Bozena Antoniu

Identification of Calcium Release-triggering and Blocking Regions of the II-III Loop of the Skeletal Muscle Dihydropyridine Receptor

In an attempt to identify and characterize functional domains of the rabbit skeletal muscle dihydropyridine receptor α subunit II-III loop, we synthetized several peptides corresponding to different regions of the loop: peptides A, B, C, C1, C2, D (cf. Fig. 1). Peptide A (Thr-Leu) activated ryanodine binding to, and induced Ca release from, rabbit skeletal muscle triads, but none of the other peptides had such effects. Peptide A-induced Ca release and activation of ryanodine binding were partially suppressed by an equimolar concentration of peptide C (Glu-Pro) but were not affected by the other peptides. These results suggest that the short stretch in the II-III loop, Thr-Leu, is responsible for triggering SR Ca release, while the other region, Glu-Pro, functions as a blocker of the release trigger. A hypothesis is proposed to account for how these subdomains interact with the sarcoplasmic reticulum Ca release channel protein during excitation-contraction coupling.

Kinetic studies of Ca2+ release from sarcoplasmic reticulum of normal and malignant hyperthermia susceptible pig muscles.

The time-course of Ca2+ release from sarcoplasmic reticulum isolated from muscles of normal pigs and those of pigs susceptible to malignant hyperthermia were investigated using stopped-flow spectrophotometry and arsenazo III as a Ca2+ indicator. Several methods were used to trigger Ca2+ release: (a) addition of halothane (e.g., 0.2 mM); (b) an increase of extravesicular Ca2+ concentration ([Ca2+0]); (c) a combination of (a) and (b), and (d) replacement of ions (potassium gluconate with choline chloride) to produce membrane depolarization. The initial rates of Ca2+ release induced by either halothane or Ca2+ alone, or both, are at least 70% higher in malignant hyperthermic sarcoplasmic reticulum than in normal. The amount of Ca2+ released by halothane at low [Ca2+0] in malignant hyperthermic sarcoplasmic reticulum is about twice as large as in normal sarcoplasmic reticulum. Membrane depolarization led to biphasic Ca2+ release in both malignant hyperthermic and normal sarcoplasmic reticulum, the rate constant of the rapid phase of Ca2+ release induced by membrane depolarization being significantly higher in malignant hyperthermic sarcoplasmic reticulum (k = 83 s-1) than in normal (k = 37 s-1). Thus, all types of Ca2+ release investigated (a, b, c and d) have higher rates in malignant hyperthermic sarcoplasmic reticulum than normal sarcoplasmic reticulum. These results suggest that the putative Ca2+ release channels located in the sarcoplasmic reticulum are altered in malignant hyperthermic sarcoplasmic reticulum.

Inhibitors of Ca2+ release from the isolated sarcoplasmic reticulum. I. Ca2+ channel blockers

The effects of Ruthenium red and tetracaine, which inhibit Ca2+-induced Ca2+ release from the isolated sarcoplasmic reticulum (e.g., Ohnishi, S.T. (1979) J. Biochem. (Tokyo) 86, 1147-1150), on several types of Ca2+ release in vitro were investigated. Ca2+ release was triggered by several methods: (1) addition of quercetin or caffeine, (2) Ca2+ jump, and (3) replacement of potassium gluconate with choline chloride to produce membrane depolarization. The time-course of Ca2+ release was monitored using stopped-flow spectrophotometry and arsenazo III as a Ca2+ indicator. Ruthenium red inhibited all of these types of Ca2+ release with the same concentration for half-inhibition C1/2 = 0.08-0.10 microM. Similarly, tetracaine inhibited these types of Ca2+ release with C1/2 = 0.07-0.11 mM. Procaine also inhibits both types of Ca2+ release induced by method 2 and 3 with C1/2 = 0.67-1.00 mM. These results suggest that Ruthenium red, tetracaine and procaine interfere with a common mechanism of the different types of Ca2+ release. On the basis of several pieces of evidence we propose that Ruthenium red and tetracaine block the Ca2+ channel of sarcoplasmic reticulum.

Effects of anti-triadin antibody on Ca2+ release from sarcoplasmic reticulum

The monoclonal antibody, mAb GE 4.90, raised against triadin, a 95 kDa protein of sarcoplasmic reticulum (SR), inhibits the slow phase of Ca2+ release from SR following depolarization of the T‐tubule moiety of the triad. The antibody has virtually no effect on the fast phase of depolarization‐induced Ca2+ release nor on caffeine‐induced Ca2+ release. Since the slow phase of depolarization‐induced Ca2+ release is also inhibited by dihydropyridines (DHP), these results suggest that triadin may be involved in the functional coupling between the DHP receptor and the SR Ca2+ channel.

[34] Measurement of calcium release in isolated membrane systems: Coupling between the transverse tubule and sarcoplasmic reticulum

Publisher Summary This chapter focuses on the measurement of calcium release in isolated membrane systems. According to the generally accepted view concerning the excitation-contraction coupling mechanism, excitation initiated at the cell surface is propagated to the inside of the muscle cell through the transverse tubule membrane system (T tubule). The depolarization is communicated to the sarcoplasmic reticulum (SR), which in turn releases the Ca 2+ accumulated in the SR lumen. Binding of the released Ca 2+ to the Ca 2+ -binding subunit of troponin located in the thin filaments releases the inhibition of the interaction between actin and myosin, which is manifested in muscle contraction. The most appropriate method to optically monitor the time course of rapid Ca 2+ release appears to be dual-wavelength stopped-flow spectrophotometry. A number of different methods that induce Ca 2+ release from the isolated SR have been described. The chapter discusses methods that permit one to induce and monitor the so-called depolarization-induced Ca 2+ release. As physiological Ca 2+ release is expected to be very rapid (t l/2 = several milliseconds) the method for monitoring Ca 2+ release should have a time resolution of the order of a few milliseconds or less.

Neomycin: A novel potent blocker of communication between T-tubule and sarcoplasmic reticulum

Ca2+ release from the sarcoplasmic reticulum (SR) was induced in isolated triads by direct stimulation of the SR moiety by polylysine, or stimulation via chemical depolarization of the transverse tubule (T‐tubule) moiety. Polylysine‐induced release was blocked by neomycin with an IC50 (the concentration for half‐maximal inhibition) of O.3, μM. However, the IC50 for neomycin block of depolarization‐induced Ca2+ release sharply decreased in a voltage‐dependent fashion, and it was 5.3 nM at a maximal extent of T‐tubule depolarization. These results suggest that the high affinity binding of neomycin to the triad leads to the specific blocking of the signal transmission from T‐tubule to SR.

Characterization of depolarization-induced calcium release from sarcoplasmic reticulum in vitro with the use of membrane potential probe

In the triad, the complex of transverse (T) tubule and sarcoplasmic reticulum (SR) Ca2+ release is induced from SR by mediation of the T-tubule. We report here evidence that this Ca2+ release is produced by depolarization of the T-tubule moiety. Thus, we found that the amount of [14C]SCN- taken up by T-tubules and triads (but not that by SR) increased upon incubation with (K, Na) gluconate, Mg ATP, indicating that the T-tubule was polarized making the lumenal side (equivalent to the extracellular side of an intact muscle fiber) more positive. Upon mixing with choline chloride, the procedure to induce Ca2+ release, [14C]SCN- uptake decreased, indicating that the T-tubule became depolarized. Activation of the T-tubule polarization by Na+ and prevention of it by digoxin [inhibitor of the (Na+, K+) pump], respectively, led to activation and inhibition of choline chloride-induced SR Ca2+ release.