Topology of the Ca ²⁺ release channel of skeletal muscle sarcoplasmic reticulum (RyR1)

Abstract

To define the topology of the skeletal muscle ryanodine receptor (RyR1), enhanced GFP (EGFP) was fused in-frame to the C terminus of RyR1, replacing a series of C-terminal deletions that started near the beginning or the end of predicted transmembrane helices M1-M10. The constructs were expressed in HEK-293 (human embryonic kidney cell line 293) or mouse embryonic fibroblast (MEF) cells, and confocal microscopy of intact and saponin-permeabilized cells was used to determine the subcellular location of the truncated fusion proteins. The fusion protein truncated after M3 exhibited uniform cytoplasmic fluorescence, which was lost after permeabilization, indicating that proposed M , M , M1, M2, and M3 sequences are not membrane-associated. The fusion protein truncated at the end of the M4 -M5 loop and containing M4 was membrane-associated. All longer truncated fusion proteins were also associated with intracellular membranes. Mapping by protease digestion and extraction of isolated microsomes demonstrated that EGFP positioned after either M5, the N-terminal half of M7 (M7a), or M8 was located in the lumen, and that EGFP positioned after either M4, M6, the C-terminal half of M7 (M7b), or M10 was located in the cytoplasm. These results indicate that RyR1 contains eight transmembrane helices, organized as four hairpin loops. The first hairpin is likely to be made up of M4a-M4b. However, it could be made up from M3-M4, which might form a hairpin loop even though M3 alone is not membrane-associated. The other three hairpin loops are formed from M5-M6, M7a-M7b, and M8 -M10. M9 is not a transmembrane helix, but it might form a selectivity filter between M8 and M10.S keletal muscle contraction is initiated by activation of a Ca 2ϩ release channel (ryanodine receptor isoform 1 or RyR1) located in the junctional terminal cisternae of the sarcoplasmic reticulum. Activation occurs through physical interaction with the dihydropyridine receptor, located in the transverse tubular membrane, where it is directly apposed to the ryanodine receptor (1, 2). Four RyR monomers, each of 565 kDa, assemble as a homotetrameric complex and transmembrane sequences from each of the four monomers interact to form the ion-conducting pore (3). Hydropathy profiles of RyR1 (4, 5), based on criteria defined by Kyte and Doolittle (6), indicate that the bulk of the RyR1 molecule is cytoplasmic and that 4-12 transmembrane sequences lie within the C-terminal one-tenth to one-fifth of the molecule. Among the predicted transmembrane sequences, four potential transmembrane sequences are clearly more hydrophobic than others, with hydropathy indices between 2.0 and 2.9. These four sequences, amino acids Phe-4564-Tyr-4580, Pro-4641-Leu-4664, Gln-4836 -Phe-4859, and Ile-4918 -Ile-4937, were designated M1-M4 and were proposed to form two hairpin loops in the topological model proposed by Takeshima et al. (4). In a second model, proposed by Zorzato et al. Earlier attempts have been made to elucidate the structure and topology of RyR. Proteolytic digestion of RyR1 ...

Ryanodine receptor type 1 gene mutations found in the Canadian malignant hyperthermia population

“…10 The p.Val4847Leu that co-segregated well with an MHS phenotype within a large French-Canadian family, C-27, is positioned within the putative transmembrane segment and maps to the HS3b ''mutation hot spot'' region in RyR1. 13,38 At least three other closely spaced MH/CCD mutations are located within the same transmembrane segment 33,39,40 (Fig. 3).…”

Role of the Sequence Surrounding Predicted Transmembrane Helix M4 in Membrane Association and Function of the Ca2+ Release Channel of Skeletal Muscle Sarcoplasmic Reticulum (Ryanodine Receptor Isoform 1)

“…The fragment was inserted into an NruI (4556) site, previously introduced (11) into a full-length RYR1 cDNA clone in pcDNA 3.1 (12), to obtain construct EGFP4556, and into an endogenous Bst1107I (4628) site in the full-length RYR1 cDNA to obtain construct EGFP4628. EGFP in EGFP4556 was located just before M5 and, in EGFP4628, just after M5, a well defined, cytosol-to-lumen TM sequence (8).…”

“…In a previous study (8), we analyzed the topology of rabbit skeletal muscle RyR1 by determining the orientation of EGFP fused to the C terminus of truncated RyR1 sequences. Compelling evidence was obtained for the existence of three hairpin loops, M5/M6, M7a/M7b, and M8/M10.…”

Ryanodine Receptors: Structure, Expression, Molecular Details, and Function in Calcium Release

“…The conformation of the open RyR has so far only been proposed to resemble the conformation of the open K þ channel (Samsó et al 2009). The pore region has been predicted to consist of between 4 and 12 transmembrane segments (Takeshima et al 1989;Zorzato et al 1990;Tunwell et al 1996;Du et al 2002). Most of these models place both amino-and carboxy-termini in the cytoplasm.…”