Effects of redox potential and Ca²⁺ on the inositol 1,4,5-trisphosphate receptor L3-1 loop region: Implications for receptor regulation

Inositol 1,4,5-trisphosphate receptor (IP₃R) is a major intracellular Ca²⁺ channel, modulated by many factors in the cytosolic and lumenal compartments. Compared with cytosolic control, lumenal-side regulation has been much less studied, and some of its mechanistic aspects have been controversial. Of particular interest with regard to lumenal regulation are whether it involves direct interactions between IP₃R and the regulators, and whether it involves conformational changes of the lumenal regions of IP₃R. To understand these lumenal-side regulation mechanisms, we studied the effects of two important lumenal regulatory factors, the redox potential and Ca²⁺, on the L3-1 lumenal loop region of IP₃R. The redox potential exerted direct and significant effects on the conformation of the loop region. By sharp contrast, Ca²⁺ showed little effect on the L3-1 conformation, suggesting that the regulation of Ca²⁺ is indirect or involves other receptor regions. GSH/oxidized glutathione-mediated oxidation introduced a unique intramolecular disulfide bond between Cys³⁴ and Cys⁴². A variety of NMR experiments revealed that oxidation also induces localized helical characteristics in the Cys³⁴-Cys⁴² region. Dynamics studies also showed reduced motions in the region upon oxidation, consistent with the conformational changes. The results raise the interesting possibility that Cys³⁴ and Cys⁴² may act together as a reduction sensor, and that Cys ⁶⁵ may function as an oxidation sensor. Overall, our studies suggest that the redox potential and Ca²⁺ can regulate IP₃R through totally different mechanisms: Ca²⁺ by the indirect effect and the redox potential by direct action causing conformational changes.