FKBP12 binds to the cardiac ryanodine receptor with negative cooperativity: implications for heart muscle physiology in health and disease

Figures

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  Figure 1.. Isolation and characterization of RyR2 channels from sheep cardiac sarcoplasmic reticulum (SR) vesicles and solubilized SR. (a–c) General considerations. Sequential centrifugation steps in isolating SR vesicles enriched in RyR2 channels and solubilized RyR2. Solute fractions are labelled S1–S4 and precipitate fractions labelled P1–P4. (b) (i) Western blot showing approximate linearity in RyR2 and FKBP12/12.6 band densities following co-immunoprecipitation (CoIP) of RyR2 from P4 vesicles, loaded at protein concentrations of approximately 3, 6 and 9 µg (left to right) (electronic supplementary material, figure S5). (ii) Blots from one lane indicating FKBP12.6 and FKBP12 and positions of molecular mass markers around the FKBPs and approximate position of 500 kDa. (c) (i) A preliminary CoIP experiment suggesting FKBP associated with RyR2 is less in P4 vesicles than in P2 vesicles (electronic supplementary material, figure S5). (ii) Less FKBP is associated with RyR2 following CoIP of solubilized protein, compared with that of P4 vesicles. (iii) The ratio of densities of FKBP12/12.6 bands to RyR2 bands (FKBP/RyR2) in P4 and solubilized fractions, normalized to FKBP/RyR2 in P4 (n = 3).…

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  Figure 2.. CoIP and Western blot analysis of FKBP12/12.6 bound to RyR2. (a) Exogenous (ex.) FKBP12 binding to RyR2 and exchange with endogenous (en.) FKBP12/12.6. (i) P4 vesicles incubated without (−GF) and with (+GF) 10 µM GST-FKBP12 before CoIP (electronic supplementary material, figure S6). (ii) The average of FKBP/RyR2 ratio relative to the −GF ratio (n = 4). For +GF, the GST-FKBP12 measure was first divided by 2 to correct for dimerization (see Methods and Results). The resulting ex. FKBP12 number was then added to en. FKBP12/12.6 to obtain the total FKBP (en. + ex. FKBP), which was then divided by the RyR2 density for that lane. The (en. + ex. FKBP)/RyR2 ratio for +BP was then normalized to the control (en. FKBP)/RyR2 ratio in the –BP lane on the same blot. (iii) eFKBP12/12.6 in –GF and +GF supernatants from the experiment in (i) and (ii) (electronic supplementary material, figure S6). (iv) Average en. FKBP12/12.6 in the supernatant normalized to the GF band (n = 4). (v) P4 incubated without (−F), and with (+F) 10 µM FKBP12 (i.e. GST-cleaved) (electronic supplementary material, figure S7). (vi) Average FKBP/RyR2 ratios normalized to the −F ratio (n = 3). * indicates significant…

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  Figure 3.. Changes in subunit occupation by FKBP12 within each tetramer might explain the changes in RyR2 channel activity with exogenous FKBP12 binding. (a,b) Patterns of FKBP12 occupation of RyR2 subunits in RyR tetramers from P4 vesicles (a) and solubilized RyR2 (b). The control horizontal panel for P4 shows examples of three control RyR2 tetramers, two with 1 FKBP-bound and one with 2 FKBP-bound molecules, approximating the mean of 1.44 (Results). The control horizontal panel for solubilized RyR2 is shown with two subunits having 0 FKBP-bound, and one RyR2 tetramer with 1 FKBP-bound. This is an overestimate of 1.44 per 10 tetramers, but is shown to indicate a finite number bound rather than 0 (Discussion). The remaining panels show increasing FKBP12 occupation with increasing time after exogenous FKBP12 addition and increasing [FKBP12]. The changes in occupation are suggested to explain the biphasic effects of FKBP12 on RyR2 channel activity presented in figure 1, in the context of data presented in figure 2, as outlined in the Discussion. Note that the activated RyR2 tetramers are illustrated as having a more open conformation, in contrast to the inhibited tetramers, which are depicted…