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Store activation mechanism for cardiac ryanodine receptors

The Ca²⁺ load in the sarcoplasmic reticulum (SR) is an important stimulator of Ca²⁺ release which is mediated by the ryanodine receptors (RyRs) in muscle. Two quite different mechanisms have been proposed, and there is no consensus on how the Ca²⁺ load in the SR alters RyR activation. The “true luminal regulation” hypothesis attributes luminal Ca²⁺-activation to Ca²⁺ regulatory sites on the luminal side of the RyR while the “feed-through” hypothesis proposes that luminal Ca²⁺ permeates the pore and binds to the cytoplasmic sites. This study proposes a resolution of the controversy based on measurements of luminal Ca²⁺ activation of isolated cardiac RyRs (RyR2 isoform) in artificial lipid bilayers. In the absence of Ca²⁺_cyt the open probability (P_o) of RyR2 had a voltage-dependent, bell-shaped dependence on [Ca²⁺]_lum. At -40 mV (favouring Ca²⁺ feed through) Ca²⁺_lum activates with a K_a = 50 μM and inhibits with a K_i = 600 μM. K_a and K_i markedly increased with membrane depolarisation. The mechanism of [Ca²⁺]_lum activation appears to be luminal-triggered, Ca²⁺ feed-through. Detailed analysis of channel gating indicated that Ca²⁺ binding sites on both the luminal (K_m =50 μM) and cytosolic (K_m = 1 μM) sides of the protein mediate Ca²⁺_lum activation. Ca²⁺ binding to either of these sites are sufficient to open RyR2. In the virtual absence of Ca²⁺_cyt (i.e. resting [Ca²⁺]) the predominant opening mechanism is the luminal site which, when bound to Ca²⁺ opens the channel briefly (P_o ∼0.3%, t_o ∼1 ms). Ca²⁺ feed-through from the luminal to cytoplasmic site prolongs channel openings (P_o ∼10%, t_o ∼10-50 ms). In this way, Ca²⁺ feed-through can produce over 90% of RyR activation yet it is completely reliant on the action of Ca²⁺ at a luminal facing site.