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. 2014 Mar 4;53(8):1373-9.
doi: 10.1021/bi401586h. Epub 2014 Feb 21.

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

Angela C Gomez  1 , Naohiro Yamaguchi
Affiliations

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

Angela C Gomez et al. Biochemistry. .

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.

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Figures

Figure 1
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
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
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
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
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
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.
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