Abstract
Ryanodine receptors are homotetrameric intracellular calcium release channels. The efficiency of these channels is underpinned by exceptional rates of cation translocation through the open channel and this is achieved at the expense of the high degree of selectivity characteristic of many other types of channel. Crystallization of prokaryotic potassium channels has provided insights into the structures and mechanisms responsible for ion selection and movement in these channels, however no equivalent structural detail is currently available for ryanodine receptors. Nevertheless both molecular modeling and cryo-electron microscopy have identified the probable pore-forming region (PFR) of the ryanodine receptor (RyR) and suggest that this region contains structural elements equivalent to those of the PFRs of potassium-selective channels. The aim of the current study was to establish if the isolated putative cardiac RyR (RyR2) PFR could form a functional ion channel. We have expressed and purified the RyR2 PFR and shown that function is retained following reconstitution into planar phospholipid bilayers. Our data provide the first direct experimental evidence to support the proposal that the conduction pathway of RyR2 is formed by structural elements equivalent to those of the potassium channel PFR.
Highlights
The cardiac ryanodine receptor (RyR2) is located in the intracellular sarcoplasmic reticulum (SR) membrane network and is responsible for the regulated release of stored calcium from the SR lumen into the cytoplasm to initiate contraction in response to cell excitation [1]. Investigations using both molecular modeling [2,3,4] and cryoelectron microscopy [5,6,7] imply that the pore forming region (PFR) of RyR channel is composed of structural elements equivalent to those found in potassium channels and that these elements are likely to have a similar topology
In accordance with results presented by Kang et al [17], lauryldimethylamine oxide (LDAO) was found to solubilise RyR2PFR with greater efficiency than 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), octyl-b-glucopyranoside (OG) and dodecyl-b-D-maltoside (DDM)
Cryo-EM and molecular modeling [3,7,24] indicate that the PFR of the RyR channel likely contains structural elements equivalent to those found in potassium channels, and correlations between experimental data and molecular dynamics simulations demonstrate that the crystal structure of KcsA provides an excellent template for the PFR of RyR2
Summary
The cardiac ryanodine receptor (RyR2) is located in the intracellular sarcoplasmic reticulum (SR) membrane network and is responsible for the regulated release of stored calcium from the SR lumen into the cytoplasm to initiate contraction in response to cell excitation [1] Investigations using both molecular modeling [2,3,4] and cryoelectron microscopy [5,6,7] imply that the pore forming region (PFR) of RyR channel is composed of structural elements equivalent to those found in potassium channels and that these elements are likely to have a similar topology. Recent studies have demonstrated that both the voltage-sensing domain (VSD) and pore domain (PD) in members of the voltage-gated ion channel (VGIC) family fold independently [9] and are capable of stand-alone function [10,11]. In the current report we provide the first demonstration that the RyR2 PFR forms functional channels following incorporation into planar phospholipid bilayers
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