Kinetics of FKBP12.6 binding to ryanodine receptors in permeabilized cardiac myocytes and effects on Ca sparks

Abstract

Rationale: FK506-binding proteins FKBP12.6 and FKBP12 are associated with cardiac ryanodine receptors (RyR2), and cAMP-dependent protein kinase A (PKA)-dependent phosphorylation of RyR2 was proposed to interrupt FKBP12.6-RyR2 association and activate RyR2. However, the function of FKBP12.6/12 and role of PKA phosphorylation in cardiac myocytes are controversial.

Objective: To directly measure in situ binding of FKBP12.6/12 to RyR2 in ventricular myocytes, with simultaneous Ca sparks measurements as a RyR2 functional index.

Methods and results: We used permeabilized rat and mouse ventricular myocytes, and fluorescently-labeled FKBP12.6/12. Both FKBP12.6 and FKBP12 concentrate at Z-lines, consistent with RyR2 and Ca spark initiation sites. However, only FKBP12.6 inhibits resting RyR2 activity. Assessment of fluorescent FKBP binding in myocyte revealed a high FKBP12.6-RyR2 affinity (K(d)=0.7+/-0.1 nmol/L) and much lower FKBP12-RyR2 affinity (K(d)=206+/-70 nmol/L). Fluorescence recovery after photobleach confirmed this K(d) difference and showed that it is mediated by k(off). RyR2 phosphorylation by PKA did not alter binding kinetics or affinity of FKBP12.6/12 for RyR2. Using quantitative immunoblots, we determined endogenous [FKBP12] in intact myocytes is approximately 1 micromol/L (similar to [RyR]), whereas [FKBP12.6] is <or=150 nmol/L.

Conclusions: Only 10% to 20% of endogenous myocyte RyR2s have FKBP12.6 associated, but virtually all myocyte FKBP12.6 is RyR2-bound (because of very high affinity). FKBP12.6 but not FKBP12 inhibits basal RyR2 activity. PKA-dependent RyR2 phosphorylation has no significant effect on binding of either FKBP12 or 12.6 to RyR2 in myocytes.

Figure 1. F-FKBP12.6 is located at junctional SR

A, Confocal image of saponin-permeabilized rat ventricular myocyte with 20 nM F-FKBP12.6; B, plot profile of striated sarcomeric pattern and sinusoidal fit; C. longitudinal line scan image from myocyte exposed to 100 nM F-FKBP12.6; D, simultaneous Ca spark measurement using rhod-2 as Ca indicator; E, Merged C-D images showing Ca sparks originate from FKBP binding sites.

Figure 2. FKBP12-RyR2 association and resting RyR2 activity

A, Time course of FKBP12 (1μM) wash-in, wash-out, and effects of cAMP and rapamycin on FKBP12 dissociation; B, Normalized effects of FKBP12 & rapamycin on CaSpF and FKBP12-RyR2 binding (n=10-20). Basal CaSpF at 100 nM [Ca]_i is 10.1 ± 0.9 sparks (s^-1 100μm^-1). C, Normalized effects of cAMP-dependent phosphorylation on CaSpF and FKBP12-RyR2 binding (n=10-20). D-F, The effects of FKBP12, rapamycin & cAMP on the Ca spark amplitude, width (FWHM) and duration (FDHM).

Figure 3. FKBP12.6-RyR2 association and resting RyR2 activity

A, Time course of FKBP12.6 (100 nM) wash-in and influence of cAMP and rapamycin; B, Normalized FKBP12.6 and cAMP effects on CaSpF, FKBP12.6-RyR2 binding (n=25,17) & SR Ca load (assessed by 10mM caffeine, n=6). Basal control CaSpF is 8.7± 0.6 sparks (s^-1 100μm^-1). C, Normalized graph of rapamycin effects on CaSpF, FKBP12.6-RyR2 binding, and SR load (n=45,10,7). D-F, FKBP12.6, rapamycin & cAMP effects on Ca spark amplitude, width (FWHM) and duration (FDHM).

Figure 4. Steady-state K_d and B_max of F-FKBP in myocytes

A, Confocal myocyte image with 20 nM bath [FKBP12.6]; B, Same as A but myocyte was pretreated with 20 μM rapamycin; C, Dependence of bath F-FKBP12.6 fluorescence on [F-FKBP12.6]. D, Background-corrected (bath & auto-fluorescence) fluorescence intensity from Z-line, M-line and average, plotted vs. bath [FKBP12.6]. E, Calibrated F-FKBP12.6 binding using known bath concentration and fluorescence (fit with one-site isotherm; n=4 rats). F, Influence of cAMP/OA pretreatment on apparent B_max and K_d (n=4 rats).

Figure 5. FRAP measurement of k_on and k_off of F-FKBP in myocytes

A, Three regions of interest (ROI) from a permeabilized myocyte equilibrated with 1 nM F-FKBP12.6 were photobleached (time 0 and 30 min after bleach). B, Time course of FRAP at 3 ROIs. C, Dependence of FRAP rate constant (k_FRAP) on [F-FKBP12.6], and FRAP block by rapamycin. D, Same as in C, but for F-FKBP12.

Figure 6. Wash-in and wash-out of FKBP

A. FKBP12.6 wash-in (1 nM) and wash-out (33 nM non-fluorescent FKBP12.6), with exponential fits (rising: y_max(1- exp(-k_in t)); falling: y₀exp(-k_off t), where k_in = k_on [FKBP12.6] + k_off). B, F-FKBP12.6 wash-in (1 and 100 nM). C-D, Neither cAMP nor rapamycin changes the k_off of FKBP12.6/12-RyR2 binding (τ_washout=1/k_off). E, FKBP12.6 and 12 have comparable k_on, and cAMP does not alter k_on of FKBP12.6/12-RyR2 binding.

Figure 7. Endogenous FKBP12.6/12 quantification

A, Apparent K_d and B_max measurement in myocytes from WT and FKBP12.6-KO mice (n=3 mice, 50-100 myocyte/ mouse). B, Immunoblot quantification of endogenous total FKBP12/12.6 from intact rat myocytes (n=4 rats). C. RyR2 phosphorylation detected by P-S2808 specific antibody, and normalized to RyR protein, for myocytes treated as in Figure 3A (before and 5 min after saponin exposure, and 30 min after cAMP/OA and FKBP12.6 exposure).