Lanthanides Report Calcium Sensor in the Vestibule of Ryanodine Receptor.

Ca regulates ryanodine receptor's (RyR) activity through an activating and an inhibiting Ca-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 (Ln) as probes to investigate the Ca sensors of RyR, because they specifically bind to Ca-binding proteins and they are impermeable to the channel. Eu's and Sm's action was tested on single RyR1 channels reconstituted into planar lipid bilayers. When the activating binding site was saturated by 50 μM Ca, Ln potently inhibited RyR's open probability (K Eu = 167 ± 5 nM and K Sm = 63 ± 3 nM), but in nominally 0 [Ca], low [Eu] activated the channel. These results suggest that Ln acts as an agonist of both Ca-binding sites. More importantly, the voltage-dependent characteristics of Ln's action led to the conclusion that the activating Ca 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 [Ca] from 50 μM to 100 nM when the toxin occupied the vestibule. These results suggest that maurocalcine mechanically prevented Ca from dissociating from its binding site and support our vestibular Ca sensor-model further.