Rapamycin is a Deceptive Tool for Probing Functional Interactions Between FK506-Binding Proteins and Ryanodine Receptors

Abstract

Although dissociation of FKBP12/FKBP12.6 from RyR channels is widely accepted to increase open probability (Po) and induce sub-conductance gating, these effects are not observed by all investigators. Since rapamycin is frequently used to dissociate FKBP12/FKBP12.6 from RyR channels, we examined whether rapamycin itself could affect RyR function. When rabbit skeletal SR vesicles were pre-treated with 20 µM rapamycin, the Po of RyR1, in the presence of 10 µM cytosolic Ca2+, was significantly higher than the Po of channels that had not been exposed to rapamycin (0.150±0.055 vs 0.016±0.005; SEM; n=7; p<0.05). Cytosolic addition of 1 µM FKBP12 did not reverse the effects of rapamycin (0.142±0.043; SEM; n=3). Similarly, the Po of RyR2 significantly increased after cytosolic addition of 20 µM rapamycin in the presence of 10 µM cytosolic Ca2+, from 0.039±0.009 to 0.564±0.072 (SEM; n=7; p<0.0001). Again, the effect was not reversed after perfusing away the rapamycin and after subsequent addition of 200 nM FKBP12.6. Although FKBP12 and FKBP12.6 were not able to lower the Po of rapamycin-treated channels, lowering free [Ca2+] to <1 nM completely shut all channels demonstrating that sensitivity to Ca2+ was retained. We observed that rapamycin treatment slightly increased the frequency of resolvable sub-conductance gating states (events>3 ms duration) but, for both RyR1 and RyR2, this appeared to be correlated with elevations in Po. Similarly, slight changes in sub-conductance state gating after additions of FKBP12/FKBP12.6 were correlated with Po changes. Our data shows that dissociation of FKBP12/FKBP12.6 from RyR1/RyR2 does not influence the incidence of sub-conductance state gating. Moreover, we suggest that the direct actions of rapamycin on RyR channel gating have led to erroneous conclusions regarding the effects of FKBP12/FKBP12.6 on RyR channel function. British Heart Foundation funding