Promiscuous attraction of ligands within the ATP binding site of RyR2 promotes diverse gating behaviour

Mano Sitsapesan,Rebecca Sitsapesan,Elisa Venturi,William Welch,Wei Mun Chan,Chris Lindsay,Maria Musgaard

doi:10.1038/s41598-018-33328-8

Mano Sitsapesan, Rebecca Sitsapesan + Show 5 more

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https://doi.org/10.1038/s41598-018-33328-8

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Abstract

ATP is an essential constitutive regulator of cardiac ryanodine receptors (RyR2), enabling small changes in cytosolic Ca2+ to trigger large changes in channel activity. With recent landmark determinations of the full structures of RyR1 (skeletal isoform) and RyR2 using cryo-EM, and identification of the RyR1 ATP binding site, we have taken the opportunity to model the binding of fragments of ATP into RyR2 in order to investigate how the structure of the ATP site dictates the functional responses of ligands attracted there. RyR2 channel gating was assessed under voltage-clamp conditions and by [3H]ryanodine binding studies. We show that even the triphosphate (PPPi) moiety alone was capable of activating RyR2 but produced two distinct effects (activation or irreversible inactivation) that we suggest correspond to two preferred binding locations within the ATP site. Combinations of complementary fragments of ATP (Pi + ADP or PPi + AMP) could not reproduce the effects of ATP, however, the presence of adenosine prevented the inactivating PPPi effects, allowing activation similar to that of ATP. RyR2 appears to accommodate diverse types of molecules, including PPPi, deep within the ATP binding site. The most effective ligands, however, have at least three phosphate groups that are guided into place by a nucleoside.

Highlights

Ryanodine receptors (RyRs) are the principal ion channels responsible for controlling the sarcoplasmic reticulum (SR) Ca2+ release that is required for cardiac cell contraction
The effects of PPPi on RyR2 gating were investigated by adding PPPi to the cytosolic side of a single RyR2 channel reconstituted into a bilayer (Fig. 2B)
The maximum Po obtained was 0.647 ± 0.126 (SEM; n = 7) and was observed at 10 mM PPPi. This is lower than the maximum Po attainable with ATP under identical experimental conditions[3] but is higher than that observed with adenine nucleotides containing fewer than three phosphates or a reduced negative charge in the region of the phosphate groups as with AMP-PCP (Po approximately 0.4)[10]

Summary

Introduction

Ryanodine receptors (RyRs) are the principal ion channels responsible for controlling the sarcoplasmic reticulum (SR) Ca2+ release that is required for cardiac cell contraction. The availability of RyR structures enables us to model the binding of ligands into the ATP site so that the structural features of ATP-like molecules that govern their ability to activate RyR2 can be investigated. We have previously shown that many adenine based compounds can activate RyR2 but that the triphosphate group of ATP (Fig. 2A) is the key portion of the molecule that enables ATP to cause high open probability (Po)[6,10]. Our results demonstrate that electrostatic interactions generated by the triphosphate region of the ATP molecule are the most important element for inducing long open states in RyR2 and that the triphosphate groups alone are capable of activating RyR2. The adenine base plays a crucial role, by encouraging the correct positioning of the phosphates in the binding site This is essential for creating high affinity binding and for preventing channel inactivation

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