Different Regions in Skeletal and Cardiac Muscle Ryanodine Receptors Are Involved in Transducing the Functional Effects of Calmodulin

Naohiro Yamaguchi,Le Xu,Kelly E Evans,Daniel A Pasek,Gerhard Meissner

doi:10.1074/jbc.m405834200

Naohiro Yamaguchi, Le Xu + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m405834200

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Abstract

Calmodulin (CaM) inhibits the skeletal muscle ryanodine receptor-1 (RyR1) and cardiac muscle RyR2 at micromolar Ca(2+) but activates RyR1 and inhibits RyR2 at submicromolar Ca(2+) by binding to a single, highly conserved CaM-binding site. To identify regions responsible for the differential regulation of RyR1 and RyR2 by CaM, we generated chimeras encompassing and flanking the CaM-binding domain. We found that the exchange of the N- and C-terminal flanking regions differentially affected RyR1 and RyR2. A RyR1/RyR2 chimera with an N-terminal flanking RyR2 substitution (RyR2 amino acid (aa) 3537-3579) was activated by CaM in single channel measurements at both submicromolar and micromolar Ca(2+). A RyR2/RyR1 chimera with a C-terminal flanking the 86-amino acid RyR1 substitution (RyR1 aa 3640-3725) bound (35)S-CaM but was not inhibited by CaM at submicromolar Ca(2+). In this region, five non-conserved amino acid residues (RyR1 aa 3680 and 3682-3685 and RyR2 aa 3647 and 3649-3652) differentially affect RyR helical probability. Substitution of the five amino acid residues in RyR1 with those of RyR2 showed responses to CaM comparable with wild type RyR1. In contrast, substitution of the five amino acid residues in RyR2 with those of RyR1 showed loss of CaM inhibition, whereas substitution of the five RyR2 sequence residues in the RyR2 chimera containing the RyR1 calmodulin-binding domain and C-flanking sequence restored wild type RyR2 inhibition by CaM at submicromolar Ca(2+). The results suggest that different regions are involved in CaM modulation of RyR1 and RyR2. They further suggest that five non-conserved amino acids in the C-terminal region flanking the CaM-binding domain have a key role in CaM inhibition of RyR2.

Highlights

Of four 565-kDa subunits and four small FK506-binding proteins
Three different concentrations of metabolically labeled 35S-CaM were used at three different Ca2ϩ concentrations of 5 mM EGTA (Ca2ϩ-free), 0.4 ␮M free Ca2ϩ (a Ca2ϩ concentration that results in activation of ryanodine receptor-1 (RyR1) but inhibition of RyR2 by CaM [10]), and 100 ␮M Ca2ϩ (Ca2ϩ-CaM)
RyR1 and RyR2 each have a single highly conserved CaM-binding region; the two ryanodine receptors (RyR) isoforms are differentially regulated by CaM, which suggests that isoform-specific regions are involved in transducing the functional effects of CaM

Summary

Introduction

Of four 565-kDa subunits and four small FK506-binding proteins. Multiple small physiological molecules such as Ca2ϩ, Mg2ϩ, and ATP and proteins such as protein kinases and calmodulin (CaM) modulate RyR ion channel function [1,2,3]. They further suggest that five non-conserved amino acids in the C-terminal region flanking the CaMbinding domain have a key role in CaM inhibition of RyR2.

Results

Conclusion