Investigations of the Contribution of a Putative Glycine Hinge to Ryanodine Receptor Channel Gating

Joanne Euden,Sammy A Mason,Cedric Viero,N Lowri Thomas,Alan J Williams

doi:10.1074/jbc.m113.465310

Abstract

Ryanodine receptor channels (RyR) are key components of striated muscle excitation-contraction coupling, and alterations in their function underlie both inherited and acquired disease. A full understanding of the disease process will require a detailed knowledge of the mechanisms and structures involved in RyR function. Unfortunately, high-resolution structural data, such as exist for K(+)-selective channels, are not available for RyR. In the absence of these data, we have used modeling to identify similarities in the structural elements of K(+) channel pore-forming regions and postulated equivalent regions of RyR. This has identified a sequence of residues in the cytosolic cavity-lining transmembrane helix of RyR (G(4864)LIIDA(4869) in RyR2) analogous to the glycine hinge motif present in many K(+) channels. Gating in these K(+) channels can be disrupted by substitution of residues for the hinge glycine. We investigated the involvement of glycine 4864 in RyR2 gating by monitoring properties of recombinant human RyR2 channels in which this glycine is replaced by residues that alter gating in K(+) channels. Our data demonstrate that introducing alanine at position 4864 produces no significant change in RyR2 function. In contrast, function is altered when glycine 4864 is replaced by either valine or proline, the former preventing channel opening and the latter modifying both ion translocation and gating. Our studies reveal novel information on the structural basis of RyR gating, identifying both similarities with, and differences from, K(+) channels. Glycine 4864 is not absolutely required for channel gating, but some flexibility at this point in the cavity-lining transmembrane helix is necessary for normal RyR function.

Highlights

In many Kϩ channels, gating requires flexing of inner helices at glycine residues within a “hinge motif.” An analogous motif is present in the ryanodine receptor channel
Expression of WT and Mutant RyR2 Channels in HEK293 Cells—The efficiency of transfection of WT and mutant RyR2 channels was determined by monitoring the endogenous fluorescence of the enhanced green fluorescent protein (eGFP)-tagged protein
Molecular modeling of the pore-forming region (PFR) of Ryanodine receptor channels (RyR) isoforms has demonstrated that these regions are likely composed of structural elements analogous to those of Kϩ channels [4, 5]

Summary

Background

In many Kϩ channels, gating requires flexing of inner helices at glycine residues within a “hinge motif.” An analogous motif is present in the ryanodine receptor channel. In the absence of these data, we have used modeling to identify similarities in the structural elements of K؉ channel pore-forming regions and postulated equivalent regions of RyR This has identified a sequence of residues in the cytosolic cavity-lining transmembrane helix of RyR (G4864LIIDA4869 in RyR2) analogous to the glycine hinge motif present in many K؉ channels. In the model presented by Ramachandran et al [9], ligand-mediated transitions between closed and open conformations of RyR1 are proposed to occur as the result of flexing at glycine 4934 This residue is equivalent to glycine 4864 in RyR2 and is the putative glycine hinge identified in the G4864LIIDA4869 motif of the inner helix of the RyR2 PFR model [4]. A comparison of the results obtained in this study with data from earlier studies in Kϩ channels highlights both similarities and differences in the roles played by glycine hinge residues in these different species of channel

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION