Sarcoplasmic reticulum interacts with the Ca(2+) indicator precursor fura-2-am

Area of Science:

• Biochemistry
• Cell Biology
• Physiology

Background:

• Fura-2 is a widely used fluorescent indicator for measuring intracellular calcium (Ca2+).
• Fura-2-acetoxymethyl ester (fura-2-am) is the cell-permeant precursor used for fura-2 loading.
• Sarcoplasmic reticulum (SR) is critical for calcium regulation in muscle cells.

Purpose of the Study:

• To investigate the interaction between fura-2-am and isolated skeletal muscle sarcoplasmic reticulum (SR) vesicles.
• To characterize the modifications that fura-2-am undergoes upon exposure to SR.
• To assess the implications of these modifications for intracellular calcium measurements.

Main Methods:

• Incubation of fura-2-am with isolated skeletal muscle SR vesicles.
• Spectrofluorometric analysis of modified fura-2-am.
• Characterization of fura-2-am binding to SR membranes.
• Enzymatic conversion of modified fura-2-am to fura-2.

Main Results:

• Fura-2-am is modified by SR, resulting in enhanced fluorescence.
• The modified fura-2-am exhibits reduced Ca2+ sensitivity.
• The modified fura-2-am partially binds to the SR membrane.
• SR-induced modification involves selective enzymatic hydrolysis of fura-2-am ester groups.

Conclusions:

• Skeletal muscle SR selectively modifies fura-2-am via enzymatic hydrolysis.
• This modification alters fura-2-am's fluorescent properties and Ca2+ sensitivity.
• The SR-mediated modification of fura-2-am can introduce complications in accurate in vivo intracellular free [Ca2+] measurements.