Lanthanides Report Calcium Sensor in the Vestibule of Ryanodine Receptor

Abstract

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.

Figure 1

3D model of RyR1 structure (PDB:3J8E). RyR1 modeling was performed in the software Jmol. Side view of RyR is given. For better visibility, two subunits were removed from the structure. E4032 is labeled by blue dots, the EF-hand domain is colored in red, and the putative Ca²⁺ binding site is in green (16, 17, 18, 32). To see this figure in color, go online.

Figure 2

Voltage-dependent activity of the RyR1 Ca²⁺ release channel. (A and B) Average open probabilities ± SEM together with individual data points of single RyR1 channels are plotted as the function of membrane potential. Single channel currents were recorded in a symmetric 250 mM KCl, 50 μM Ca²⁺ solution. Differences between data of similar symbols are statistically significant (^∗, #, $, §; p < 0.05). Representative records are shown. The closed state of the channel is marked by c. (B) Schematic diagram of our working hypothesis constructed to guide our further investigation, which were designed to explain the voltage-dependent activity of RyR.

Figure 3

Modified Ca²⁺ sensitivity of RyR in the presence of MCA. Shown here is the representative current record under control conditions in (A) 100 nM Ca²⁺ and (B) 50 μM Ca²⁺; (C) during MCA (20 nM) treatment at −60 mV; (D) then immediately after being switched to +60 mV and (E) 10 s later. Closed states are labeled by C and the subconductive states are labeled with S. The cartoons describe the proposed model for the voltage-dependent behavior of MCA and Ca²⁺ in the vestibule.

Figure 4

The effect of Ln³⁺ on the [3H] ryanodine binding properties of HSR vesicles. (A) Relative ryanodine binding of HSR vesicles as a function of [Eu³⁺] (open squares) is shown. (Inset) Shown here is relative ryanodine binding in control (solid squares), and then at 7.5 μM (shaded diamonds), at 18 μM (open triangles), and at 20 μM (open spheres) Eu³⁺. (B) Relative ryanodine binding of HSR vesicles as a function of [Sm³⁺] (open squares) is given. Inset shows the relative ryanodine binding in control (solid squares), and then at 4 μM (shaded diamonds), at 11 μM (open triangles), and at 45 μM (open spheres) Sm³⁺. (C and D) Shown here is the relative ryanodine binding as a function of [Ca²⁺] in the absence (solid squares) and in the presence of different Eu³⁺ (C) and Sm³⁺ (D) concentrations. Eu³⁺ was applied at 7.5 μM (shaded triangles) and 20 μM (open spheres). Sm³⁺ was applied at 4 μM (shaded triangles) and 45 μM (open spheres).

Figure 5

RyR is impermeable to Ln³⁺. (A) Representative current records under control conditions and in the presence of Eu³⁺are given. Long-lasting closed events of RyR evoked by cis Eu³⁺ at −80 mV are shown in the bottom trace. Closed state is labeled by c. (B) RyR activity at 1 μM Eu³⁺cis, +80 mV with no long-lasting closures, is shown. (C) Long-lasting closed events of RyR evoked by 90 nM Sm³⁺cis at −80 mV are shown. (D) Long-lasting closed events of RyR at 250 and 500 nM Sm³⁺ at +80 mV are given. The closed events were reversible by the addition of EGTA. At the end of the experiment, ryanodine was added to the cis chamber to verify RyR. Cartoons of RyR in the middle show the direction of current at the corresponding voltages.

Figure 6

Dose-response relationship and voltage sensitivity of Eu³⁺ action on RyR at nominally 0 μM Ca²⁺. (A) Biphasic concentration-dependent effect of cis [Eu³⁺] on the open probability (P_o) of RyR1 is given. Inset shows representative current records in control and at 250 nM Eu³⁺. (B) Voltage dependence of the activating action of 250 nM Eu³⁺ is shown. Relative effect of Eu³⁺ in five individual experiments were plotted at −60 and +60 mV membrane potentials. Cartoons of RyR on the sides show the direction of currents at the negative and positive voltages, respectively.

Figure 7

Dose-response relationship and voltage sensitivity of Ln³⁺ action on RyR in 50 μM Ca²⁺ is shown. Relative RyR1 open probabilities as a function of cis [Eu³⁺] (A) and [Sm³⁺] (B) are given. Hill fit revealed dissociation constants of 167 ± 5 nM for Eu³⁺ and 63 ± 3 nM for Sm³⁺. Hill coefficients were “2” in both cases. (C). The voltage dependence of Ln³⁺ action is given. P_o values in the presence of 0.5–1.5 μM cis Eu³⁺ were normalized to their own control P_o values (solid squares). Sm³⁺ data at ±60 mV are given (open spheres). (Insets) Schematic illustrations of the RyR channel show the current direction through the pore.

Figure 8

In silico studies of the activating Ca²⁺ binding sites. Shown here is the solvent-excluded surface of the C-terminal end of RyR1. The surface color represents the electrostatic potential at the surface points calculated by the software APBS (http://www.poissonboltzmann.org/). The negative to positive electrostatic potential values are shown by red to blue colors, respectively. The tetramer structure was cut in half by a plane, which includes the main axis of the pore. The Ca²⁺ binding site determined by cryoelectron microscopy is circled with green. To see this figure in color, go online.