[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).

denaturing gel and then immunoblotted, using antibody to NOS2. NOS2 is normally found in cells as a monomer (approximate Mr 140,000) and as a homo-dimer (approximate Mx 280,000). However, coexpression of NAP-110 alters the relative mobility of NOS2: no NOS2 monomers are detected, and a novel NAP-NOS2 complex is detected—and the proportion of NOS2 homodimers are greatly decreased.

These experiments demonstrate that NAP-110 interacts with NOS2: NAP-110 inhibits NOS2 by inhibiting formation of the active NOS2 homodimer (Fig. 2).

Conclusions

The use of the yeast two-hybrid system has facilitated the identification of polypeptides that potentially interact with the NOS isoforms. Because the yeast two-hybrid system can produce false positives, demonstration of an interaction in mammalian cells is necessary. The functional consequences of any protein interacting with NOS can be explored by assessing NOS localization, NOS activity, and NOS dimerization in the absence and presence of the interacting protein. Identification of the biological effects of NOS interactors may reveal novel physiological mechanisms of regulating NOS.

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