The contribution of hydrophobic residues in the pore-forming region of the ryanodine receptor channel to block by large tetraalkylammonium cations and Shaker B inactivation peptides

Abstract

Although no high-resolution structural information is available for the ryanodine receptor (RyR) channel pore-forming region (PFR), molecular modeling has revealed broad structural similarities between this region and the equivalent region of K+ channels. This study predicts that, as is the case in K+ channels, RyR has a cytosolic vestibule lined with predominantly hydrophobic residues of transmembrane helices (TM10). In K+ channels, this vestibule is the binding site for blocking tetraalkylammonium (TAA) cations and Shaker B inactivation peptides (ShBPs), which are stabilized by hydrophobic interactions involving specific residues of the lining helices. We have tested the hypothesis that the cytosolic vestibule of RyR fulfils a similar role and that TAAs and ShBPs are stabilized by hydrophobic interactions with residues of TM10. Both TAAs and ShBPs block RyR from the cytosolic side of the channel. By varying the composition of TAAs and ShBPs, we demonstrate that the affinity of both species is determined by their hydrophobicity, with variations reflecting alterations in the dissociation rate of the bound blockers. We investigated the role of TM10 residues of RyR by monitoring block by TAAs and ShBPs in channels in which the hydrophobicity of individual TM10 residues was lowered by alanine substitution. Although substitutions changed the kinetics of TAA interaction, they produced no significant changes in ShBP kinetics, indicating the absence of specific hydrophobic sites of interactions between RyR and these peptides. Our investigations (a) provide significant new information on both the mechanisms and structural components of the RyR PFR involved in block by TAAs and ShBPs, (b) highlight important differences in the mechanisms and structures determining TAA and ShBP block in RyR and K+ channels, and (c) demonstrate that although the PFRs of these channels contain analogous structural components, significant differences in structure determine the distinct ion-handling properties of the two species of channel.

Arrhythmogenesis in a catecholaminergic polymorphic ventricular tachycardia mutation that depresses ryanodine receptor function

“…In KcsA channels, the inner α-helix equivalent to S6 undergoes a hinge-type movement that allows channel gating (28); it is therefore possible that substitution of RyR2-A4860 by the helix-breaker residue Gly may directly interfere with channel opening. In fact, experimental tests of the 3D model also indicate that mutations close to the A4860 residue and forming part of the same α-helix (I4867A, F4870A) profoundly affect channel gating (29). We found that the naturally occurring RyR2-A4860G mutation does not affect channel expression (Fig.…”

Functional Characterization of the Cardiac Ryanodine Receptor Pore-Forming Region

“…We have examined the influence of representative blockers of the full-length RyR2 on hRyR2-PFR. Tetrabutylammonium (TBA) and tetrapentylammonium (TPeA) are organic cations that interact with residues in the cytosolic vestibule of the full-length RyR2 channel [22]. When bound these molecules partially occlude the conduction pathway producing well-resolved blocking events with characteristic residual currents.…”

Effect of flecainide derivatives on sarcoplasmic reticulum calcium release suggests a lack of direct action on the cardiac ryanodine receptor

“…Previous work from our group has established that a wide range of large monovalent and polyvalent cations are concentration‐ and voltage‐dependent blockers of cytosolic to luminal cation flux through the open RyR2 channel (Tinker et al ., 1992a, 1992b; Tinker and Williams, 1993; Mead and Williams, 2004; Mason et al ., 2012). Given this, it is logical to conclude that the open channel block reported here arises from the interaction of the cationic component of flecainide, QX‐FL and NU‐FL, with a site within the cytosolic vestibule of RyR2 channels.…”