Topology of the Ca2+ 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.

Fig 1.

Prediction of transmembrane sequences of RyR1 and illustration of constructs. (A) The amino acid sequence of rabbit skeletal muscle RyR1 was analyzed by MACVECTOR 6.5.3 (Accelrys, Burlington, MA) with the Argos transmembrane algorithm (28) in the default setting. (Upper) The analysis of the full-length sequence of RyR1. (Lower) The expanded C-terminal region, including 10 potential transmembrane sequences corresponding to those identified in the Zorzato model (5). (B) Scheme for construction of a series of RyR1–EGFP fusion vectors. The white bar represents RyR1 and the black bar represents EGFP. (C) The boundaries of the transmembrane sequences tested in this study are shown and the relative positions for each truncation of RyR1 in the Zorzato model are indicated by X. The construction of these vectors is described in Experimental Procedures.

Fig 2.

Confocal microscopy of MEF cells expressing EGFP and RyR–EGFP fusion proteins. (Left) Transfected MEF cells before treatment with saponin. (Right) Cells after treatment with saponin, which permeabilizes the cell membrane so that soluble components can leave the cell (e.g., B, F, H, and J), leaving aggregated (F, H, and J) and membrane proteins in situ (D, L, and N). The designation for each construct is on the left side of the image.

Fig 3.

Immunoblotting of RyR1–EGFP fusion proteins. (A) Samples of microsomal preparations from transfected HEK-293 cells were probed with anti-EGFP antibody, as described in Experimental Procedures. Postmitochondrial supernatants from cell homogenates were made 0.6 M in KCl and centrifuged at 186,000 × g to obtain pellet and supernatant fractions. Aliquots of 10 μl from a total of 200 μl of microsomes was dissolved in loading buffer, whereas 100 μl from a total of 5 ml of supernatants was precipitated by 6% trichloroacetic acid (TCA) and resuspended in loading buffer for SDS/PAGE. (B) The microsomes isolated from the transfected HEK-293 cells, described in A, were subjected to extraction with 0.1 M Na₂CO₃, pH 11.0. Lanes 1–7 show the proteins remaining in the microsomal pellet and lanes 8–14 show proteins in the alkaline extract of the corresponding samples. For the pellet, one-fifth of the sample was used for SDS/PAGE, and for the supernatant, one-half of the sample was precipitated by 6% TCA, resuspended in loading buffer, and used for SDS/PAGE.

Fig 4.

Localization of EGFP in HEK-293 cells transfected with RyR1 fusion protein constructs. Microsomes from transfected HEK-293 cells were digested with trypsin and centrifuged to separate membrane-associated proteins from soluble digests. Proteins in the supernatant (A) and microsomes (B) were subjected to 12% SDS/PAGE and immunoblotted with anti-EGFP antibody. The cartoon on the left in A indicates that EGFP will be released to the cytosol with a normal mass, after tryptic digestion, if it lies in a helical hairpin loop facing the cytosolic side. The cartoon on the left in B indicates that EGFP will remain membrane-bound with an increased mass, after tryptic digestion, if it lies at the luminal end of a single transmembrane helix. In PostM8, PostM7a, and PostM5, EGFP remained mainly in membranes, with only trace amounts in the supernatants. The mass of the EGFP-containing fragment in the pellet was increased in proportion to the mass of the transmembrane and luminal sequence attached. EGFP from other fusion proteins was exclusively in the supernatant. EGFP and EGFP after tryptic digestion are included in the 12th and 11th lanes of A, respectively.

Fig 5.

Proposal for the transmembrane topology of rabbit skeletal muscle RyR1. Possible boundaries for eight transmembrane sequences with cytosolic N and C termini are shown. The first two cylinders with dashed lines indicate the tentative nature of the composition of the first predicted helical hairpin loop (M3–M4 or M4a–M4b) in the transmembrane sector of RyR1. The numbers, M3–M10, inside each transmembrane sequence are those proposed in the Zorzato model (5). The long M7 sequence is now designated as M7a and M7b. The proposed selectivity filter between M8 and M10 is designated as “(M9),” even though it is clearly not a transmembrane sequence.