Halothane and cyclopiazonic acid modulate Ca-ATPase oligomeric state and function in sarcoplasmic reticulum

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Sarcoplasmic reticulum Ca-ATPase (SR Ca-ATPase) is crucial for muscle contraction.
• Cyclopiazonic acid (CPA) is a known inhibitor of Ca-ATPase.
• Understanding CPA's mechanism requires investigating its effects on enzyme structure and conformation.

Purpose of the Study:

• To investigate the impact of CPA and halothane on Ca-ATPase enzymatic activity, oligomeric state, and conformational equilibrium.
• To elucidate the relationship between CPA-induced structural changes and enzyme inhibition.
• To determine how halothane and solubilization affect CPA's interaction with Ca-ATPase.

Main Methods:

• Enzyme kinetics to assess CPA inhibition and ATP concentration dependence.
• Time-resolved phosphorescence anisotropy to quantify Ca-ATPase monomer, dimer, and aggregate fractions.
• Fluorescence spectroscopy (FITC) and enzyme phosphorylation assays to monitor conformational states (E1, E2, E2-P).

Main Results:

• CPA inhibits Ca-ATPase activity competitively with ATP and increases the proportion of Ca-ATPase dimers and aggregates.
• Halothane or detergent solubilization reduces CPA inhibition and favors the monomeric state.
• CPA stabilizes the E2 conformation, an effect partially reversed by halothane and solubilization.

Conclusions:

• CPA inhibits Ca-ATPase partly by stabilizing dimers/oligomers and the E2 conformation.
• Halothane and solubilization modulate CPA's effects by favoring monomers and other conformations.
• These findings provide insights into Ca-ATPase regulation and CPA's inhibitory mechanism.