Probing the Role of Negatively Charged Amino Acid Residues in Ion Permeation of Skeletal Muscle Ryanodine Receptor

Abstract

Sequence comparison suggests that the ryanodine receptors (RyRs) have pore architecture similar to that of the bacterial K+ channel KcsA. The lumenal loop linking the two most C-terminal transmembrane spanning segments in the RyRs has a predicted pore helix and an amino acid motif (GGGIG) similar to the selectivity filter (TVGYG) of KcsA identified by x-ray analysis. The RyRs have many negatively charged amino acid residues in the two regions linking the GGGIG motif and predicted pore helix with the two most C-terminal transmembrane spanning segments. We tested the role of these residues by generating single-site mutants, focusing on amino acid residues conserved among the mammalian RyRs. Replacement of two acidic residues immediately after the GGGIG motif in skeletal muscle ryanodine receptor (RyR1-D4899 and -E4900) with asparagine and glutamine profoundly affected ion permeation and selectivity. By comparison, mutagenesis of aspartate and glutamate residues in the putative linker regions showed a K+ conductance and selectivity for Ca2+ compared to K+ (P(Ca)/P(K)) close to wild-type. The results show that the negatively charged carboxyl oxygens of D4899 and E4900 side chains are major determinants of RyR ion conductance and selectivity.

Single Channel Properties of Heterotetrameric Mutant RyR1 Ion Channels Linked to Core Myopathies

“…The model is in reasonable agreement with two cryoelectron microscopy studies that determined the pore structure of the closed channel at a resolution of ϳ10 Å (13,14). We identified by mutagenesis, in close proximity to the selectivity filter motif GGGIG, a conserved lumenal D4899/ E4900 motif that has a critical role in RyR ion permeation and selectivity (15). Charge neutralization of two acidic residues (Asp-4938 and Asp-4945) in putative cytosolic vestibule reduced K ϩ conductance, with D4938N also having a reduced Ca 2ϩ selectivity (11).…”

“…Replacement of aspartate residues immediately following the GGGIG motif with glutamine residues greatly reduces ion permeation and selectivity, without loss of regulation by Ca 2ϩ (15). It is therefore unlikely that the mutation has a major impact on the global structure of the channel.…”

Inositol Trisphosphate Receptor Ca2+Release Channels

“…The magnitude of the conductance was correlated with the amount of negative charge in the inner vestibule. Recent mutagenesis studies suggest that this mechanism may also play a role in contributing to the large K ϩ conductance of the RyR (504,524). However, it remains to be determined whether similar mechanisms apply to the InsP 3 R. Cyclic nucleotide-gated (CNG) channels are similar to InsP 3 R/RyR channels in having divalent permeability with considerable monovalent permeability, with little selectivity among monovalent alkali cations (see references in Ref.…”

“…The lack of more substantial data in this area begs for additional experimental effort. A number of mutations in the pore region of the RyR have either been described (central core disease mutations) or engineered (114,116,117,155,217,304,500,501,504,522,524,546). The pore region of the cardiac RyR2 channel has been modeled onto the KcsA pore structure (507).…”

IP3 Receptors: Toward Understanding Their Activation

“…A conserved acidic residue (D2550 in IP 3 R1) at the luminal end of the selectivity filter (Fig. 2C) contributes to the modest Ca 2þ selectivity of IP 3 R (Boehning et al 2001b;Dellis et al 2008) and RyR (Gao et al 2000;Wang et al 2005;Gillespie 2008), but the structural determinants of ion selectivity and permeation by IP 3 R are otherwise poorly understood. The changes in pore structure that allow it to open are also minimally understood.…”