Fluorescence Lifetime Spectroscopy Reveals Distinct Allosteric Mechanisms of SERCA Inhibitors

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• The calcium pump SERCA (sarcoplasmic reticulum Ca2+-ATPase) is crucial for muscle function.
• Understanding SERCA inhibition mechanisms is key to developing targeted therapies.
• Existing structural studies show similar effects of thapsigargin (TG) and cyclopiazonic acid (CPA) on SERCA states.

Purpose of the Study:

• To characterize the differential effects of TG and CPA on SERCA conformational dynamics using TCSPC spectroscopy.
• To elucidate the distinct mechanisms by which these chemically different inhibitors modulate SERCA function.
• To investigate how TG and CPA affect Ca2+ and ATP-dependent transitions in SERCA.

Main Methods:

• Development and application of time-correlated single photon counting (TCSPC) spectroscopy.
• Characterization of SERCA inhibitors thapsigargin (TG) and cyclopiazonic acid (CPA).
• Analysis of SERCA conformational dynamics and modulation by inhibitors.

Main Results:

• TCSPC revealed fundamentally distinct inhibition mechanisms for TG and CPA, despite structural similarities.
• TG traps SERCA in a nonproductive 'dead-end' state, blocking Ca2+ and ATP-dependent transitions.
• CPA attenuates Ca2+ and nucleotide-dependent transitions, reducing the closed, activation-associated state population in a concentration-dependent manner.
• CPA's effect on Ca2+ affinity is ATP-dependent, suggesting an allosteric mechanism.

Conclusions:

• TCSPC spectroscopy can resolve subtle mechanistic differences between structurally similar SERCA inhibitors.
• TG and CPA employ distinct allosteric strategies to inhibit SERCA.
• These findings provide a framework for understanding how different ligands modulate SERCA function and conformational dynamics.