Lobe-dependent regulation of ryanodine receptor type 1 by calmodulin

Abstract

Calmodulin activates the skeletal muscle Ca2+ release channel RYR1 at nM Ca2+ concentrations and inhibits the channel at μM Ca2+ 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 μM Ca2+ concentrations and are required for maximum inhibition of the channel at μM Ca2+ concentrations. In contrast, the addition of three amino acids to the N terminus of calmodulin increased the affinity for RYR1 at both nM and μM 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. Ca2+ 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.