Identification of an amino-terminus determinant critical for ryanodine receptor/Ca2+ release channel function

Figure 1. Deletion of the β8–β9 loop perturbs RyR2 N-terminus tetramerization. Chemical cross-linking assays of HEK293 cell homogenates expressing NT (RyR2 residues 1–906) WT or deletion mutants: NTΔβ8–β9 (residues 167–178 removed), NTβ13–β14/4Ala (residues 240–243 substituted by alanine), NTΔβ20–β21 (residues 335–358 removed), NTΔβ23–β24 (residues 399–401 removed), and NTΔβ31–β32 (residues 748–752 removed). (A) Cell homogenates were incubated with glutaraldehyde for the indicated time points under reducing (10 mM DTT) conditions and analysed by western blotting using AbcMyc; monomer (M) and tetramer (T) are indicated with the arrows. (B) Densitometry analysis (n ≥ 6) was carried out on the bands corresponding to tetramer and monomer moieties and used to calculate tetramer formation. Data are given as mean value ± SEM; statistical analysis was carried out using Kruskal–Wallis test with Dunn’s multiple comparison test. SEM, standard error of the mean.

Figure 2. Aspartate 179 and aspartate 180 are indispensable for RyR2 N-terminus tetramerization. Chemical cross-linking assays (n = 8) of NTWT, NTK167A + Q168A, NTE173A + K174A, and NTD179A + D180A as described in the legend to Figure 1. Data are given as mean value ± SEM; statistical analysis was carried out using Kruskal–Wallis test with Dunn’s multiple comparison test.

Figure 3. The β8–β9 loop is dispensable for RyR2 N-terminus interaction with C-terminus. Co-immunoprecipitation assays from HEK293 cells co-expressing NTWT or NTΔβ8–β9 together with HA-tagged RyR2-CT (residues 3529–4967) in the presence of 1 mM EGTA or 100 μM CaCl2 as indicated. (A) HA-RyR2-CT was immunoprecipitated with AbHA from CHAPS-solubilized cell lysates and the presence of co-precipitated NTWT/NTΔβ8–β9 was analysed by western blotting using AbcMyc (top). To detect immuno-isolated HA-RyR2-CT, one-tenth of IP samples was analysed by western blotting using AbHA (bottom). Non-immune rabbit IgG served as negative control (non-specific binding). An aliquot of HEK293 cell lysate corresponding to 1% of the amount processed in the co-IP assay was included in the gels to assess protein expression. (B) Data summary (n = 6) for NT-specific binding (non-immune IgG IP signal subtracted from anti-HA IP signal) following densitometry analysis and normalization to each construct’s respective lysate (taken as 100%). Data are given as mean value ± SEM; statistical analysis was carried out using one-way analysis of variance with Bonferroni’s multiple comparisons test.

Figure 4. Deletion of the β8–β9 loop impairs RyR2 oligomerization. Protein distribution profile of RyR2WT and RyR2Δβ8–β9 assessed by sucrose density gradient centrifugation. (A) RyR2WT and RyR2Δβ8–β9 were expressed in HEK293 cells, CHAPS-solubilized microsomal membranes were subjected to density-gradient centrifugation and collected sucrose fractions were analysed by western blotting using RyR2 Ab1093. Microsomes (25 μg, M) were also included to assess protein expression; sucrose concentration as indicated. (B) Cumulative data (n = 4) following densitometry analysis and normalization against the amount of input protein in the microsomes; data are given as mean value ± SEM.

Figure 5. RyR2Δβ8–β9 displays drastically altered intracellular Ca2± release dynamics. Single-cell Ca2+ imaging using confocal laser scanning microscopy to monitor spontaneous Ca2+ release transient events. (A) Traces from Fluo-3 loaded single HEK293 cells expressing RyR2WT or RyR2Δβ8–β9 showing spontaneous Ca2+ transients and Ca2+ release induced by caffeine (1 mmol/L, 10 mmol/L) or thapsigargin (1 μmol/L). (B) Ca2+ transient characteristics, including amplitude, duration, and frequency of events, were analysed for 84 cells expressing RyR2WT and 81 cells expressing RyR2Δβ8–β9 from four separate experiments, whereas the caffeine response for at least 20 cells. The ER Ca2+ store content was measured by integration of the (1 μmol/L) thapsigargin-induced Ca2+ release (following 1 mmol/L caffeine application) at the end of each experiment. Data are normalized for RyR2WT and expressed as mean value ± SEM; statistical analysis was carried out using Mann–Whitney test.

Figure 6. The pathogenic R176Q mutation decreases N-terminus tetramerization but it has no effect on N-terminus interaction with C-terminus or RyR2 tetramer assembly. (A) Chemical cross-linking assays (n = 8) of NTWT and NTR176Q as described in the legend to Figure 1. Data are given as mean value ± SEM; statistical analysis was carried out using Mann–Whitney test. (B) Co-immunoprecipitation assays (n ≥ 8) of HA-RyR2-CT with NTWT or NTR176Q as described in the legend to Figure 3. Data are given as mean value ± SEM; statistical analysis was carried out using Student’s t-test. (C) Sucrose density gradient centrifugation (n = 4) of RyR2WT and RyR2R176Q as described in the legend to Figure 4; data are given as mean value ± SEM.

Figure 7. RyR2R176Q displays hypersensitive channel characteristics. Single-cell Ca2+ imaging using confocal laser scanning microscopy to monitor spontaneous Ca2+ release transient events in HEK293 cells expressing RyR2WT or RyR2R176Q as described in the legend to Figure 5. Data from 66 cells expressing RyR2WT and 222 cells expressing RyR2R176Q from four separate experiments, are normalized for RyR2WT and expressed as mean value ± SEM; statistical analysis was carried out using Mann–Whitney test.