Abstract
Calmodulin activates the skeletal muscle Ca(2+) release channel RYR1 at nm Ca(2+) concentrations and inhibits the channel at microm Ca(2+) concentrations. Using a deletion mutant of calmodulin, we demonstrate that amino acids 2-8 are required for high affinity binding of calmodulin to RYR1 at both nm and microm Ca(2+) concentrations and are required for maximum inhibition of the channel at microm Ca(2+) concentrations. In contrast, the addition of three amino acids to the N terminus of calmodulin increased the affinity for RYR1 at both nm and microm Ca(2+) concentrations, but destroyed its functional effects on RYR1 at nm Ca(2+). Using both full-length RYR1 and synthetic peptides, we demonstrate that the calmodulin-binding site on RYR1 is likely to be noncontiguous, with the C-terminal lobe of both apocalmodulin and Ca(2+)-calmodulin binding to amino acids between positions 3614 and 3643 and the N-terminal lobe binding at sites that are not proximal in the primary sequence. Ca(2+) binding to the C-terminal lobe of calmodulin converted it from an activator to an inhibitor, but an interaction with the N-terminal lobe was required for a maximum effect on RYR1. This interaction apparently depends on the native sequence or structure of the first few amino acids at the N terminus of calmodulin.
Highlights
The skeletal muscle calcium release channel RYR1 is regulated by calmodulin (CaM)1 in both its Ca2ϩ-free and Ca2ϩ-bound (Ca2ϩ-CaM) states
The addition of three amino acids to the N terminus of calmodulin increased the affinity for RYR1 at both nM and M Ca2؉ concentrations, but destroyed its functional effects on RYR1 at nM Ca2؉. Using both full-length RYR1 and synthetic peptides, we demonstrate that the calmodulin-binding site on RYR1 is likely to be noncontiguous, with the C-terminal lobe of both apocalmodulin and Ca2؉-calmodulin binding to amino acids between positions 3614 and 3643 and the N-terminal lobe binding at sites that are not proximal in the primary sequence
In the course of analyzing the interaction of CaM with RYR1, we found that the addition of three amino acids (Gly-Ser-His) to the N terminus of Drosophila CaM (designated (Nϩ3)dCaM) increases its affinity for RYR1 compared with wild-type CaM
Summary
CaM, calmodulin; mCaM, mammalian calmodulin; dCaM, Drosophila calmodulin; SR, sarcoplasmic reticulum; MOPS, 4-morpholinepropanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. There are at least two possible explanations for the affinity increase: 1) the extension alters the conformation of the molecule; and/or 2) the N-terminal amino acids of CaM make specific and essential contacts with RYR1, and these residues are more accessible in the mutant. The deletion of seven amino acids (DQLTEEQ, residues 2– 8) from the N terminus of mammalian CaM ((N2– 8)mCaM) abolishes CaM-dependent activation of skeletal muscle myosin light chain kinase This mutant CaM still binds the kinase with high affinity [7]. Small-angle x-ray scattering structural analysis indicates that this mutant CaM has a different location in the complex with the kinase than native CaM [8] These findings with the N-terminally altered CaMs suggest that the addition or deletion of a few amino acids at the N terminus can greatly change CaM interaction with a subset of targets. Our findings suggest a complex role of both lobes of CaM in regulating the activity of RYR1
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