Kinetics of rapid calcium release by sarcoplasmic reticulum. Effects of calcium, magnesium, and adenine nucleotides

Abstract

A radioisotope flux-rapid-quench-Millipore filtration method is described for determining the effects of Ca2+, adenine nucleotides, and Mg2+ on the Ca2+ release behaviour of "heavy" sarcoplasmic reticulum (SR) vesicles. Rapid 45Ca2+ efflux from passively loaded vesicles was blocked by the addition of Mg2+ and ruthenium red. At pH 7 and 10(-9) M Ca2+, vesicles released 45Ca2+ with a low rate (k = 0.1 s-1). An increase in external Ca2+ concentration to 4 microM or the addition of 5 mM ATP or the ATP analogue adenosine 5'-(beta,gamma-methylenetriphosphate) (AMP-PCP) resulted in intermediate 45Ca2+ release rates. The maximal release rate was observed in media containing 4 microM Ca2+ and 5 mM AMP-PCP and had a first-order rate constant of 30-100 s-1. Mg2+ partially inhibited Ca2+- and nucleotide-induced 45Ca2+ efflux. In the absence of AMP-PCP, 45Ca2+ release was fully inhibited at 5 mM Mg2+ or 5 mM Ca2+. The composition of the release media was systematically varied, and the flux data were expressed in the form of Hill equations. The apparent n values of activation of Ca2+ release by ATP and AMP-PCP were 1.6-1.9. The Hill coefficient of Ca2+ activation (n = 0.8-2.1) was dependent on nucleotide and Mg2+ concentrations, whereas the one of Mg2+ inhibition (n = 1.1-1.6) varied with external Ca2+ concentration. These results suggest that heavy SR vesicles contain a "Ca2+ release channel" which is capable of conducting Ca2+ at rates comparable with those found in intact muscle. Ca2+, AMP-PCP (ATP), and Mg2+ appear to act at noninteracting or interacting sites of the channel.

Allosterically coupled calcium and magnesium binding sites are unmasked by ryanodine receptor chimeras

“…1D. Theses results provide more direct biochemical evidence in support of the dual mechanism of Mg 2+ inhibition originally proposed by Meissner and Laver based on experiments with WT RyR [4], [16] and [32].…”

“…Ca 2+ and Mg 2+ titrations in [ 3 H]ryanodine binding and Ca 2+ release experiments have revealed Hill coefficients >1, indicating multiple, coordinated regulation of CICR channels by multiple cation binding sites [15]. Mg 2+ inhibition studied with channels reconstituted in lipid bilayer membranes [16] and Ca 2+ release from SR membrane vesicles [4] and [11] suggest dual mechanisms of Mg 2+ inhibition through competition with Ca 2+ for high affinity (H) activation sites and binding at low affinity (L) cation inhibition sites. At physiological concentrations, free Mg 2+ (1-2mM) is likely to occupy both H and L sites, providing a basal level of WT RyR inhibition that must be overcome for EC-coupling to occur [17] and [18].…”

Thimerosal modulates the agonist‐specific cytosolic Ca2+ oscillatory patterns in single pancreatic acinar cells of mouse

“…IICR is inhibited by heparin, whereas this s~,bstance has no effect on the CICR [20,21]. CICR is inhibit~d by ruthenium red, which has no effect on the IICR [20,22]. To test whether thimerosal mimicked the agonist-spec;fic cytosolic Ca 2÷ oscillation patterns mediated by IICR or (ICR, we have employed intracellularly heparin and ruthenium red.…”

Intracellular aluminium inhibits acetylcholine‐ and caffeine‐evoked Ca2+ mobilization

“…It is known that the Ca*+ release channel in muscle which is specifically activated by micromolar or submicromolar Ca*+ concentrations [20,21] can be inhibited by millimolar concentrations of either Mg*+ or Ca*+ [21]. This inhibition is therefore not specific and may well be exerted also by other ions such as A13+.…”