IMMUNOPHILIN FKBP MODULATES CA2+ RELEASE CHANNEL CLOSURE AND ADAPTATION IN HEART

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Ca++-induced Ca++ release (CICR) from intracellular stores is a ubiquitous Ca++ signalling process that amplifies a Ca++ stimulus via positive feedback. The nature of the signal that terminates the release of Ca++ from the cardiac sarcoplasmic reticulum has remained elusive. This study was intended to examine whether the immunophilin FK506-binding protein (FKBP), a 12.6 KDa peptide that is tightly associated to ryanodine receptor (RyR)/Ca++ release channel, plays a role in the termination of CICR in heart. Confocal microscopy and the Ca++ indicator fluo-3 were used to visualize the in situ gating of RyRs as "Ca++ sparks". We observed that inhibition of FKBP by FK506 or rapamycin turned brief Ca++ sparks into long-lasting "Ca++ glows" in rat ventricular myocytes, increasing the mean open time of RyRs from 14.6 to 93 ms. Ca++ sparks with two-level amplitudes, resembling RyRs full and sub-conductance openings, were also observed. In voltage-clamped cells, Ca++ sparks evoked by depolarization pulses had a 6.8-fold increase in duration in the presence of FK506. The frequency of Ca++ spark occurrence was unaffected by FK506. In addition, FK506 potentiated and prolonged the electrically stimulated [Ca++]i transient and contraction, and subsequently induced spontaneous oscillations of [Ca++]i that are potentially arrythmogenic. These observations in situ were extended by single-channel recordings or rat cardiac RyRs incorporated in planar lipid bilayers. FK506 prolonged the lifetime of RyR openings and induced the occurrence of sub-conductance states in bilayers at both 0.1 and 10 muMM [Ca++]. Furthermore, in bilayers, FK506 virtually abolished RyR ~adaptation~, the spontaneous decay of RyR activity elicited by step increases of [Ca++] (from 0.1 to 10 muM) produced by photolysis of Ca++-caged compound NP-EGTA. Altogether, we conclude that FKBP, by facilitating the closure of RyR and enabling RyR to adapt to sustained Ca++ stimuli, may afford an intrinsic mechanism to terminate RyR openings and thus to exert a negative feedback regulation of CICR in heart cells.