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Am J Physiol Cell Physiol 255: C694-C698, 1988;
0363-6143/88 $5.00
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AJP - Cell Physiology, Vol 255, Issue 5 C694-C698, Copyright © 1988 by American Physiological Society

ARTICLES

Continuous monitoring of Ca2+ uptake in membrane vesicles with fura-2

M. E. Kargacin, C. R. Scheid and T. W. Honeyman
Department of Physiology, University of Massachusetts Medical School, Worcester 01655.

The Ca2+-selective, fluorescent dye, fura-2, was used to monitor ATP-dependent Ca2+ uptake by membrane vesicles isolated from rabbit skeletal muscle. Micromolar fura-2 concentrations, added outside the vesicles, served as a high-affinity, low-capacity Ca2+ buffer. Changes in fura-2 fluorescence resulted from the decline in free Ca2+ concentration [( Ca2+]free) owing to active Ca2+ accumulation by the vesicles. Ca2+ uptake (delta[Ca2+]total) was calculated from changes in [Ca2+]free and from the Kd value for the fura-2-Ca2+ complex. The velocity of Ca2+ uptake determined in this manner had an apparent [Ca2+]0.5 of approximately 200 nM. The Hill coefficient for dependence of uptake velocity on [Ca2+]free was congruent to 2. Changes in [Ca2+]free and Ca2+ uptake expected for Ca2+ transport by skeletal muscle sarcoplasmic reticulum were determined theoretically from known kinetic parameters and found to be similar to experimental values. This method of directly monitoring Ca2+ uptake can be used to determine the kinetic parameters for Ca2+ transport with small amounts of vesicles and with greater precision than possible with radiometric techniques.


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