Looking for the Calcium-Binding Site in the Ryanodine Receptor's Vestibule

Abstract

Ca2+ regulates ryanodine receptor's (RyR) activity through a high-affinity (activating) and a low-affinity (inhibiting) Ca2+-binding site. Their altered sensitivity plays an important role in the pathology of malignant hyperthermia and heart failure. Although, the function of Ca2+-binding sites has been extensively studied, their location within the channel protein remains elusive. We used Eu3+ as a tool to learn more about the Ca2+-regulation of RyR, because it has high affinity to Ca2+-binding proteins and due to its high valency it is more susceptible to be moved across an electric field than Ca2+.Eu3+ was tested on single RyR1 channels reconstituted into planar lipid bilayers. When the activating binding site was saturated by 50 μM Ca2+, Eu3+ potently inhibited RyR's open probability (kd=167±5 nM nHill=2±0.1), but in nominally 0 [Ca2+], low [Eu3+] activated the channel. These results suggest that Eu3+ acts as an agonist of both Ca2+-binding sites. More interestingly, the action of Eu3+ was voltage-dependent: at negative membrane potentials, when the electrical force tends to drive Eu3+ into the pore, >1 μM Eu3+ caused long closed states by occluding the pore, but unexpectedly, ≤1 μM Eu3+ induced significantly weaker inhibition compared to positive potentials (which drives positively charged ions out from the outer vestibule). These findings led to the hypothesis that negative potentials prevent Eu3+-inhibition by increasing relative [Eu3+] in the vicinity of the activating site located within the vestibule. This idea was further tested with the peptide-toxin maurocalcine. We found that RyR open probability was insensitive to lowering [Ca2+] to 100 nM as long as the toxin occluded the vestibule. These results suggest, that maurocalcine mechanically prevented Ca2+ from dissociating from its binding site, therefore the high-affinity binding site (but not the low-affinity) might be located in the RyR¯s vestibule.