In smooth muscle, FK506-binding protein modulates IP₃ receptor-evoked Ca²⁺ release by mTOR and calcineurin

Ca²⁺ release from the sarcoplasmic reticulum (SR) by the IP₃ receptors (IP₃Rs) crucially regulates diverse cell signalling processes from reproduction to apoptosis. Release from the IP₃R may be modulated by endogenous proteins associated with the receptor, such as the 12 kDa FK506-binding protein (FKBP12), either directly or indirectly by inhibition of the phosphatase calcineurin. Here, we report that, in addition to calcineurin, FKPBs modulate release through the mammalian target of rapamycin (mTOR), a kinase that potentiates Ca²⁺ release from the IP₃R in smooth muscle. The presence of FKBP12 was confirmed in colonic myocytes and co-immunoprecipitated with the IP₃R. In aortic smooth muscle, however, although present, FKBP12 did not co- immunoprecipitate with IP₃R. In voltage-clamped single colonic myocytes rapamycin, which together with FKBP12 inhibits mTOR (but not calcineurin), decreased the rise in cytosolic Ca²⁺ concentration ([Ca²⁺]_c) evoked by IP₃R activation (by photolysis of caged IP₃), without decreasing the SR luminal Ca²⁺ concentration ([Ca²⁺]_l) as did the mTOR inhibitors RAD001 and LY294002. However, FK506, which with FKBP12 inhibits calcineurin (but not mTOR), potentiated the IP₃-evoked [Ca²⁺]_c increase. This potentiation was due to the inhibition of calcineurin; it was mimicked by the phosphatase inhibitors cypermethrin and okadaic acid. The latter two inhibitors also prevented the FK506-evoked increase as did a calcineurin inhibitory peptide (CiP). In aortic smooth muscle, where FKBP12 was not associated with IP₃R, the IP₃-mediated Ca²⁺ release was unaffected by FK506 or rapamycin. Together, these results suggest that FKBP12 has little direct effect on IP₃-mediated Ca²⁺ release, even though it is associated with IP₃R in colonic myocytes. However, FKBP12 might indirectly modulate Ca²⁺ release through two effector proteins: (1) mTOR, which potentiates and (2) calcineurin, which inhibits Ca²⁺ release from IP₃R in smooth muscle.