Abstract
Global conformational changes in the three-dimensional structure of the Ca(2+) release channel/ryanodine receptor (RyR) occur upon ligand activation. A number of ligands are able to activate the RyR channel, but whether these structurally diverse ligands induce the same or different conformational changes in the channel is largely unknown. Here we constructed a fluorescence resonance energy transfer (FRET)-based probe by inserting a CFP after residue Ser-2367 and a YFP after residue Tyr-2801 in the cardiac RyR (RyR2) to yield a CFP- and YFP-dual labeled RyR2 (RyR2(Ser-2367-CFP/Tyr-2801-YFP)). Both of these insertion sites have previously been mapped to the "clamp" region in the four corners of the square-shaped cytoplasmic assembly of the three-dimensional structure of RyR2. Using this novel FRET probe, we monitored the extent of conformational changes in the clamp region of RyR2(Ser-2367-CFP/Tyr-2801-YFP) induced by various ligands. We also monitored the extent of Ca(2+) release induced by the same ligands in HEK293 cells expressing RyR2(Ser-2367-CFP/Tyr-2801-YFP). We detected conformational changes in the clamp region for the ligands caffeine, aminophylline, theophylline, ATP, and ryanodine but not for Ca(2+) or 4-chloro-m-cresol, although they all induced Ca(2+) release. Interestingly, caffeine is able to induce further conformational changes in the clamp region of the ryanodine-modified channel, suggesting that ryanodine does not lock RyR in a fixed conformation. Our data demonstrate that conformational changes in the clamp region of RyR are ligand-dependent and suggest the existence of multiple ligand dependent RyR activation mechanisms associated with distinct conformational changes.
Highlights
Global structural changes occur in the ryanodine receptor (RyR) upon ligand activation
As expected based on their close proximity in the three-dimensional structure of residue Tyr2801 in the cardiac RyR (RyR2), HEK293 cells expressing RyR2Ser-2367-CFP/Tyr-2801-YFP displayed a significant level of fluorescence resonance energy transfer (FRET) with a FRET efficiency of 14.4 Ϯ 1.1% (n ϭ 20) (Fig. 1D, a and c)
The store Ca2ϩ level remained depleted after removing caffeine and unbound ryanodine (Fig. 5, B and C). These observations are in agreement with the view that ryanodine interacts with the caffeine-opened RyR2 and keeps the channel in the open state, leading to store Ca2ϩ depletion. These results indicate that ryanodine induces conformational changes in the clamp region of RyR2 and that the ryanodine-induced conformation can still be altered by caffeine
Summary
Global structural changes occur in the ryanodine receptor (RyR) upon ligand activation. Global conformational changes in the three-dimensional structure of the Ca2؉ release channel/ryanodine receptor (RyR) occur upon ligand activation. Both of these insertion sites have previously been mapped to the “clamp” region in the four corners of the square-shaped cytoplasmic assembly of the three-dimensional structure of RyR2 Using this novel FRET probe, we monitored the extent of conformational changes in the clamp region of RyR2Ser-2367-CFP/Tyr-2801-YFP induced by various ligands. Ikemoto and co-workers [12, 13, 16] labeled RyR with a thiol-reacting fluorescent probe and showed that channel ligands such as Ca2ϩ and ATP changed the fluorescence intensity of the labeled RyR, suggesting ligand-induced conformational changes in RyR Consistent with these biochemical studies, cryo-electron microscopy (cryo-EM) and single particle image analysis revealed major structural rearrangements in the three-dimensional structure of RyR upon binding of ryanodine and Ca2ϩ or binding of a non-hydrolyzable analog of ATP, AMP-PCP, and Ca2ϩ
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