Model for luminal-triggered Ca²⁺ feedthrough. (A) Three Ca²⁺-sensing sites on each subunit have been linked to regulation of cardiac RYRs by luminal Ca²⁺: the luminal activation site (L-site), the cytoplasmic activation site (A-site), and the cytoplasmic Ca²⁺-inactivation site (I₂-site). In addition, we show here that the low affinity Ca²⁺/Mg²⁺ inhibition site (I₁-site) has a small effect on RYR2 activity under physiological conditions. Cardiac RYR activation by luminal Ca²⁺ (•) occurs by a multistep process in which Ca²⁺ binding to the L-site initiates brief (1-ms) openings at rates up to 1 per second. Once the pore is open, luminal Ca²⁺ has access to the A-site–producing prolongation of openings and to the I₂-site–causing inactivation at high levels of Ca²⁺ feedthrough. (B) Optimal conditions for measuring L-site properties. Cytoplasmic [Ca²⁺] (≤0.1 μM) is at subactivating levels and (1) during intervals when the channel is shut (×) or (2) when there is a sufficiently large electrochemical gradient in opposition to Ca²⁺ feedthrough (e.g., +40 mV and luminal [Ca²⁺] ≤100 μM) can effectively prevent luminal Ca²⁺ from binding to the cytoplasmic sites. (C) Optimal conditions for measuring A- and I₂-site activation by cytoplasmic Ca²⁺ (○). Luminal [Ca²⁺] (≤10 μM) is at subactivating levels and when electrochemical gradient opposes Ca²⁺ feedthrough.