A Calmodulin Binding Domain of RyR Increases Activation of Spontaneous Ca2+ Sparks in Frog Skeletal Muscle

George G Rodney,Gerald M Wilson,Martin F Schneider

doi:10.1074/jbc.m408189200

George G Rodney, Gerald M Wilson + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m408189200

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Abstract

The calmodulin C lobe binding region (residues 3614-3643) on the sarcoplasmic reticulum Ca2+ release channel (RyR1) is thought to be a region of contact between subunits within RyR1 homotetramer Ca2+ release channels. To determine whether the 3614-3643 region is a regulatory site/interaction domain within RyR in muscle fibers, we have investigated the effect of a synthetic peptide corresponding to this region (R3614-3643) on Ca2+ sparks in frog skeletal muscle fibers. R3614-3643 (0.2-3.0 microM) promoted the occurrence of Ca2+ sparks in a highly cooperative dose-dependent manner, with a half-maximal activation at 0.47 microM and a maximal increase in frequency of approximately 5-fold. A peptide with a single amino acid substitution within R3614-3643 (L3624D) retained the ability to bind Ca(2+)-free calmodulin but did not increase Ca2+ spark frequency, suggesting that R3614-3643 does not modulate Ca2+ sparks by removal of endogenous calmodulin. Our data support a model in which the calmodulin binding domain of RyR1 modulates channel activity by at least two mechanisms: direct binding of calmodulin as well as interactions with other regions of RyR.

Highlights

The sarcoplasmic reticulum (SR)1 Ca2ϩ release channel is a homotetramer with a subunit molecular mass of ϳ560 kDa
To determine whether the 3614 –3643 region is a regulatory site/interaction domain within RyR in muscle fibers, we have investigated the effect of a synthetic peptide corresponding to this region (R3614 –3643) on Ca2؉ sparks in frog skeletal muscle fibers
We found that R3614 –3643 increases Ca2ϩ spark occurrence in permeabilized frog skeletal muscle fibers in a highly cooperative dose-dependent manner

Summary

Activation of RyR by a Calmodulin Binding Domain

A single amino acid mutation within R3614 –3643 (L3624D) abolished the activating effect of the peptide Both R3614 –3643 and L3624D bind CaM with similar affinity. The maximum increase in Ca2ϩ spark frequency produced by exogenous recombinant CaM was the same in the presence or absence of R3614 –3643, even though R3614 –3643 binds to CaM. Addition of either exogenous R3614 –3643 or exogenous CaM disrupts the native interaction, thereby destabilizing the closed state of the channel, with CaM having a stronger destabilizing effect than R3614 –3643. Some of these data have been presented in abstract form [12, 13]

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

TABLE I Equilibrium simulations