In skeletal muscle the activity of ryanodine receptor (RyR) calcium release channels in the sarcoplasmic reticulum is regulated by the dihydropyridine receptor (DHPR) voltage sensors in the t-tubule membrane. Ca2+, Mg2+ and ATP are potent intracellular regulators of RyRs. The effects of these substances on isolated RyRs are well characterised yet it is not clear how they regulate RyR opening under voltage-sensor control. RyRs are activated by μM cytoplasmic Ca2+ and mM ATP while physiological [Mg2+] (∼1 mM) in the cytoplasm fully inhibits them. It is proposed that during muscle contraction, DHPRs transiently relieve Mg2+ inhibition which then permits activation of RyRs by ATP (Lamb et al., 1991).
Mg2+ is thought to inhibit RyRs by binding both to low affinity sites that show little specificity between divalent ions (I-sites) and to high affinity sites for Ca2+ (A-sites) thus preventing Ca2+ from activating the channel (Laver et al., 1997). However, ATP is known to activate RyRs in the absence of cytoplasmic Ca2+ so it is not clear how Mg2+ at the A-sites affects channel opening under physiological conditions. Here we investigate the mechanism of Mg2+ inhibition in the presence of ATP and two drugs, 4,4'-diisothiocyano-stilbene-2,2'-disulfonic acid (DIDS) and ryanodine, which also activate RyRs in the absence of Ca2+.
RyRs were isolated from rabbit skeletal muscle and incorporated into lipid bilayers using standard techniques (O'Neill et al., 2003). Skeletal muscle was removed from dead rabbits. Cytoplasmic solutions contained 250 mM Cs+ (230 mM CsCH3O3S and 20 mM CsCl) 10 mM TES at pH 7.4. Luminal solutions contained 50 mM Cs (30 mM CsCH3O3S and 20 mM CsCl), 10 mM TES, pH 7.4.
DIDS decreased I-site affinity for Mg2+ and Ca2+ by 10 fold and ryanodine abolished binding completely. Cytoplasmic Mg2+ inhibited RyRs via the Ca2+ activation site even in the absence of Ca2+ indicating that Mg2+ inhibition is not merely due to the prevention of Ca2+ binding. In the case of ryanodine modified RyRs, monovalent ions (Cs+) could also activate the channel. RyR activity in the virtual absence of Ca2+ (∼1 nM) was not due to sensitisation of the channel to Ca2+ as previously thought (Du et al., 2001; Masumiya et al., 2001) but was due to Ca2+-independent channel opening by ryanodine. The apparent Mg2+ affinity at the A-site was decreased by cytoplasmic Cs+ and Ca2+ as well as by luminal Ca2+ in a way which suggests that cytoplasmic Mg2+, Cs+ and Ca2+ compete for a site near the cytoplasmic entrance. Ions at this site may progress to the A-site further into the pore. Binding of these ions at the A-site is in competition with luminal Ca2+ and leads to either activation (2 × Cs+ or Ca2+) or inhibition (Mg2+) of RyRs.
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Laver, D.R., T.M. Baynes & A.F. Dulhunty (1997). Magnesium inhibition of ryanodine-receptor calcium channels: evidence for two independent mechanisms. Journal of Membrane Biology, 156: 213-229.
Masumiya, H., Li, P., Zhang, L., & Chen, S.R. (2001) Ryanodine sensitizes the Ca2+ release channel (ryanodine receptor) to Ca2+ activation. Journal of Biological Chemistry, 276: 39727-35.
O'Neill E.R., Sakowska, M.M., & Laver D.R. (2003) Regulation of the calcium release channel from skeletal muscle by suramin and the disulphonate stilbene derivatives DIDS, DBDS, and DNDS. Biophysical Journal, 84: 1-16,