Molecular Identification of the Ryanodine Receptor Pore-forming Segment

Abstract

A sequence motif, GXRXGGGXGD, located in the putative channel-forming domain, is conserved in all known ryanodine receptors and inositol 1,4,5-trisphosphate receptors. The functional significance of this conserved region was investigated by using site-directed mutagenesis together with functional assays consisting of Ca 2؉ Ryanodine receptors (RyRs) 1 are members of a superfamily of intracellular Ca 2ϩ channels that include the inositol 1,4,5-trisphosphate receptors (IP 3 Rs). These channels play an essential role in intracellular Ca 2ϩ signaling by virtue of releasing Ca 2ϩ from the lumen of sarco(endo)plasmic reticulum to the cytosol of muscle and non-muscle cells (1, 2).RyR is a homotetrameric structure composed of four identical subunits, each having ϳ5000 amino acids. Sequence analysis reveals that one-fifth of the COOH terminus of the molecule is likely to form the channel conducting pore. The remaining ϳ4000 amino acid residues apparently constitute the cytoplasmic "foot" domain (3-7). A truncated RyR in which the foot domain has been deleted has been shown to function as a Ca 2ϩ release channel. The truncated RyR channel was still regulated by Ca 2ϩ , was modified by ryanodine, and exhibited a single channel conductance similar to that of the full-length RyR (8). These studies indicate that the sites for Ca 2ϩ activation and ryanodine binding, and the ion conduction pathway are located within the COOH-terminal ϳ1000 amino acid residues. A glutamate residue located in the putative transmembrane sequence M2 has recently been identified as the Ca 2ϩ sensor of RyR (9). The locations of the ryanodine binding site and the pore-forming segment of RyR, however, have yet to be defined.RyRs and IP 3 Rs share some sequence homology, in particular in the COOH-terminal channel-forming domain (10). Considering their sequence homology and similar conduction properties (11-13), RyRs and IP 3 Rs are likely to share similar structural features in the channel pore. A hydrophobic region between the M5 and M6 transmembrane sequences of the mouse type 1 IP 3 R has been proposed to be the pore-forming region (14). The equivalent region in RyR, corresponding to the M9 transmembrane sequence proposed by Zorzato et al. (15), is also hydrophobic. Sequence alignment of these regions reveals a GXRXGGGXGD motif that can be found in all known RyRs and IP 3 Rs (Fig. 1). To investigate its role in RyR function, we have introduced point mutations into this highly conserved region and examined the functional consequences of these point mutations. Our data indicate that this region is critical for ryanodine binding and ion conduction and probably constitutes the pore-forming segment of RyRs. EXPERIMENTAL PROCEDURES Materials-Ryanodine was obtained from Calbiochem. [3 H]Ryanodine was from NEN Life Science Products. Monoclonal antibody 34C was a generous gift from Dr. John L. Sutko (16).Cloning of the Mouse Cardiac RyR cDNA-Total RNA from mouse heart tissue, isolated by the method of Chomczynski and Sacchi (17), was used to generate firs...

Methanethiosulfonate Ethylammonium Block of Amine Currents through the Ryanodine Receptor Reveals Single Pore Architecture

“…The six conductance groups were then correlated with six possible arrangements of mutant and wild type RyR subunits. The conclusion from this study was that the conduction pathway was a single pore created by an equal contribution from each subunit (12). Finally images using negative stain electron microscopy showed that the RyR complex exists as a four-subunit structure encircling a central "hole" (3).…”

Residue Gln4863 within a Predicted Transmembrane Sequence of the Ca2+ Release Channel (Ryanodine Receptor) Is Critical for Ryanodine Interaction

“…Each mutation was confirmed by DNA sequencing. The generation of mutations G4824A and G4826C within the proposed poreforming segment of RyR2 has been described earlier (23,24). HEK293 cells grown on 100-mm tissue culture dishes in supplemented Dulbecco's modified Eagle's medium for 18 -20 h after subculture were transfected with 12-16 g of wild type (wt) or mutant RyR2 cDNA using the method of Ca 2ϩ phosphate precipitation (29).…”

“…3A, b-e), indicating that these mutant channels remain sensitive to ryanodine modulation in HEK293 cells despite their lack of [ 3 H]ryanodine binding in cell lysates. On the other hand, no measurable caffeine-or ryanodine-induced intracellular Ca 2ϩ release was detected in HEK293 cells expressing mutants D4847A [4], F4850A [7], F4851A [8], L4858A [15], L4859A [16], or I4866A [23] (Fig. 3B).…”

“…I4861A [18], I4862A [19], G4864A [21], L4865A [22], and I4867A [24] all had binding capacities in excess of 25% of the wt RyR2. In contrast, mutants F4846A [3], D4847A [4], T4849A [6], F4850A [7], F4851A [8], I4855A [12], V4856A [13], L4858A [15], L4859A [16], Q4863A [20], and I4866A [23] displayed binding capacities of 5% or less of wt RyR2 ( Fig. 2 and Table I).…”

“…Evidence in support of a pore localization of the high affinity ryanodine binding site in RyR comes from recent sitedirected mutagenesis in a motif located in a luminal loop linking transmembrane domains 8 and 10 (TM8 and TM10) (22), 4820 GVRAGGGIGD 4829 . Mutations in this highly conserved motif abolish or markedly reduce high affinity [ 3 H]ryanodine binding to RyR, suggesting that this region constitutes an essential determinant of ryanodine interaction (23)(24)(25)(26).…”