Ryanodine receptor point mutant E4032A reveals an allosteric interaction with ryanodine

The ryanodine receptor (RyR) family of proteins constitutes a unique type o f calcium channel that mediates Ca2+ release from endoplasmic reticulum/sar coplasmic reticulum stores. Ryanodine has been widely used to identify cont ributions made by the RyR to signaling in both muscle and nonmuscle cells, Ryanodine, through binding to high- and law-affinity sites, has been sugges ted to block the channel pore based on its ability to induce partial conduc tance states and irreversible inhibition. We examined the effect of ryanodi ne on an RyR type 1 (RyR1) point mutant (E4032A) that exhibits a severely c ompromised phenotype. When expressed in 1B5 (RyR null/dyspedic) myotubes, E 4032A is relatively unresponsive to stimulation by cell membrane depolariza tion or RyR agonists, although the full-length protein is correctly targete d to junctions and interacts with dihydropyridine receptors (DHPRs) inducin g their arrangement into tetrads. However, treatment of E4032A-expressing c ells with 200-500 muM ryanodine, concentrations that rapidly activate and t hen inhibit wild-type (wt) RyR1, restores the responsiveness of E4032A-expr essing myotubes to depolarization and RyR agonists. Moreover, the restored E4032A channels remain resistant to subsequent exposure to ryanodine. In si ngle-channel studies, E4032A exhibits infrequent (channel-open probability, P-o < 0.005) and brief (<250 mus) gating events and insensitivity to Ca2+. Addition of ryanodine restores Ca2+-dependent channel activity exhibiting full, 3/4, 1/2, and 1/4 substates. This evidence suggests that, whereas rya nodine does not occlude the RyR pore, it does bind to sites that allosteric ally induce substantial conformational changes in the RyR. In the case of E 4032A, these changes overcome unfavorable energy barriers introduced by the E4032A mutation to restore channel function.