Spectroscopic determination of sarcoplasmic reticulum Ca2+ uptake and Ca2+ release.
In this report we describe the application of spectroscopic methods to the study of Ca2+ release by isolated native sarcoplasmic reticulum (SR) membranes from rabbit skeletal muscle. To date, dual-wavelength spectroscopy of arsenazo III and antipyrylazo III difference absorbance have been the most common spectroscopic methods for the assay of SR Ca2+ transport. The utility of these methods is the ability to manipulate intraluminal Ca2+ loading of SR vesicles. These methods have also been useful for studying the effect of both agonists and antagonists upon SR Ca2+ release and Ca2+ uptake. In this study, we have developed the application of Calcium Green-2, a long-wavelength excitable fluorescent indicator, for the study of SR Ca2+ uptake and release. With this method we demonstrate how ryanodine receptor Ca2+ channel opening and closing is regulated in a complex manner by the relative distribution of Ca2+ between extraluminal and intraluminal Ca2+ compartments. Intraluminal Ca2+ is shown to be a key regulator of Ca2+ channel opening. However, these methods also reveal that the intraluminal Ca2+ threshold for Ca2+-induced Ca2+ release varies as a function of extraluminal Ca2+ concentration. The ability to study how the relative distribution of a finite pool of Ca2+ across the SR membrane influences Ca2+ uptake and Ca2+ release may be useful for understanding how the ryanodine receptor is regulated, in vivo.
Gilchrist
J S
JS
Department of Oral Biology, University of Manitoba, Winnipeg, Canada.
Palahniuk
C
C
Bose
R
R
D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D005456:Fluorescent Dyes; D018482:Muscle, Skeletal / Q000378:metabolism; D009930:Organic Chemicals; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D005456:Fluorescent Dyes; D009930:Organic Chemicals; C071336:calcium green; D002118:Calcium