Effect of FKBP12.6 addition to channels previously incubated by PKA and rapamycin. (A) Single-channel recording of RyR2s that were subjected to in vitro dephosphorylation upon PKA phosphorylation (PKA-PP1, described in Fig. 2) and rapamycin (RyR2s were subjected to 20 μM rapamycin for 1 h at room temperature prior to channel recordings) and then exposed to 10 μM dantrolene after addition of 2 μM FKBP12.6 to the cytoplasmic bath for 2 min. The traces are presentative of three experiments where dantrolene inhibition was measured both before and after incubation with FKBP12.6 in the same channel. Single-channel recording conditions are the same as in Fig. 1, and channel openings are downward current jumps from the baseline. (B) Maximum likelihood amplitude distributions generated by the HMM (see Materials and methods) of channel activity from the bottom trace in A from regions marked by the dashed and solid lines beneath. The peaks indicate the open and closed current levels on the recordings.