Evidence for novel caffeine and Ca²⁺ binding sites on the lobster skeletal ryanodine receptor

Abstract

1 The e ects of Ca 2+ , ATP and ca eine on the gating of lobster skeletal muscle ryanodine receptors (RyR) was investigated after reconstitution of the channels into planar phospholipid bilayers and by using [ 3 H]-ryanodine binding studies. 2 The single channel studies reveal that the EC 50 (60 mM) for activation of the lobster skeletal RyR by Ca 2+ as the sole ligand is higher than for any other isoform of RyR studied. 3 Inactivation of the channel by Ca 2+ (EC 50 =1 mM) occurs at concentrations slightly higher than those required to inactivate mammalian skeletal RyR (RyR1) but lower than those required to inactivate mammalian cardiac RyR (RyR2). 4 Lifetime analysis demonstrates that cytosolic Ca 2+ , as the sole activating ligand, cannot fully open the lobster skeletal RyR (maximum Po approximately 0.2). The mechanism for the increase in open probability (Po) is an increase in both the frequency and the duration of the open events. 5 ATP is a very e ective activator of the lobster RyR and can almost fully open the channel in the presence of activating cytosolic [Ca 2+ ]. In the presence of 700 mM Ca 2+ , 1 mM ATP increased Po to approximately 0.8. 6 Ca eine, often used as a tool to identify the presence of RyR channels, is relatively ine ective and cannot increase Po above the level that can be attained with Ca 2+ alone. 7 The results reveal that ca eine increases Po by a di erent mechanism to that of cytosolic Ca 2+ demonstrating that the mechanism for channel activation by ca eine is not`sensitization' to cytosolic Ca 2+ . 8 By studying the mechanisms involved in the activation of the lobster RyR we have demonstrated that the channel responds in a unique manner to Ca 2+ and to ca eine. The results strongly indicate that these ligand binding sites on the channel are di erent to those on mammalian isoforms of RyR.

Effects of Mg2+ on Ca2+ release from sarcoplasmic reticulum of skeletal muscle fibres from yabby (crustacean) and rat

“…Several groups have now reported that the crustacean SR Ca 2+ ‐release channels display the two cationic modulatory sites known to be present in their mammalian counterparts, with the activation site being sensitive to micromolar Ca 2+ , and millimolar ATP and caffeine (Lea, 1996; Quinn et al 1998; Xiong et al 1998; Zhang et al 1999). Results concerning the sensitivity of the inhibitory site for Ca 2+ are more controversial, with reports that 50 % inhibition is achieved between 3 μ m Ca 2+ (Quinn et al 1998) and 1 m m Ca 2+ (Xiong et al 1998; Zhang et al 1999). This discrepancy may be explained by the presence of mammalian skeletal‐like and mammalian cardiac‐like Ca 2+ ‐release channels in the crustacean skeletal muscles with respect to sensitivity of the inhibitory site for divalent cations.…”

“…These characteristics are similar to those of the Ca 2+ ‐release channels in mammalian cardiac muscle, which also display a robust CICR mechanism (Fabiato, 1985) and become only marginally activated at pCa > 8 in the presence of 5 m m ATP (Meissner & Henderson, 1987). Concerning the inhibition at the Ca 2+ /Mg 2+ inhibition site, Xiong et al (1998) and Zhang et al (1999) have shown that the SR Ca 2+ ‐release channels from the abdominal muscle of decapod crustacea, containing predominantly short sarcomere fibres (Hayes et al 1971), were about 50 % inhibited by 1 m m Ca 2+ . This form of channel inhibition is known to be non‐specific for divalent cations (Laver & Lamb, 1998).…”

Requirement of Ryanodine Receptor Subtypes 1 and 2 for Ca2+-induced Ca2+ Release in Vascular Myocytes

“…Looking at the protein expression level, we used BODIPY ® -labeled ryanodine to detect RYRs, since this has been previously reported on several cell types (27,28) Fig. 2A) with some spots of fluorescence in discrete areas probably expressing more RYRs (Fig.…”

Depressing Effect of Caffeine at Crayfish Neuromuscular Synapses II. Initial Search for Possible Sites of Action

“…This extra Ca ++ could modulate the Ca ++ sensitive Ca ++ pumps and channels to increase their activity, bringing overall internal Ca ++ levels down to a new level sooner that with only the ionophore present. However, one could also envision another scenario based on the fact that ryanodine-sensitive channels in crustaceans appear not to be identical to the well-studied vertebrate ones (Seok et al, 1992;Zhang et al, 1999). In crustacean opener muscle, 2 mM caffeine failed to open Ca ++ stores in mitochondria after tetanic enhancement, stores regulated by ryanodine receptors (Tang and Zucker, 1997).…”