Ryanodine Receptor Point Mutant E4032A Reveals an Allosteric Interaction with Ryanodine

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 98; no. 5; pp. 2865 - 2870
• Main Authors: Fessenden, James D., Chen, Lili, Wang, Yaming, Paolini, Cecilia, Franzini-Armstrong, Clara, Allen, Paul D., Pessah, Isaac N.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 27.02.2001; National Acad Sciences; The National Academy of Sciences
• Subjects: Agonists; Alkaloids; Allosteric Regulation; Base Sequence; Binding sites; Biological Sciences; Calcium; Cell membranes; Cells, Cultured; Complementary DNA; DNA Primers; Functional responses; Imaging; Microscopy, Electron; Muscle fibers; Mutation; Pharmacology; Point Mutation; Protein Binding; Proteins; Receptors; Ryanodine - metabolism; Ryanodine Receptor Calcium Release Channel - genetics; Ryanodine Receptor Calcium Release Channel - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.041608898

The ryanodine receptor (RyR) family of proteins constitutes a unique type of calcium channel that mediates Ca2+ release from endoplasmic reticulum/sarcoplasmic reticulum stores. Ryanodine has been widely used to identify contributions made by the RyR to signaling in both muscle and nonmuscle cells. Ryanodine, through binding to high- and low-affinity sites, has been suggested to block the channel pore based on its ability to induce partial conductance states and irreversible inhibition. We examined the effect of ryanodine on an RyR type 1 (RyR1) point mutant (E4032A) that exhibits a severely compromised phenotype. When expressed in 1B5 (RyR null/dyspedic) myotubes, E4032A is relatively unresponsive to stimulation by cell membrane depolarization or RyR agonists, although the full-length protein is correctly targeted to junctions and interacts with dihydropyridine receptors (DHPRs) inducing their arrangement into tetrads. However, treatment of E4032A-expressing cells with 200-500 µM ryanodine, concentrations that rapidly activate and then inhibit wild-type (wt) RyR1, restores the responsiveness of E4032A-expressing myotubes to depolarization and RyR agonists. Moreover, the restored E4032A channels remain resistant to subsequent exposure to ryanodine. In single-channel studies, E4032A exhibits infrequent (channel-open probability, Po < 0.005) and brief (<250 µs) 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 ryanodine does not occlude the RyR pore, it does bind to sites that allosterically induce substantial conformational changes in the RyR. In the case of E4032A, these changes overcome unfavorable energy barriers introduced by the E4032A mutation to restore channel function.