Abstract
Changes in FKBP12.6 binding to cardiac ryanodine receptors (RyR2) are implicated in mediating disturbances in Ca2+-homeostasis in heart failure but there is controversy over the functional effects of FKBP12.6 on RyR2 channel gating. We have therefore investigated the effects of FKBP12.6 and another structurally similar molecule, FKBP12, which is far more abundant in heart, on the gating of single sheep RyR2 channels incorporated into planar phospholipid bilayers and on spontaneous waves of Ca2+-induced Ca2+-release in rat isolated permeabilised cardiac cells. We demonstrate that FKBP12 is a high affinity activator of RyR2, sensitising the channel to cytosolic Ca2+, whereas FKBP12.6 has very low efficacy, but can antagonise the effects of FKBP12. Mathematical modelling of the data shows the importance of the relative concentrations of FKBP12 and FKBP12.6 in determining RyR2 activity. Consistent with the single-channel results, physiological concentrations of FKBP12 (3 µM) increased Ca2+-wave frequency and decreased the SR Ca2+-content in cardiac cells. FKBP12.6, itself, had no effect on wave frequency but antagonised the effects of FKBP12.We provide a biophysical analysis of the mechanisms by which FK-binding proteins can regulate RyR2 single-channel gating. Our data indicate that FKBP12, in addition to FKBP12.6, may be important in regulating RyR2 function in the heart. In heart failure, it is possible that an alteration in the dual regulation of RyR2 by FKBP12 and FKBP12.6 may occur. This could contribute towards a higher RyR2 open probability, ‘leaky’ RyR2 channels and Ca2+-dependent arrhythmias.
Highlights
The cardiac ryanodine receptor (RyR2) is the main pathway for the release of intracellular Ca2+ during excitation-contraction (EC) coupling in cardiac muscle [1]
For single-channel studies this treatment is preferable to treatment with drugs such as rapamycin or FK-506 as we found, in line with other investigators [29,30], that these compounds produced direct and irreversible effects on RyR2 channel gating that were not related to the dissociation of FKBPs
We show that FKBP12.6 has no intrinsic ability to reduce RyR2 Po but perhaps may have extremely low efficacy as an activator of RyR2
Summary
The cardiac ryanodine receptor (RyR2) is the main pathway for the release of intracellular Ca2+ during excitation-contraction (EC) coupling in cardiac muscle [1]. Several studies have shown that FKBP12.6, a member of the FK506-binding protein family, binds with high affinity to RyR2 [2,3,4] but the functional consequences of this interaction has remained a highly controversial subject. The dissociation of FKBP12.6 from RyR2 has been linked with heart failure and arrhythmia generation [5,6] and it has been proposed that the ensuing dysfunctional RyR2 channel behaviour contributes to the defective Ca2+ homeostasis that is characteristic of heart failure [7]. The dissociation of FKBP12.6 from RyR2 has been reported to induce marked changes to RyR2 function which include pronounced sub-conductance state gating, high open probability (Po) and channel gating that is unregulated by Ca2+ [5,8,9]. On the basis of this work, FKBP12.6 has become widely accepted as a ‘stabiliser’ of RyR2 channel function but there is an underlying impression that this is an over-simplification of the role of FKBP12.6 as some investigators find that FKBP12.6 appears not to influence RyR2 gating [10,11,12]
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