Two Regions of the Ryanodine Receptor Calcium Channel Are Involved in Ca²⁺-Dependent Inactivation

Skeletal (RyR1) and cardiac muscle (RyR2) isoforms of ryanodine receptor calcium channels are inhibited by millimollar Ca²⁺, but the affinity of RyR2 for inhibitory Ca²⁺ is ∼10 times lower than that of RyR1. Previous studies demonstrated that the C-terminal quarter of RyR has critical domain(s) for Ca²⁺ inactivation. To obtain further insights into the molecular basis of regulation of RyRs by Ca²⁺, we constructed and expressed 18 RyR1–RyR2 chimeras in HEK293 cells and determined the Ca²⁺ activation and inactivation affinities of these channels using the [³H]­ryanodine binding assay. Replacing two distinct regions of RyR1 with corresponding RyR2 sequences reduced the affinity for Ca²⁺ inactivation. The first region (RyR2 amino acids 4020–4250) contains two EF-hand Ca²⁺ 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 Ca²⁺ inactivation, whereas another chimera channel carrying only EF1 from RyR2 had a significantly reduced level of Ca²⁺ inactivation. The results suggest that EF1 is a more critical determinant for RyR inactivation by Ca²⁺. In addition, activities of the chimera carrying RyR2 EF-hands were suppressed at 10–100 μM Ca²⁺, and the suppression was relieved by 1 mM Mg²⁺. 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 Ca²⁺ inactivation affinity comparable to that of RyR2, indicating that these regions are sufficient to confer RyR2-type Ca²⁺-dependent inactivation on RyR1.