Programme Contents

Role of phosphorylation in the Ca²⁺ regulation of Ca²⁺ release channels in skeletal muscle

During excitation contraction coupling, the action potential depolarises the voltage-dependent L-type Ca²⁺ channels in the sarcolemma and T-tubules, leading to a release of Ca²⁺ from the sarcoplasmic reticulum (SR, the main intracellular Ca²⁺ store) and a rise in cytoplasmic Ca²⁺ which stimulates muscle contraction. In skeletal muscle, the Ca²⁺ release channels in the SR (RyR1 isoform) are stimulated via a direct protein-protein interaction with the L-type Ca²⁺ channels whereas in cardiac muscle it is the inflow of Ca²⁺ into the cytoplasm through the L-type Ca²⁺ channels that activates the cardiac ryanodine receptors (RyR2). Cardiac and skeletal RyR isoforms are modulated differently by intracellular Ca²⁺. Cardiac RyRs have a complete reliance on Ca²⁺ for opening in which Ca²⁺ in the cytoplasm and SR lumen produce a synergistic activation of the channel (Laver & Honen, 2008). On the other hand, skeletal RyRs can open in the complete absence of Ca²⁺ or any other activating ligand and intracellular Mg²⁺ is required to inhibit RyR1 and prevent SR Ca²⁺ release during muscle relaxation (Laver et al., 2004). In this study, we explore the effect of endogenous phosphorylation of RyR1 on its ability to open in the absence of Ca²⁺.

RyR1 was isolated from rabbit skeletal muscle as described previously (Laver et al., 1995). RyRs were incorporated into artificial lipid bilayers and channel gating was measured by single channel recording. RyR open and closed times were measured in the presence various concentrations of cytoplasmic and luminal [Ca²⁺] and in the presence of cytoplasmic ATP or AMP-PCP (2 mmol/l). The effect of endogenous phosphorylation of RyR1 was assessed by incubating SR vesicles containing RyR1 with Protein Phosphatase1 (PP1, 20 units/mg SR protein) for 5 min at 30°C. PP1 removes phosphate groups from serine residues.

RyR1 in their endogenous phosphorylation state were quite active in the virtual absence of cytoplasmic and luminal Ca²⁺ (1 nmol/l cytoplasmic and < 10 μmol/l luminal). Under these conditions they had an opening rate of 100 ± 50 s⁻¹ (n=4). The opening rate showed no significant dependence on luminal [Ca²⁺] between 10 μmol/l and 2 mmol/l. The opening rate increased with increasing cytoplasmic [Ca²⁺] up to 1000 s⁻¹ with half activation at 1 μmol/l. RyR1 treated with PP1 had zero opening rate in the absence cytoplasmic and luminal Ca²⁺. Opening rates increased to 2 ± 0.5 s^-1 in the presence of 0.1 mmol/l luminal Ca²⁺ and 170 ± 80 s⁻¹ in the presence of 0.1 mmol/l cytoplasmic Ca²⁺.

The results indicate that endogenous phosphorylation is important for maintaining the level of activity of RyR1 in skeletal muscle. Interestingly, dephosphorylation of RyR1 makes them reliant on Ca²⁺ for their activation and in that regard makes them very similar cardiac RyRs. This suggests that a key functional difference between cardiac and skeletal muscle may depend on phosphorylation of a serine residue in RyR1.

Laver DR, Roden LD, Ahern GP, Eager KR, Junankar PR & Dulhunty AF. (1995). Cytoplasmic Ca²⁺ inhibits the ryanodine receptor from cardiac muscle. Journal of Membrane Biology 147, 7-22.

Laver DR, O'Neill ER, and Lamb GD (2004) Luminal Ca²⁺-regulated Mg²⁺ inhibition of skeletal RyRs reconstituted as isolated channels or coupled clusters. Journal of General Physiology 124: 741-758

Laver DR, Honen BN (2008) Luminal Mg²⁺, a key factor controlling RyR2-mediated Ca²⁺ release: Cytoplasmic and luminal regulation modelled in a tetrameric channel. Journal of General Physiology, 132: 429-446.