Lanthanides Report Calcium Sensor in the Vestibule of Ryanodine Receptor

Ca²⁺ regulates ryanodine receptor's (RyR) activity through an activating and an inhibiting Ca²⁺-binding site located on the cytoplasmic side of the RyR channel. Their altered sensitivity plays an important role in the pathology of malignant hyperthermia and heart failure. We used lanthanide ions (Ln³⁺) as probes to investigate the Ca²⁺ sensors of RyR, because they specifically bind to Ca²⁺-binding proteins and they are impermeable to the channel. Eu³⁺'s and Sm³⁺'s action was tested on single RyR1 channels reconstituted into planar lipid bilayers. When the activating binding site was saturated by 50 μM Ca²⁺, Ln³⁺ potently inhibited RyR's open probability (K_d Eu³⁺ = 167 ± 5 nM and K_d Sm³⁺ = 63 ± 3 nM), but in nominally 0 [Ca²⁺], low [Eu³⁺] activated the channel. These results suggest that Ln³⁺ acts as an agonist of both Ca²⁺-binding sites. More importantly, the voltage-dependent characteristics of Ln³⁺'s action led to the conclusion that the activating Ca²⁺ binding site is located within the electrical field of the channel (in the vestibule). This idea was tested by applying the pore blocker toxin maurocalcine on the cytoplasmic side of RyR. These experiments showed that RyR lost reactivity to changing cytosolic [Ca²⁺] from 50 μM to 100 nM when the toxin occupied the vestibule. These results suggest that maurocalcine mechanically prevented Ca²⁺ from dissociating from its binding site and support our vestibular Ca²⁺ sensor-model further.