Gating of the Pore of the Human Ryanodine Receptor Type 2 does not require Glycine Hinges

Abstract

Many potassium channels use a conserved glycine residue to allow flexibility of the inner helices and facilitate gating. A KcsA-based analogy model of the RyR2 pore-forming region (Welch et al Biophys J 87: 2335-2351) indicates that this region is composed of structural elements equivalent to those found in potassium channels and includes a potentially equivalent inner-helix hinge motif (GXXXXA).We studied the functional consequences of substitutions of the conserved glycine residue at position 4864 in recombinantly expressed human RyR2 GFP-tagged proteins. Whilst wild type (WT) RyR2 and the mutants G4864A and G4864V were expressed at equivalent levels in HEK293 cells, G4864P was less tolerated with a reduced expression. This was confirmed by western blot analysis in HEK293 membrane preparations.Caffeine-induced intracellular calcium release was observed in Fluo3-loaded HEK293 cells expressing WT, G4864A and G4864P RyR2 channels. In contrast cells expressing G4864V were not sensitive to caffeine. [3H]-ryanodine binding to HEK293 membrane preparations resulted in similar specific binding for WT and G4864A at 0.192 ± 0.005 and 0.190 ± 0.002 pmol/mg, while G4864V and G4864P showed comparable low values of 0.006 ± 0.001 and 0.002 ± 0.002 pmol/mg, respectively. Characteristic single channel current fluctuations were observed following the incorporation of purified WT and G4864A RyR2 proteins into planar phospholipid bilayers and G4864A channels displayed ion handling, and calcium-dependent gating properties comparable to WT channels. No equivalent activity was observed for G4864V and G4864P.Our investigation indicates that whilst functionally sensitive to mutagenesis, a glycine residue at 4864 is not essential for RyR2 channel gating. Rather, there is a requirement for amino acids with small side chains that allow close packing of helices during transitions from closed to open states. The BHF supported this research.