Old dog, new trick: Charybdotoxin as a novel ryanodine receptor blocker

Figure 1. Schematic illustration of ion channel‐toxin interactions A, schematic illustration of charybdotoxin (ChTX) binding to the extracellular (EC) side of a BK channel and blocking it by inserting the side chain of lysine 27 into the ion‐conducting pore. The channel gate is formed by the constriction of four S6 transmembrane helices on the intracellular (IC) side. Physiological flow of K+ (yellow sphere) is shown by the yellow arrow. B, a single RyR1 channel recording in the presence of MCa showing a long‐lasting subconductance state. The hyphen on the right indicates the closed state of the channel. C, schematic illustration of maurocalcine (MCa) binding to the cytoplasmic (cis) side of the ryanodine receptor (RyR) vestibule, outside the constriction of the channel gate.

Figure 2. Single‐channel recordings of ryanodine receptor 1 (RyR1) under control conditions and in the presence of charybdotoxin (ChTX) Recording conditions: 50 µm Ca2+, +60 mV holding potential. Hyphens indicate closed channel states on the right side. Some examples of a subconductance states (SS) and long‐lasting closed event (LLCE) induced by 40 nm ChTX are highlighted by a dashed grey or green outlines. Another example of a SS is demonstrated in the bottom trace with higher resolution along with its amplitude histogram.

Figure 3. Evaluation of charybdotoxin (ChTX)‐induced long lasting closed events (LLCE) A, representative current recording of a single RyR1 channel at +60 mV membrane potential, showing that 400 nm ChTX induced LLCEs. The closed state current levels are indicated by hyphens on the side. B, representative traces under similar conditions, but at −60 mV, demonstrating the absence of LLCEs. C, reversibility of LLCE at +60 mV (green) by a rapid change of the membrane potential to −60 mV (indicated by the green arrow). Insets illustrate corresponding current direction at +60 and −60 mV (reverse vs. physiological current direction, respectively). D, closed time histograms of RyR1 currents under control conditions and increasing concentrations of ChTX at +60 mV. The inset is an enlargement of the framed detail (closed events >0.5 s), showing that the number and duration of single LLCEs increases with ChTX concentration. E, closed time histograms of RyR1 currents under control conditions and increasing concentrations of ChTX at −60 mV, showing the absence of LLCEs at this membrane potential (box). F and G, average long‐lasting closed event (LLCE) times (s min−1) of several channels as the function…

Figure 4. Voltage dependence of charybdotoxin block on RyR1 A, representative single channel current recordings of a ChTX‐modified channel at different positive voltages. Closed state current levels are labelled by hyphens on the right side. B, average long‐lasting closed events (LLCE) times measured in 400 nm ChTX are plotted as the function of membrane potential (mV). Cartoons indicate current directions through RyR at corresponding membrane potentials: SR lumen‐to‐cytosolic (i.e. physiological) and reverse current directions at negative and positive voltages, respectively.

Figure 5. Combined treatment of RyR1 with charybdotoxin (ChTX) and ryanodine or maurocalcine (MCa) A, top trace shows a Mca‐induced long lasting subconductance state (red hyphen), which was followed by ChTX treatment. Bottom trace shows that subconductance states were replaced by long closed events upon ChTX treatment of the Mca‐modified channel, indicating competitive binding of the two peptides. B, top trace shows a ChTX‐modified channel current with ChTX‐induced long lasting closed events. Bottom trace represents the same channel's current after the addition of ryanodine. Ryanodine‐stabilized subconductance state is shown by a red hyphen, closed channel state is indicated by black hyphens.

Figure 6. The effect of charybdotoxin (ChTX)‐variant K27N on the cardiac ryanodine receptor (RyR2) Representative recording of single RyR1 (A) and RyR2 (B) channel currents under control conditions and in the presence of 200 nm K27N ChTX at +60 mV holding potential.

Figure 7. The impact of basic amino acid residues of charybdotoxin (ChTX) on RyR1 block Ribbon representation of ChTX backbone structure (top left). Amino acid substitutions introduced into the peptide are illustrated with coloured spheres (top right). Average time spent in long lasting closed events (LLCE) (s min−1) was plotted as the function of the concentration of different ChTX‐variants. Single channel currents were recorded at +60 mV holding potential. Colours are used consequently.

Figure 8. The effect of different α‐KTx peptides on the ryanodine receptor (RyR1) Representative current traces under control conditions and kaliotoxin, noxiustoxin, slotoxin and iberiotoxin treatments. Top: primary structures of the peptides.

Figure 9. Overview of molecular interactions between RyR1 and ChTX vs. RyR1 and IpCa A, two oppositely oriented chains of the RyR1 tetramer, highlighting the pore and vestibule, are shown as blue ribbon representations, in complex with ChTX positioned in the vestibule and displayed as a white ribbon with a red α‐helix. The key interacting RyR1 residues, D4945 and Q4949 (grey), are highlighted as thick stick representations interacting with R25 and R34 of ChTX (orange). B, the same RyR1 region as shown in (A) in complex with IpCa positioned in the vestibule and displayed as a purple ribbon. The key interacting residues, D4945 (grey) of RyR1 interacting with R23 of IpCa (orange), are highlighted as thick sticks.