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

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journal contribution
posted on 2015-12-17, 00:51 authored by Angela C. Gomez, Naohiro Yamaguchi
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.