PMC

FRET efficiency values are shown from each of 5 labeled D-FKBP positions to 3 μM Cy3NTA (red bars) or Cy5NTA (blue) targeted to HEK-293T cells expressing His¹³⁵⁸ (DR2) (A), His¹⁹¹⁵ (DR3) (B), or His⁴⁴²⁹(DR1)(C) RyR1 constructs. Values represent mean ± SEM. See also and .

(A) Side-view of the RyR1 cryo-EM map (grey) indicating the trilateration results of Cy3NTA bound to His₁₀ at position 4429 in DR1. The trilateration locus (red) was defined from datasets lacking the D49 FRET results (-D49). The portion of the RyR1 cryo-EM map within 15 Å of this locus is indicated in yellow. (B) Distances from either GFP fused at the RyR1 N-terminus (GFP(1)) or position 620 (GFP(620)) to the His⁴⁴²⁹ (DR1) locus are shown. (C) Trilateration results using either Cy3NTA (red) or Cy5NTA (blue) as acceptors are shown. Both datasets identify His⁴⁴²⁹ within RyR1 subdomain 3. See also .

(A) Side-view of the RyR1 cryo-EM map (grey) indicating the trilateration result of Cy3NTA bound to a His₁₀ tag at position 1915 (red), and the map portion within 15 Å of this locus (DR3, pink). FKBP is docked to one RyR1 subunit as indicated (yellow, with red dots indicating attachment sites of AF488 donors). (B) Distances from either GFP fused at the RyR1 N-terminus (GFP(1)) or position 620 (GFP(620)) to the Cy3NTA-His¹⁹¹⁵ locus are shown. (C) Trilateration results using either Cy3NTA (red) or Cy5NTA (blue) as acceptors are shown. Both datasets identify His¹⁹¹⁵ within RyR1 subdomain 9.

(A) HEK-293T cells expressing either WT, His¹³⁵⁸(DR2), His¹⁸⁶³(DR3), His¹⁹¹⁵(DR3) or His⁴⁴²⁹(DR1) RyR1 constructs are shown after incubation with 10 nM D14-FKBP (left panels) and subsequent analysis of RyR1 expression using immunocytochemistry (middle panels). Right panels indicate overlay of the two channels. (B) D-FKBP binding maximum (B_max) of indicated His₁₀-tagged RyR construct normalized to WT-RyR1 level. The number of cells analyzed is indicated for each construct. Significance (*) was determined by one-way ANOVA, followed by Dunnett’s post test (p<0.01).

(A) Strategy for FRET-based measurements from GFP fused at either the N-terminus (GFP(1)) or position 620 (GFP(620)) (green ovals) to Cy3NTA (red rectangle) targeted to His₁₀ tags inserted in each divergent region in the RyR1 protein sequence (black bar). (B) FRET efficiency levels are shown for GFP(1)-RyR1 or GFP(620)-RyR1 fusion constructs lacking (-His) or containing His₁₀ tags at either the N- terminus (N-term) or positions 620, 1358(DR2), 1863(DR3), 1915(DR3) or 4429(DR1). Asterisks indicate significant differences in FRET relative to non His-tagged constructs, determined using one-way ANOVA followed by Dunnett’s post test (p<0.01). See also .

(A) Side view of RyR1 cryo-EM map (grey) indicating the position of FKBP12.6 (yellow, with numbered red dots indicating attachment sites of AF488 donors) docked to one subunit. Loci based on sets of four distances are shown lacking measurements from FKBP positions D14 (-D14, dark blue), D32 (-D32, red), or D44 (-D44, cyan). Note: -D14b and –D44b are inside the RyR1 cryo-EM map, which is rendered partially transparent. (B) RyR1 cryo-EM map from the boxed region in (A) is shown indicating distances from the -D14b and -D44b loci to GFP fused to the N-terminus of RyR1 (GFP(1), green ribbon diagram). Distances from the –D14a and –D44a loci to the surface of the RyR are indicated. (C) Inset indicates distances from either GFP(1) or GFP fused at position 620 (GFP(620)) of RyR1 to the -D32 locus (red). The RyR1 cryo-EM map surface within 15 Å of this locus is shaded (cyan). (D) The -D32 loci based on FRET measurements to either Cy3NTA (red) or Cy5NTA (blue) are indicated. The RyR1 cryo-EM map surface within 15 Å of the Cy3NTA-derived locus (cyan) is within RyR1 subdomain 5.

(A) Representative Fluo-4 based Ca²⁺ imaging traces are shown for WT-RyR1 and each His₁₀-tagged construct. Indicated caffeine concentrations were perfused during the time intervals indicated (black bars). Calibration: 0.5 F/F₀ ratio units vs. one minute. (B) Concentration dependence of normalized caffeine-induced Ca²⁺ release in HEK-293T cells expressing each RyR1 construct. Values represent mean ± SEM. (C) Summary of EC₅₀ values, 95% confidence intervals and number of cells analyzed (n) for caffeine activation of each His₁₀-tagged construct. No significant differences were observed between these EC₅₀ values as determined by oneway ANOVA, followed by Dunnett’s post test (p<0.05).

(A) FRET-based method used in this study. FKBP (green oval) acts as a FRET donor transferring energy to Cy3NTA or Cy5NTA, FRET acceptors that bind specifically to His₁₀ tags inserted into either divergent region DR2 (blue), DR3 (pink) or DR1 (yellow) within the RyR1 protein sequence (black bar). (B) Cryo-EM reconstruction of RyR1 as viewed from the “top” (i.e. the side facing the t-tubule membrane in situ) showing the clamp and handle regions, key subdomains within the clamp region (numbered), and FKBP (yellow structure, FK) docked to each RyR1 subunit. (C) Ribbon diagram of FKBP in same orientation as “FK” atomic structure in (B), indicating numbered AF488-labeled amino acid positions (red spheres) and optimized AF488 donor position (green spheres). (D) Sequence positions of each of the divergent regions within RyR1, and the locations of the inserted His₁₀ tags.

(A) Summary of trilateration results to either DR2 (cyan patch), DR3 (magenta) or DR1 (yellow). Docking of FKBP to the RyR1 cryo-EM structure (yellow, with numbered red dots indicating attachment sites of AF488 donors) and locations of RyR1 subdomains 3,5,6,9 and 10 are indicated. (B) Stepwise rotated view of RyR1 cryo-EM structure is shown with a putative location of the Ca_v1.1 cryo-EM structure (tan). The distance from our DR2 trilateration result (cyan) to GFP fused at position 1350 from a previously published study (green ball), () is indicated. (C) The DR3 trilateration result (magenta) is shown relative to a GFP fusion at position 1908 (green ball) (). We propose a location of the RyR1 polyacidic sequence spanning residues 1873-1920 as indicated (orange). (D) DR1 trilateration result (yellow patch) relative to GFP fused at position 4413 (green ball) (), and Ca²⁺- calmodulin (blue ball) () are indicated.

(A) Characterization of D14-FKBP binding to His⁵⁰⁰-RyR1. Panels show AF488 D14-FKBP fluorescence (upper left), His⁵⁰⁰-RyR1 expression (upper right) and overlay (lower left) for HEK-293T cells expressing His⁵⁰⁰-RyR1 treated with 10 nM D14-FKBP followed by anti-RyR immunocytochemistry. No significant difference in FKBP binding to His⁵⁰⁰-RyR1 was observed, relative to WT-RyR1 (lower right panel). (B) Functional analysis of His⁵⁰⁰-RyR1. Concentration-dependence of caffeine-induced Ca²⁺ release from either WT-RyR1 or His⁵⁰⁰-RyR1 is shown. (C) FRET analysis of His⁵⁰⁰-RyR1. FRET efficiency values are shown from each of 5 labeled FKBP positions to Cy3NTA (red) or Cy5NTA (blue) after each FRET acceptor was targeted to HEK-293T cells expressing His⁵⁰⁰-RyR1. Values represent mean ± SEM. (D,E) Trilateration of Cy3/5NTA bound to His⁵⁰⁰ within the cryo-EM map (gray). Loci determined using either the Cy3NTA (red) or Cy5NTA dataset (blue) are depicted relative to FKBP (yellow, with numbered red dots indicating attachment sites of AF488 donors) and the insertion site of the His₁₀ tag (green sphere) within the RyR1 “C” domain (purple ribbon structure) docked to the RyR1 cryo-EM map. See also, .