Time-resolved charge movements in the sarcoplasmatic reticulum Ca-ATPase

Area of Science:

• Biochemistry
• Molecular Biology
• Membrane Transport

Background:

• The sarcoplasmic reticulum Ca-ATPase (SERCA) is vital for muscle contraction by pumping calcium ions.
• Understanding its partial reactions, particularly Ca(2+) translocation, is key to elucidating its function.
• Previous studies have suggested complex conformational changes during the SERCA cycle.

Purpose of the Study:

• To investigate the time-resolved kinetics of the Ca(2+)-translocating partial reaction of SERCA.
• To identify and characterize the cation movements involved in the Ca(2+) transport mechanism.
• To correlate these movements with specific conformational transitions within the enzyme.

Main Methods:

• Utilized ATP-concentration jump experiments initiated by UV light.
• Employed the fluorescent styryl dye 2BITC to detect charge movements in the membrane domain.
• Analyzed pH-dependent kinetics and substrate concentration independence of observed processes.

Main Results:

• Identified two oppositely directed cation movements: Ca(2+) release and H(+) binding.
• Characterized a fast process (30 ms) attributed to the E(1) --> E(2) conformational transition, preceding Ca(2+) release.
• Observed a slow, pH-dependent process (1-3 s) assigned to conformational relaxation (P-E(2)) before H(+) binding.

Conclusions:

• The study elucidates the kinetic steps of Ca(2+) translocation by SERCA.
• Confirms the involvement of distinct conformational changes (E(1) --> E(2) and P-E(2)) in the pump cycle.
• High activation energies suggest these conformational changes are rate-limiting steps.