter Ca2+-Pump from Sarco-Endoplasmic Reticulum by the Antitumor Agent Dimethyl-Celecoxib

Death pathways associated with photodynamic therapy

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Inhibition of sarcoplasmic reticulum Ca²⁺-ATPase by miconazole

Inhibition of sarcoplasmic reticulum Ca²⁺-ATPase by miconazole