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+]ex-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.
Saiki
Y
Y
Boston Biomedical Research Institute, Harvard Medical School, Boston, Massachusetts 02114, USA.
Ikemoto
N
N
D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D011107:Polylysine / Q000494:pharmacology; D011817:Rabbits; D012430:Ruthenium Red / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000502:physiology; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D019837:Ryanodine Receptor Calcium Release Channel; D012430:Ruthenium Red; C072323:Mag-Fura-2-acetoxymethyl ester; D011107:Polylysine; D002118:Calcium; D016257:Fura-2