Calmodulin Oxidation and Methionine to Glutamine Substitutions Reveal Methionine Residues Critical for Functional Interaction with Ryanodine Receptor-1

Edward M Balog,Laura E Norton,Rachel A Bloomquist,Razvan L Cornea,D.J Black,Charles F Louis,David D Thomas,Bradley R Fruen

doi:10.1074/jbc.m209180200

Abstract

Calmodulin (CaM) binds to the skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1) with high affinity, and it may act as a Ca(2+)-sensing subunit of the channel. Apo-CaM increases RyR1 channel activity, but Ca(2+)-CaM is inhibitory. Here we examine the functional effects of CaM oxidation on RyR1 regulation by both apo-CaM and Ca(2+)-CaM, as assessed via determinations of [(3)H]ryanodine and [(35)S]CaM binding to skeletal muscle sarcoplasmic reticulum vesicles. Oxidation of all nine CaM Met residues abolished functional interactions of CaM with RyR1. Incomplete CaM oxidation, affecting 5-8 Met residues, increased the CaM concentration required to modulate RyR1, having a greater effect on the apo-CaM species. Mutating individual CaM Met residues to Gln demonstrated that Met-109 was required for apo-CaM activation of RyR1 but not for Ca(2+)-CaM inhibition of the channel. Furthermore, substitution of Gln for Met-124 increased the apo- and Ca(2+)-CaM concentrations required to regulate RyR1. These results thus identify Met residues critical for the productive association of CaM with RyR1 channels and suggest that oxidation of CaM may contribute to altered regulation of sarcoplasmic reticulum Ca(2+) release during oxidative stress.

Highlights

Calmodulin (CaM) binds to the skeletal muscle ryanodine receptor Ca2؉ release channel (RyR1) with high affinity, and it may act as a Ca2؉-sensing subunit of the channel
Our results define CaM Met residues that are critical for the functional interaction between CaM and RyR1 and suggest that CaM oxidation may contribute to altered regulation of sarcoplasmic reticulum (SR) Ca2ϩ release during oxidative stress
Partial oxidation of CaM by incubation in 50 mM H2O2 for 30 min did not fully abolish CaM modulation of ryanodine binding; both the half-activating (EC50 Ͼ 1000 nM) and half-inhibiting (IC50 ϭ 104 Ϯ 8, nH ϭ 1.3 Ϯ 0.2) CaM concentrations were increased (Fig. 1, A and B), with a larger effect occurring at 100 nM Ca2ϩ. These results suggest that the oxidation of critical CaM Met residues alters the productive association of CaM with the RyR1

Summary

EXPERIMENTAL PROCEDURES

Pigs were obtained from the University of Minnesota Experimental Farm. Tran35S-labeled Met and Cys were obtained from ICN Radiochemicals (Costa Mesa, CA). [3H]Ryanodine was purchased from PerkinElmer Life Sciences. SR vesicles (0.2 mg/ml) were incubated at 37 °C in medium containing 120 mM potassium propionate, 10 mM PIPES, pH 7.0, 3 mM AMPPCP, 100 nM [3H]ryanodine, and a Ca-EGTA buffer set to give the desired free Ca2ϩ concentration [12]. Estimates of maximal [3H]ryanodine binding capacity of each SR vesicle preparation were determined in medium that in addition contained 500 mM KCl, 6 mM ATP, and 100 ␮M Ca2ϩ. Ca2ϩ titrations were performed by the addition of small aliquots of concentrated CaCl2 to the sample in the apo buffer (120 mM KCl, 20 mM PIPES, 1.0 mM EGTA, pH 7.0). The CaM concentration dependence of SR vesicle [3H]ryanodine binding and the inhibition of [35S]CaM binding by unlabeled CaM were fit with the Hill equation.

RESULTS

42 Ϯ 5 31 Ϯ 7 41 Ϯ 11 60 Ϯ 13 60 Ϯ 12 47 Ϯ 16 26 Ϯ 2 204 Ϯ 24a 36 Ϯ 13 68 Ϯ 17

73 Ϯ 4 65 Ϯ 2 110 Ϯ 4 37 Ϯ 2 2304 Ϯ 532a 641 Ϯ 168a

DISCUSSION