A Region Involved in Isoform-Specific Regulation of Skeletal Muscle Ryanodine Receptor by Calmodulin

Abstract

Calmodulin (CaM) regulates both skeletal (RyR1) and cardiac (RyR2) muscle ryanodine receptor calcium channels by binding to a single highly conserved CaM binding domain (RyR1 amino acid (aa) 3614-3643; RyR2 aa 3581-3610). CaM inhibits both isoforms at an elevated Ca2+ concentration (micromollar), whereas at cellular resting Ca2+ concentration CaM has opposite effects on two isoforms (activation of RyR1 and inhibition of RyR2). This implies another region of RyR is involved in isoform-specific CaM regulation at submicromollar Ca2+ concentration. To identify the regions we constructed and analyzed a series of RyR1/RyR2 chimera. RyR1/RyR2 chimera carrying RyR1 aa 1-3725 is inhibited by CaM at 0.4 µM Ca2+ (RyR2-type), whereas chimera carrying RyR1 aa 1-4301 is activated (RyR1-type). The results suggest that RyR1 aa 3726-4301 contains a region that is responsible for CaM activation. The region overlaps with a domain resembling the sequence of CaM (CaM-like domain). Replacement of RyR1 3726-4301 with the corresponding RyR2 sequence confers CaM inhibition at [Ca2+]<1 µM (RyR2 type). Furthermore, substitution of 5 non-conserved amino acids in RyR1 CaM-like domain with those of RyR2 (M4122T, I4123L, N4124D, F4125Y, N4130K) is sufficient for RyR2-type CaM inhibition. However, the reverse chimera and mutant RyR2 were not activated but inhibited by CaM. Taken together, the results suggest that 5 non-conserved amino acids of RyR1 are crucial for RyR1-specific CaM activation at submicromollar Ca2+ concentration. On the other hand, CaM inhibition of RyR2 is likely controlled by a different region. Supported by NIH (AR018687 and HL073051), NSF (EPS-0903795) and AHA (10SDG3500001).