Objectives: 

It is generally thought that FKBP12.6 and FKBP12 bind tightly to cardiac (RyR2) and skeletal (RyR1)ryanodine receptors, respectively, and maintain low open probability (Po). Dissociation of FKBP12/FKBP12.6 is suggested to be involved in heart disease and skeletal muscle disorders. Despite the high structural similarity and partial sequence identity (85%) shared by FKBP12 and FKBP12.6, we find that FKBP12 selectively activates RyR2 while FKBP12.6 selectively activates RyR1. To investigate the relationship between FKBP structure and the ability to activate RyR1/RyR2 we mutated three amino acid residues found in FKBP12 to the corresponding residues found in FKBP12.6.

Materials: 

A triple FKBP12 mutant (FKBP12E31Q/D32N/W59F), where the amino acids Glu-31, Asp-32 and Trp-59 were mutated to the respective residues found in FKBP12.6 (Gln, Asn and Phe), was cloned and recombinantly expressed. The effects of FKBP12E31Q/D32N/W59F on the gating of single RyR1 and RyR2 channels incorporated into planar phospholipid bilayers was investigated and compared to the effects of FKBP12 and FKBP12.6.

Results: 

FKBP12E31Q/D32N/W59F lost the ability of FKBP12 to activate RyR2 and behaved like FKBP12.6 instead. No significant change in RyR2 Po was observed. For example, Po was 0.206±0.08 before and 0.223±0.10 after addition of 1 mM FKBP12E31Q/D32N/W59F (SEM; n=5). In contrast, when FKBP12E31Q/D32N/W59F was added to RyR1 channels, an increase in Po was observed (Po=0.021±0.008 and 0.110±0.026 after 1 mM FKBP12E31Q/D32N/W59F (SEM; n=7; p<0.01)). This effect was irreversible and similar to that observed with FKBP12.6.

Conclusions: 

Our results demonstrate that the ability of FKBP12 and FKBP12.6 to selectively activate RyR2 and RyR1, respectively, is determined by just three amino acid residues. Since RyR1 and RyR2 respond so differently to addition of FKBP12, FKBP12.6 and FKBP12E31Q/D32N/W59F, it is likely that the FKBP binding sites on RyR1 and RyR2 must also be significantly different.