Two Regions are Involved in Ca2+-Dependent Inactivation of Ryanodine Receptor Calcium Channels

Abstract

Skeletal (RyR1) and cardiac muscle (RyR2) isoforms of ryanodine receptor calcium channels are ∼65 % homologous in amino acid sequences; however they differ in their regulation by endogenous molecules and proteins. Both RyR1 and RyR2 are inhibited by millimollar Ca2+, but RyR2 affinity for inhibitory Ca2+ is ∼10 times lower than RyR1. Previous studies demonstrated that C-terminus quarter of RyR has critical domain(s) for Ca2+ inactivation (Du and MacLennan (1999) J. Biol. Chem.274, 26120; Nakai et al. (1999) FEBS Lett.459, 154). We pursued these observations to obtain further insights into RyR regulation by Ca2+. We constructed and expressed 8 RyR1/RyR2 chimeras in HEK293 cells and determined Ca2+ activation and inactivation affinities of these channels by [3H]ryanodine ligand binding method. We found that replacing two regions of RyR1 with corresponding RyR2 sequence reduced the affinity for Ca2+ inactivation. The first region (RyR2 amino acid 4020-4250) contains two EF hand Ca2+ binding motifs (EF1: 4036-4047, EF2: 4071-4082). Another chimera containing only EF2 of RyR2 (RyR2 4053-4250) has only a modest (not significant) change in Ca2+ inactivation. The results suggest that EF1 is a critical determinant for RyR inactivation by Ca2+. Consistently, a chimera channel carrying only RyR2-EF1 (RyR2 3692-4052) showed significantly reduced Ca2+ inactivation, whereas a chimera carrying RyR2 3692-4019 (no EF hands) behaved essentially as RyR1. Second, preliminary studies indicate that substitution of the last ∼450 amino acids of RyR1 with corresponding RyR2 (4521-4968) results in Ca2+ inactivation affinity between RyR1 and RyR2. Replacing both regions in RyR1 with RyR2 sequences (4020-4250 and 4521-4968) further reduced Ca2+ inactivation affinity, which suggests that in addition to EF1, C-terminal ∼450 amino acids have a role in Ca2+-dependent inactivation of RyRs. Supported by NIH, AHA and NSF.