Two regions of the ryanodine receptor calcium channel are involved in Ca(2+)-dependent inactivation

Abstract

Skeletal (RyR1) and cardiac muscle (RyR2) isoforms of ryanodine receptor calcium channels are inhibited by millimollar Ca(2+), but the affinity of RyR2 for inhibitory Ca(2+) is ~10 times lower than that of RyR1. Previous studies demonstrated that the C-terminal quarter of RyR has critical domain(s) for Ca(2+) inactivation. To obtain further insights into the molecular basis of regulation of RyRs by Ca(2+), we constructed and expressed 18 RyR1-RyR2 chimeras in HEK293 cells and determined the Ca(2+) activation and inactivation affinities of these channels using the [(3)H]ryanodine binding assay. Replacing two distinct regions of RyR1 with corresponding RyR2 sequences reduced the affinity for Ca(2+) inactivation. The first region (RyR2 amino acids 4020-4250) contains two EF-hand Ca(2+) binding motifs (EF1, amino acids 4036-4047; EF2, amino acids 4071-4082), and the second region includes the putative second transmembrane segment (S2). A RyR1-backbone chimera containing only EF2 from RyR2 had a modest (not significant) change in Ca(2+) inactivation, whereas another chimera channel carrying only EF1 from RyR2 had a significantly reduced level of Ca(2+) inactivation. The results suggest that EF1 is a more critical determinant for RyR inactivation by Ca(2+). In addition, activities of the chimera carrying RyR2 EF-hands were suppressed at 10-100 μM Ca(2+), and the suppression was relieved by 1 mM Mg(2+). The same effects have been observed with wild-type RyR2. A mutant RyR1 carrying both regions replaced with RyR2 sequences (amino acids 4020-4250 and 4560-4618) showed a Ca(2+) inactivation affinity comparable to that of RyR2, indicating that these regions are sufficient to confer RyR2-type Ca(2+)-dependent inactivation on RyR1.

Figure 1

Two regions are involved in isoform-specific Ca²⁺-dependent inactivation of RyRs. (A) Ca²⁺-dependent changes in the activities of WT RyR1 (●) and WT RyR2 (○) were measured in [³H]ryanodine binding assays in the absence (left) or presence (right) of 1 mM Mg²⁺. Data are means ± SE (n = 4–9). (B) Schematic of R21 and R0 chimeras together with the R1 chimera, which was shown to have RyR2-type Ca²⁺-dependent inactivation.^, IC₅₀ values are means ± SE of the number of experiments indicated in parentheses. *p < 0.05 compared with WT RyR1 (ANOVA followed by Tukey’s test among four groups). (C) Ca²⁺-dependent regulation of R21 (●) and R0 (○) chimeras in the absence (left) or presence (right) of 1 mM Mg²⁺. Solid red and blue lines represent mean values of WT RyR1 and WT RyR2, respectively, from panel A. Data are means ± SE (n = 4–5).

Figure 2

EF-hand Ca²⁺ binding motifs are critical for Ca²⁺-dependent inactivation and RyR2-specific Mg²⁺ activation. (A) Schematic of R41, R51, R41′, and R51′ chimera channels. IC₅₀ values are means ± SE of the number of experiments indicated in parentheses. *p < 0.05 compared with WT RyR1 (ANOVA followed by Tukey’s test among seven groups). (B) Ca²⁺-dependent regulation of R41 (●), R51 (○), R41′ (▼), and R51′ (△) chimeras in the absence (left) or presence (right) of 1 mM Mg²⁺. Solid red and blue lines represent mean values of WT RyR1 and WT RyR2 from Figure 1A, respectively. Data are means ± SE (n = 4–6). (C) Effect of 1 mM Mg²⁺ on WT and chimera RyRs. Data are means ± SE (n = 4–12). *Significant activation (p < 0.05) compared to no Mg²⁺.

Figure 3

C-Terminal end that involves another important region for Ca²⁺-dependent inactivation. (A) Schematic of R61 and R71 chimera channels. IC₅₀ values are means ± SE of the number of experiments shown in parentheses. *p < 0.05 compared with WT RyR1 (ANOVA followed by Tukey’s test among five groups). (B) Ca²⁺-dependent regulation of R61 (●) and R71 (○) chimeras in the absence of Mg²⁺. Solid red, blue, and green lines represent mean values of WT RyR1, WT RyR2, and the R0 chimera, respectively (from Figure 1A,C). Data are means ± SE (n = 4–5).

Figure 4

Second putative transmembrane region that is a critical determinant for Ca²⁺-dependent inactivation of RyRs. (A) Proposed six-transmembrane model of RyRs. The replaced regions in a series of the R81 chimeras are highlighted with a different color. (B) Schematic of the series of R81, R91, and R101 chimeras. (C) IC₅₀ values of chimeras are means ± SE (n = 4–8). *p < 0.05 compared with WT RyR1 (ANOVA followed by Tukey’s test among nine groups).

Figure 5

Two RyR2 domains are sufficient for RyR2-type Ca²⁺-dependent inactivation. (A) Schematic of R121b and R131b chimeras. (B) Ca²⁺-dependent activity changes of R121b (●) and R131b (○) chimeras in the absence of Mg²⁺. Solid red and blue lines represent mean values of WT RyR1 and WT RyR2, respectively, from Figure 1A. IC₅₀ values of R121b and R131b are 11.2 ± 1.3 and 12.3 ± 0.7 mM, respectively. Data are means ± SE (n = 5). (C) Effect of 1 mM Mg²⁺ on R121b and R131b chimeras. Data are means ± SE (n = 4–5). *Significant activation (p < 0.05) compared with no Mg²⁺.

Figure 6

Diagram of domains for Ca²⁺-dependent inactivation. Sequence domains suggested by RyR1–RyR2 chimera analyses in previous work^,, and this study are shown as open boxes together with other identified regulatory domains.^, EF denotes the position of two EF-hand Ca²⁺ binding sites. P indicates three potential phosphorylation sites. CaM represents calmodulin.