The FK506 binding protein (FKBP) family are thought to regulate the activity of ryanodine receptors (RyR) but the mechanisms by which they do so are currently unclear.
Smooth muscle cells were isolated from the colon of guinea-pigs (~500 g), which were killed humanely by stunning followed by exsanguination. FKBP12 but not FKBP12.6 co-immunoprecipitated RyR2 from these cells. The effects of FKBP12 on Ca2+ release in these cells were examined in single, voltage-clamped myocytes using the dye fluo-3 to monitor [Ca2+]c. All values are given as the mean ± S.E.M. and statistical significance was assessed using Student’s paired t test.
FK506 (10 µM), which inhibits the interaction between RyR and FKBPs as well as the phosphatase action of calcineurin, significantly increased the [Ca2+]c rise evoked by the RyR agonist caffeine from ΔF/F0 of 0.8 ± 0.16 to 1.34 ± 0.19 (n = 9). Rapamycin (10 µM), which also inhibits the RyR-FKBP complex but does not inhibit calcineurin, also significantly increased the caffeine-evoked [Ca2+]c rise from ΔF/F0 of 0.63 ± 0.14 to 1.08 ± 0.33 (n = 6). Cyclosporin A (CsA) inhibits calcineurin but not the interaction between RyR and FKBP and was shown to have no effect on the [Ca2+]c rise evoked by caffeine, ΔF/F0 of 2 ± 0.42 for control cells and 2.14 ± 0.44 when treated with 10 µM CsA (n = 7, P > 0.05).
These results indicate that the interaction between FKBP12 and RyR reduces the ability of the receptor to release Ca2+ in colonic smooth muscle cells.
This work was supported by The Wellcome Trust and the British Heart Foundation.