Involvement of Mg²⁺ in terminating Ca²⁺ release in cultured rat skeletal muscle

Abstract

Combined patch-clamp and fura-2 measurements were performed to investigate the mechanism that terminates Ca 2+ release in rat skeletal myoballs. When cells were intracellularly perfused with solution containing 1 mM free Mg 2+, the caffeine (10 mM)-induced Ca 2÷ transient was abruptly terminated by membrane repolarization (-70 mV). With low intracellular Mg 2÷ (e.g. 50 pM) perfusion, however, repolarization failed to terminate the caffeine transient. The results show that intracellular Mg 2÷ is necessary for repolarization-induced closing of the Ca 2+ release channel.Key words: Skeletal muscle; Excitation-contraction coupling; Ryanodine receptor; Ca 2+ release channel; Caffeine; Fura-2 is estimated to be around 1 mM [12][13][14][15][16] and appears to be maintained rather constant [12,13,15,16]. The physiological [Mg2+]i effectively inhibited Ca 2÷ release channel activity in cut muscle fibers [17], fragmented and reconstituted SR vesicles [18][19][20], and reconstituted RyRs in lipid bilayer membranes [20]. Furthermore, 10 mM Mg 2+ completely inhibited depolarization-induced contraction in skinned fibers with sealed Ttubules [21,22]. Therefore, Mg 2÷ binding to the RyR induced by repolarization may be a possible step in closing the release channel, as was recently proposed by . The present study was conducted to further address this idea with regard to the RISC phenomenon. Materials and methods

RISC (Repolarization-induced stop of caffeine-contracture) is not due to store depletion in cultured murine skeletal muscle

“…Sylgardcoated patch-pipettes had resistances between 1.3 and 2.2 M52 after filling with internal solution which contained (in mM): 145 Csglutamate, 0.1-0.2 Fura-2 (potassium salt), 7 MgATP, 1 Na2ATP, 6 NaCI, and 10 Hepes-CsOH (pH=7.2). The free-Mg 2 concentration of this solution was calculated to be 0.6 mM, from an assumed apparent affinity constant of 6.9x 10 3 M-I for Mgt binding to ATP [5]. In some experiments in which 8 MgATP and 0 Na2ATP were used, [Mg 2+], was around 1 mM.…”

“…The procedures for preparation of primary cultures of rat and mouse myoballs were essentially as described previously [4,5]. Skeletal myotubes were cultured at least for 5 days after initiation of fusion because skeletal type E-C coupling predominates over cardiac type E-C coupling at later stages of cell culture [7].…”

“…Therefore, interactions between VOCR and CICR open modes may be observable in native mammalian skeletal muscle. In the presence of physiological intracellular Mgt concentration ([Mg 2+] ;), caffeine-activated Cat transient (Calf) can be terminated by membrane repolarization at all stages of the CafT [4,5]. This phenomenon, termed RISC (repolarizationinduced stop of caffeine-contracture), was present only when the release channels appeared to be functionally in a "voltage-activated" state [4].…”

Activation of Ca2+ release by caffeine and voltage in frog skeletal muscle.

“…Most of the releasable Ca2+ is bound to calsequestrin, which is localized to the terminal cisternae (Meissner, 1975;Franzini-Armstrong, Kenney & Varriano-Marston, 1987 Justifiably, Suda & Penner (1994) and Suda (1995) inferred from their observation that the voltage-sensing device in the T tubule dominates all release channels, even if they are also susceptible to CICR. Their model also includes elements of hysteresis (repolarization rendered the release channel insensitive to caffeine, even though depolarization was not necessary to make it sensitive).…”

Involvement of dihydropyridine receptors in terminating Ca2+ release in rat skeletal myotubes.

“…The present study extends the previous study by investigating the effect of DHP antagonists on RISC. Some aspects of this work have been presented in abstract form Suda, 1995).…”