Effects of Rapamycin on Ryanodine Receptor/Ca ²⁺ -Release Channels From Cardiac Muscle

Abstract

Ryanodine receptors (RyRs) are intracellular channels that regulate the release of Ca2+ from the endoplasmic reticulum of many cell types. The RyRs are physically associated with FK506-binding proteins (FKBPs); immunophilins, with cis-trans peptidyl-prolyl isomerase activity. FKBP12 copurifies with RyR1 (skeletal isoform) and modulates its gating. A different form of FKBP with a slightly higher molecular weight copurifies with RyR2 (cardiac isoform). Previous studies have demonstrated that FKBP stablizes gating of the skeletal Ca(2+)-release channel. In the present study, we measured the activity of cardiac RyRs incorporated into planar lipid bilayers to show that rapamycin, a drug that inhibits the prolyl isomerase activity of FKBP and dissociates FKBP from the RyR, increases the open probability and reduces the current amplitude of cardiac muscle Ca(2+)-release channels. These experiments show for the first time that submicromolar concentrations of rapamycin can alter channel function. Our results provide support for the hypotheses that FKBP functionally associates with the RyR and that the immunosuppressant drug, rapamycin, alters the function of both cardiac and skeletal muscle isoforms of the Ca(2+)-release channel. Our findings suggest that FKBP-dependent modulation of channel function may be generally applicable to all members of the intracellular Ca(2+)-release channel family and that FKBPs may play important regulatory roles in many cell processes, ranging from long-term depression in neurons to contractility in cardiomyocytes.

Enhancing calstabin binding to ryanodine receptors improves cardiac and skeletal muscle function in heart failure

“…We have previously shown that calstabin-2 stabilizes the RyR2 channel in the closed state (16,22) and that PKA phosphorylation of RyR2 at Ser-2808 (Ser-2809 in humans) causes dissociation of calstabin-2 from the channel complex (7,22). Compared with RyR2 channels from sham-operated mice, RyR2 were significantly PKA-hyperphosphorylated in mice with MI ( Fig.…”

“…RyR1 or RyR2 was phosphorylated with PKA catalytic subunit (40 units; Sigma) in the presence or absence of the PKA inhibitor PKI [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] in phosphorylation buffer (8 mM MgCl 2 ͞10 mM EGTA͞50 mM Tris͞Pipes, pH 6.8). 35 S-labeled calstabin-1 or calstabin-2 were generated by using the TNT Quick Coupled Transcription͞Translation system from Promega (19).…”

“…The dissociation constants (K d values) for calstabin-1 binding to nonphosphorylated or PKA-phosphorylated mouse skeletal muscle RyR1 channels were as follows: 93.8 Ϯ 4.0 nM for RyR1 plus PKA and PKI [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] (PKA inhibitor); 1,068.5 Ϯ 77.5 nM for RyR1 plus PKA; and 110.3 Ϯ 1.8 nM for RyR1 plus PKA and JTV519 (each n ϭ 3; P Ͻ 0.05) (Fig. 6B, which is published as supporting information on the PNAS web site).…”

“…In cardiac muscle, RyR2 constitutes a homotetrameric channel comprised of four RyR2 monomers, each binding a Ca 2ϩ channel-stabilizing subunit, calstabin-2 (also known as FKBP12.6) (16). Accordingly, skeletal muscle RyR1 comprises a homotetrameric channel binding four calstabin-1 (FKBP12) subunits (17).…”

Coupled Gating Between Cardiac Calcium Release Channels (Ryanodine Receptors)

“…10,11 In the absence of FKBP12/ 12.6, a homotetrameric RyR1 or RyR2 channel can be examined in planar lipid bilayers but reveals subconductance states, consistent with a defect in coordinated activity of the four subunits that form the RyR channel. 12,13 The addition of FKBP12 to recombinant RyR1 stabilizes the channel complex, resulting in channels with full conductance. 12 These stabilizing effects are reversed by treating the channels with rapamycin or FK506 to remove FKBP12/12.6 from the RyR channels.…”