[22 Detection of Redox Sensor of Ryanodine Receptor Complexes

By Wei Feng and Isaac N. Pessah

Introduction

A prominent property of the three genetic isoforms of ryanodine receptors (RyRs) is their exquisite sensitivity to functional modification by sulfhydryl-modifying reagents.1 Sensitivity to sulfhydryl-modifying agents appears to be a general property of Ca2+ channels targeted to sarcoplasmic and endoplasmic reticulum (SR/ER), including all three genetic isoforms of RyR and the inositol 1,4,5-trisphosphate receptor (IP3R). A large number of chemically dissimilar sulfhydryl-oxidizing, -reducing, and -arylating reagents have been used to activate and inhibit RyR channel activity. The net influence of sulfhydryl modification is highly dependent on the concentration of the reagent utilized, the length of time the reaction is permitted to proceed, and the nature of the chemical reaction the

1 I. N. Pessah and W. Feng, Antioxidants Redox Signal. 2, 17 (2000).

CPM-thioether adduct

Fig. 1. CPM forms a highly fluorescent thioether adduct with protein thiols by Michael addition. For clarity a single adduct is shown. However, by severely limiting the mole ratio of CPM to protein in the reaction (i.e., 0.02-1 pmol//ig), CPM adducts will form rapidly with the most reactive class of thiol, if present (shown as -S- in the example).

SR protein

50|ig

CPM-thioether adduct

Fig. 1. CPM forms a highly fluorescent thioether adduct with protein thiols by Michael addition. For clarity a single adduct is shown. However, by severely limiting the mole ratio of CPM to protein in the reaction (i.e., 0.02-1 pmol//ig), CPM adducts will form rapidly with the most reactive class of thiol, if present (shown as -S- in the example).

reagent undertakes with sulfhydryl groups. What has proved most challenging is to gain understanding of how specific sulfhydryl moieties ascribe specific aspects of channel function, and to ascribe their physiologic or pathophysiologic importance. In this respect, one methodological strategy has aimed to define whether RyR complexes possess a class of cysteine moieties that can be distinguished on the basis of their exceptional chemical reactivity2 and to understand their contribution to channel function. The following sections present detailed biochemical and functional methods that have been employed to detect the presence of hyperreactive sulfhydryl moieties within RyR complexes and to define their role in redox sensing.

Measurement and Localization of Hyperreactive Sulfhydryls Associated with Junctional Sarcoplasmic Reticulum

CPM [7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin; Molecular Probes, Eugene, OR] is a membrane-permeable coumarin maleimide that readily undergoes Michael addition with protein sulfhydryl residues.3 CPM has low intrinsic fluorescence until it forms a thioether adduct on protein cysteines (Fig. 1). Although CPM lacks ligand specificity for a particular binding domain on the RyR complex, its chemical properties impart high specificity toward cysteines within both hydrophilic and hydrophobic regions of structure. Therefore CPM, when utilized with isolated SR/ER membrane vesicles, will readily form fluorescent CPM-thioether adducts with cysteines residing within both accessible (e.g., cytoplasmic) and inaccessible (e.g., lumenal) domains of intrinsic membrane proteins.

2 G. Liu, J. J. Abramson, A. C. Zable, and I. N. Pessah, Mol. Pharmacol. 45, 189 (1994).

3 T. O. Sipple, J. Histochem. Cytochem. 29, 314 (1981).

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