Reconstitution of local Ca²⁺ signaling between cardiac L-type Ca²⁺ channels and ryanodine receptors: insights into regulation by FKBP12.6

Abstract

Ca+-induced Ca2+ release (CICR) in the heart involves local Ca2+ signaling between sarcolemmal L-type Ca2+ channels (dihydropyridine receptors, DHPRs) and type 2 ryanodine receptors (RyR2s) in the sarcoplasmic reticulum (SR). We reconstituted cardiac-like CICR by expressing a cardiac dihydropyridine-insensitive (T1066Y/Q1070M) α1-subunit (α1CYM) and RyR2 in myotubes derived from RyR1-knockout (dyspedic) mice. Myotubes expressing α1CYM and RyR2 were vesiculated and exhibited spontaneous Ca2+ oscillations that resulted in chaotic and uncontrolled contractions. Coexpression of FKBP12.6 (but not FKBP12.0) with α1CYM and RyR2 eliminated vesiculations and reduced the percentage of myotubes exhibiting uncontrolled global Ca2+ oscillations (63% and 13% of cells exhibited oscillations in the absence and presence of FKBP12.6, respectively). α1CYM/RyR2/FKBP12.6-expressing myotubes exhibited robust and rapid electrically evoked Ca2+ transients that required extracellular Ca2+. Depolarization-induced Ca2+ release in α1CYM/RyR2/FKBP12.6-expressing myotubes exhibited a bell-shaped voltage dependence that was fourfold larger than that of myotubes expressing α1CYM alone (maximal fluorescence change was 2.10 ± 0.39 and 0.54 ± 0.07, respectively), despite similar Ca2+ current densities. In addition, the gain of CICR in α1CYM/RyR2/FKBP12.6-expressing myotubes exhibited a nonlinear voltage dependence, being considerably larger at threshold potentials. We used this molecular model of local α1C-RyR2 signaling to assess the ability of FKBP12.6 to inhibit spontaneous Ca2+ release via a phosphomimetic mutation in RyR2 (S2808D). Electrically evoked Ca2+ release and the incidence of spontaneous Ca2+ oscillations did not differ in wild-type RyR2- and S2808D-expressing myotubes over a wide range of FKBP12.6 expression. Thus a negative charge at S2808 does not alter in situ regulation of RyR2 by FKBP12.6.

Disparate Ryanodine Receptor Association with the FK506-binding Proteins in Mammalian Heart

“…FKBP12 failed to modulate recombinant RyR2 when co-expressed in CHO cells or skeletal myotubes (George et al, 2003;Goonasekera et al, 2005). Similarly, exogenous FKBP12 had no effects in permeabilised cardiomyocytes (Guo et al, 2010).…”

“…FKBP12.6 is considered to inhibit RyR2 activity, mediating channel closure. These FKBP12.6 effects have been investigated in heterologous systems expressing recombinant RyR2 channels, where FKBP12.6 coexpression suppressed spontaneous or agonist-induced Ca 2+ release (George et al, 2003;Goonasekera et al, 2005). FKBP12.6 deficiency in cardiomyocytes (upon FK506 or rapamycin treatment, or gene-targeted deletion) results in increased intracellular Ca 2+ transients and contractions, and also increased Ca 2+ spark frequency, amplitude and duration (McCall et al, 1996;Xiao et al, 1997;Xin et al, 2002).…”

“…Adenovirusmediated FKBP12 overexpression in isolated cardiomyocytes increases SR Ca 2+ content and decreases Ca 2+ spark frequency (Seidler et al, 2007). However, FKBP12 fails to modulate recombinant RyR2 in heterologous expression systems (George et al, 2003;Goonasekera et al, 2005) or in permeabilised cardiomyocytes (Guo et al, 2010).…”

Triadin Binding to the C-Terminal Luminal Loop of the Ryanodine Receptor is Important for Skeletal Muscle Excitation–Contraction Coupling

“…The additional variable k' is a scaling factor that varies with (∆F/F) max (Sheridan et al, 2003;Goonasekera et al, 2005). Pooled current-voltage (I-V) and fl uorescence-voltage (∆F/F-V) data in Table I are expressed as mean ± SEM.…”

Trafficking and stability of voltage-gated calcium channels

“…The ensuing increase in intracellular calcium concentrations gives rise to a wide range of physiological responses, including calcium-dependent gene transcription, calcium release from internal stores, excitation contraction coupling, and secretion/neurotransmitter release [1][2][3][4][5][6][7][8]. Physiological roles of voltage-gated calcium channels are well summarized in other reviews [9].…”