The N-Terminal Region of the Ryanodine Receptor Affects Channel Activation

Figure 1. Activation of RyR2 by the peptide DPcpvtN2. (A) RyR2 activity at 0 mV under control conditions (left) and after addition of 1.0 μM (center) or 2.0 μM DPcpvtN2 (right). All records correspond to the same single channel. Markers indicate the closed state of the channel. (B) The concentration dependence of the effect of the domain peptide DPcpvtN2 on the open probability of the RyR2 channel (n = 9 channels). Lines illustrate the fit of the data by gating schemes GS 1A (red; Equation 1), GS 1B (green; Equation 2) and GS 2 (blue; Equation 3) with parameters from Table 1. (C) Time course of open probability changes induced by DPcpvtN2 in the RyR channel shown in (A). The average open probability measured in 5-s segments is plotted against time after channel incorporation. Concentration of DPcpvtN2 (in μM) is shown at the top. (D) Possible models of activation of the RyR2 by the peptide. Monomers with bound peptide are shown in a darker shade. Peptide-induced open state is denoted by a circle. GS 1 and GS 2 assume that the binding of peptide induces channel opening directly if any of the monomers is peptide-bound (GS 1) or if all four monomers have to be occupied by the peptide to…

Figure 2. CD spectra of the peptide DPcpvtN2. (A) 1.5, 2.0, and 3.0 μM solution of DPcpvtN2 in phosphate buffer, pH 7.0. (B) 1.5, 2.0, and 3.0 μM solution of DPcpvtN2 in ethanol.

Figure 3. Complexes of DPcpvtN2 with the NTR. (A,B) Two views on the plausible positions of DPcpvtN2 in the complex with NTR. (A) A view along the central helix. (B) Oblique view. The models are positioned in the cryo-EM map of oRyR1 (EMD 2807; Yan et al., 2015). The axis and angle of rotation of the second view are shown. Models CM 1–CM 3 are shown in blue, cyan and green, respectively. (C) Top views of the ryanodine receptor (EMD 2807) with the docked models of NTR in the open state, shown as electrostatic potential distribution (blue, positive; red, negative) of its surface, and with bound DPcpvtN2 shown as a ribbon. Left to right: CM 1, CM 2, and CM 3. The rectangles correspond to the areas shown in (D). (D) Electrostatic potential distribution of the interacting interfaces of NTR and DPcpvtN2. Rotation relative to panels in (C) is shown at the top. Left to right: CM 1, CM 2, and CM 3. In each panel, one side of the interface is shown as the electrostatic potential distribution of its surface (left—NTR; right—DPcpvtN2), while the complementary molecule is shown as a ribbon (left—DPcpvtN2; red; right—NTR, blue, with the central helix shown in a lighter shade). The complementary views…

Figure 4. Interaction networks in NTR and CM2. (A–D) the NTR hydrogen bond network E40-R417-D61-R298. (A,B) Closed state of NTR. The interactions between E40 and R417 (A) are disrupted by introduction of DPcpvtN2 (B). (C,D) Open state of NTR. Interaction between R417 and D61 (C) are disrupted but interaction between D61 and R298 is strengthened by introduction of DPcpvtN2 (D). (E–H) the CM2 hydrogen bonding network. (E,F) closed state of NTR; (G,H) open state of NTR. A hydrogen bonding network is formed in CM2 (F,H) between D318 and E320 from domain B and H1 and/or R5 from DPcpvtN2. The central helix is shown in red; DPcpvtN2 is shown in green.

Figure 5. Energies of wild type and mutant NTRs in the closed and open state. (A) Energies of individual molecules in the closed and open state; (B) opening energies of individual molecules.

Figure 6. Configuration of the interaction network D61—R417—E40—R298 in mutant NTRs. (A) Wild type (yellow) and mutant (light gray) NTR models in the closed (A) and open state (B).