Fluorescence probe study of the lumenal Ca2+ of the sarcoplasmic reticulum vesicles during Ca2+ uptake and Ca2+ release

A limited amount of information is available about the lumenal Ca2+ kinetics of the sarcoplasmic reticulum (SR). Incubation of mag-fura-2AM permitted to incorporate a sufficient amount of the probe into the SR vesicles, as determined by Mn2+ quenching. Rapid changes in the lumenal [Ca2+] ([Ca2+](lum)) during Ca2+ uptake and release could be monitored by following the signal derived from the lumenal probe while clamping the extra-vesicular Ca2+ ([Ca2+](ex)) at various desired levels with a BAPTA/Ca buffer. Changes in the [Ca2+](lum) during uptake and release show the characteristics intrinsic to the SR Ca2+ pump (the [Ca2+](exm) dependence of the activation and inhibition by thapsigargin) and the Ca2+ release channel (blocking by ruthenium red), respectively. A new feature revealed by the [Ca2+](lum) measurement is that during the uptake reaction the free [Ca2+](lum) showed a significant oscillation. Several pieces of evidence suggest that this is due to some interactions between the Ca2+ pump and lumenal proteins. (C) 1997 Academic Press.