Differentiation between Ca2+ transport and ATP-induced Ca2+ binding by sarcoplasmic reticulum

Area of Science:

• Biochemistry
• Cell Biology
• Muscle Physiology

Background:

• Sarcoplasmic reticulum (SR) is crucial for calcium (Ca2+) regulation in muscle cells.
• SR actively accumulates Ca2+ via ATP-dependent mechanisms.
• The precise nature of Ca2+ binding within the SR is not fully understood.

Purpose of the Study:

• To investigate the distinct fractions of Ca2+ accumulated by isolated skeletal muscle sarcoplasmic reticulum.
• To determine the role of ATP and ATP hydrolysis in the accumulation of membrane-bound Ca2+.
• To differentiate between intravesicular and membrane-bound Ca2+ fractions.

Main Methods:

• Isolation of sarcoplasmic reticulum from skeletal muscle.
• Measurement of Ca2+ retention in the presence and absence of the Ca2+ ionophore X-537A.
• Utilizing EGTA to distinguish intravesicular Ca2+.

Main Results:

• Sarcoplasmic reticulum Ca2+ accumulation consists of intravesicular free Ca2+ and membrane-bound Ca2+.
• Both fractions are ATP-dependent.
• The Ca2+ ionophore X-537A revealed that ATP-induced Ca2+ accumulation can be entirely membrane-bound.
• Membrane-bound Ca2+ was quantified at approximately 30 nmol/mg protein.
• EGTA impermeability confirmed the distinctness of membrane-bound Ca2+.

Conclusions:

• Sarcoplasmic reticulum actively accumulates Ca2+ into at least two distinct fractions: intravesicular and membrane-bound.
• ATP plays a role in both Ca2+ fractions, with evidence suggesting it induces membrane binding.
• This membrane-bound Ca2+ is a significant component of SR Ca2+ accumulation.