Bastadins relate ryanodine-sensitive and -insensitive Ca2+ efflux pathways in skeletal SR and BC3H1 cells

Abstract

Bastadins potently interact with the FK-506-binding protein of 12 kDa (FKBP12)-ryanodine receptor (Ry1R) complex in skeletal muscle to enhance a high-affinity ryanodine binding conformation (M. M. Mack, T. F. Molinski, E. D. Buck, and I. N. Pessah. J. Biol. Chem. 269: 23236-23249, 1994). Bastadins are used to examine the relationship between ryanodine-sensitive and ryanodine-insensitive Ca2+ efflux pathways that coexist in junctional sarcoplasmic reticulum (SR) vesicles from rabbit skeletal muscle and differentiated BC3H1 cells. Complete block of caffeine-sensitive Ca2+ channels with micromolar ryanodine or ruthenium red does not alter the steady-state loading capacity of SR. Inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps with thapsigargin unmasks a ryanodine- and ruthenium red-insensitive Ca2+ efflux pathway. Bastadin 5 alone does not inhibit Ca2+ efflux unmasked by inhibition of SERCA pumps, but, in combination with blocking concentrations of ryanodine or ruthenium red, it eliminates the ryanodine-insensitive Ca2+ "leak" and enhances steady-state loading capacity of SR vesicles approximately 2.5-fold. These actions of bastadins occur in the same concentration range that enhances the number of high-affinity binding sites for [3H]ryanodine (50% effective concentration of approximately 2 microM). Similar effects on SR Ca2+ transport are found with FK-506 and ryanodine in combination. Block of Ry1R in intact BC3H1 cells with ryanodine does not eliminate the prominent Ca2+ leak unmasked by thapsigargin. A membrane-permeant mixture of bastadins in combination with ryanodine nearly eliminates the Ca2+ leak unmasked by thapsigargin, even though the Ca2+ stores are replete. The requirement of both a known Ry1R blocker and bastadins in combination provides a pharmacological link between ryanodine-sensitive Ca2+ channels and ryanodine-insensitive leak pathways in isolated junctional SR and BC3H1 cells. Together, these results strongly suggest that bastadins, through their modulatory actions on the FKBP12-Ry1R complex, convert ryanodine-insensitive leak states into ryanodine-sensitive channels that recognize [3H]ryanodine with high affinity.

Malignant hyperthermia susceptibility arising from altered resting coupling between the skeletal muscle L-type Ca 2+ channel and the type 1 ryanodine receptor

“…4). This leak pathway in dysgenic myotubes appears to be distinct from the conventional activated state of RyR1 because it is blocked by bastadin 5 (or bastadin 5 in the presence of ryanodine) but not by ryanodine alone (32,33); bastadin 5 (B5) application also causes a partial reduction in resting myoplasmic Ca 2+ in dysgenic myotubes expressing Ca V 1.1 R174W ( Table 1). As shown in Fig.…”

Gene Dose Influences Cellular and Calcium Channel Dysregulation in Heterozygous and Homozygous T4826I-RYR1 Malignant Hyperthermia-susceptible Muscle

“…As for the mechanism by which the T4826I mutation causes RyR1 dysfunction, two possibilities are apparent: 1) impairment of the strong negative feedback regulation provided by protein-protein interactions within the junctional Ca 2ϩ release unit, especially the DHPR (16), and 2) increased SR Ca 2ϩ leak, possibly mediated by the RyR1 leak conformation (58). Mutation of the ␣-helical conformation of the S4-S5 linker may be sufficient to impair both of these regulatory mechanism (53).…”

Orthograde dihydropyridine receptor signal regulates ryanodine receptor passive leak

“…This ryanodine-insensitive conformation is distinct from the actively gating ryanodine-sensitive conformation that is involved in excitation-contraction coupling (10,18). Bastadin 5 (B5), a brominated macrocyclic derivative of dityrosine isolated from the marine sponge Iathella basta (19), interacts with RyR1, modulating RyR1 gating behavior in a FKBP12-dependent manner.…”

“…The comparison of the passive Ca 2+ release rate in light and heavy SR fraction vesicles (LSR and HSR, respectively) shows that the rate in HSR is at least fourfold faster than in LSR membranes (23). This observation is in good agreement with the preferential location of RyR1 in the terminal cisternae of the SR and HSR vesicles, suggesting that RyR1 could be a significant contributor to the passive Ca 2+ release from the SR. B5, an agent that directly interacts with RyR1, is able to reduce passive leak (18) and increase SR filling capacity (19 ] rest was measured as described in Materials and Methods. The measurements were done in WT (n = 12) and dysgenic (MDG) myotubes (n = 11) in control conditions, treated with 5 μM BTP2 alone (WT n = 10, dysgenic n = 11), B5 alone (WT n = 17, dysgenic n = 18), or the combination of both drugs (B5+BTP2, WT n = 10, dysgenic n = 10).…”