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 exists for K+-selective channels, is not available for RyR. In its absence we have used modelling to identify similarities in the structural elements of K+ channel pore-forming regions (PFR) and postulated equivalent regions of RyR. This has identified a sequence of residues in the cytosolic cavity-lining trans-membrane helix of RyR (G4864LIIDA4869 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 glycine4864 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 glycine4864 is replaced by either valine or proline. The former preventing channel opening, 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. Glycine4864 is not absolutely required for channel gating but some flexibility at this point in the cavity-lining trans-membrane helix is necessary for normal RyR function.