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Magnesium inhibition of skeletal muscle ryanodine receptors modified by DIDS, ryanodine and ATP

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.

Du, G.G., Guo, X, Khanna, V.K. & MacLennan, D.H. (2001) Ryanodine sensitizes the cardiac Ca²⁺ release channel (ryanodine receptor isoform 2) to Ca²⁺ activation and dissociates as the channel is closed by Ca²⁺ depletion. Proceedings of the National Academy of Sciences of the United States of America, 98: 13625-30.

Lamb, G.D. & D.G. Stephenson (1991). Effect of Mg²⁺ on the control of Ca²⁺ release in skeletal muscle fibres of the toad. Journal of Physiology, 434: 507-528.

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,