Selective Binding of FKBP12.6 by the Cardiac Ryanodine Receptor

Abstract

The calcium release channels (CRC)/ryanodine receptors of skeletal (Sk) and cardiac (C) muscle sarcoplasmic reticulum (SR) are hetero-oligomeric complexes with the structural formulas (ryanodine recepter (RyR)1 protomer) 4 (FKBP12) 4 and (RyR2 protomer) 4 (FKBP12.6) 4 , respectively, where FKBP12 and FKBP12.6 are isoforms of the 12-kDa receptor for the immunosuppressant drug FK506. The sequence similarity between the RyR protomers and FKBP12 isoforms is 63 and 85%, respectively. Using 35 S-labeled FKBP12 and 35 S-labeled FKBP12.6 as probes to study the interaction with CRC, we find that: 1) analogous to its action in skeletal muscle sarcoplasmic reticulum (SkMSR), FK506 (or analog FK590) dissociates FKBP12.6 from CSR; 2) both FKBP isoforms bind to FKBP-stripped SkMSR and exchange with endogenously bound FKBP12 of SkMSR; and 3) by contrast, only FKBP12.6 exchanges with endogenously bound FKBP12.6 or rebinds to FKBP-stripped CSR. This selective binding appears to explain why the cardiac CRC is isolated as a complex with FKBP12.6, whereas the skeletal muscle CRC is isolated as a complex with FKBP12, although only FKBP12 is detectable in the myoplasm of both muscle types. Also, in contrast to the activation of the channel by removal of FKBP from skeletal muscle, no activation is detected in CRC activity in FKBPstripped CSR. This differential action of FKBP may reflect a fundamental difference in the modulation of excitation-contraction coupling in heart versus skeletal muscle.FK506 is a potent immunosuppressive drug that binds to a family of related intracellular receptors termed FK506-binding proteins, varying in size from 12 to 54 kDa. Among these FKBPs, 1 FKBP12 is the most abundant and is involved in mediating the immunosuppressive action of FK506 in T lymphocytes. All of the known FKBP family members display cis-trans peptidyl-prolyl isomerase (PPIase) activity that is inhibited by FK506 and a structurally related compound, rapamycin (1). However, neither the immunosuppressive nor toxic side effects (including severe neuro-and nephrotoxicity) associated with FK506 therapy result from the inhibition of PPIase activity. Rather, the action of FK506 results from the specific inhibition of calcineurin by the FKBP12⅐drug complex. Calcineurin is a calcium-dependent protein phosphatase involved in the activation of T-lymphocytes (2). FKBP12 is a mere bystander protein during T-cell activation that becomes involved in blocking activation after it binds FK506. To date, the only physiological function directly associated with FKBP12 is its tight binding to (3, 4) and modulation of the ryanodine receptor (RyR-1) or calcium release channel of skeletal muscle SR (5-10). The purified SkM CRC 2 isolated from the CHAPS-solubilized TC is a hetero-oligomeric complex, with the structural formula of (RyR-1 protomer) 4 (FKBP12) 4 (5). Both FK506 and rapamycin bind to and dissociate FKBP12 from the SkM CRC (5). Both the native and FKBP-stripped CRCs have similar unitary conductance and sensitivity to ruthenium red (6). However, in co...

cADP-ribose potentiates cytosolic Ca2+ elevation and Ca2+ entry via L-type voltage-activated Ca2+ channels in NG108-15 neuronal cells

“…The β-NAD + -elicited potentiation in depolarization-induced [Ca# + ] i increases was suppressed in NG108-15 cells preincubated for 24 h with FK506, a known competitor of cADPR at RyRs [25,26]. The β-NAD + -elicited potentiation in depolarizationinduced [Ca# + ] i increases was suppressed.…”

Ryanodine Receptors: Structure, Expression, Molecular Details, and Function in Calcium Release

“…FKBP12 and FKBP12.6 (also known as calstabin 1 and 2, respectively) physically interact with all three isoforms of RyR but have different expression levels and binding affinity in different tissues (Chelu et al 2004). FKBP12 copurifies with RyR1 (Jayaraman et al 1992;Brillantes et al 1994) and FKBP12.6 copurifies with RyR2 (Timerman et al 1995;Timerman et al 1996;Barg et al 1997;Jeyakumar et al 2001;Masumiya et al 2003). Although somewhat controversial, a component of the FKBP12 binding site appears to be located between amino acids 2458 and 2468 of RyR1 (Rabbit sequence, SwissProt accession #P11716).…”

“…RyRs are modulated (see Fig. 1) directly or indirectly by the dihydropyridine receptor (DHPR; also known as L-type Ca 2þ channel, Ca V 1.1/1.2) and by various ions, small molecules and proteins, e.g., Ca 2þ , Mg 2þ , protein kinase A (PKA), FK506 binding proteins (FKBP12 and 12.6), calmodulin (CaM), Ca 2þ /calmodulin-dependent protein kinase II (CaMKII), calsequestrin (CSQ), triadin, junctin Tanabe et al 1990;Ikemoto et al 1991;Sabbadini et al 1992;Wang and Best 1992;Brillantes et al 1994;Chen and MacLennan 1994;Yang et al 1994;Ma et al 1995;Mayrleitner et al 1995;Tripathy et al 1995;Timerman et al 1996;Nakai et al 1998;Moore et al 1999b;Rodney et al 2000). RyR1 and RyR2 are crucial for E-C coupling in both skeletal and cardiac muscle, respectively.…”

Calcium Signaling in Airway Smooth Muscle

“…The open state of the RyR channels in cardiac muscle cells is regulated by FK506-binding proteins (33). Specifically, FKBP12.6 is known to associate with the RyR2 isoform in smooth muscle cells to regulate its function (23,34).…”