Complex Actions of FKBP12 on RyR1 Ion Channel Activity Consistent with Negative Co-Operativity in FKBP12 Binding to the RyR1 Tetramer

Figures

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  Figure 1. FKBP12 dissociation from RyR1 during SR processing. (A) Sequential centrifugation and sucrose gradient fractionation in isolating SR vesicles enriched in RyR1 channels and solubilized RyR1. Relevant solute fractions are labeled S1 and S5, the relevant pellet is labeled P2, and sucrose gradient bands are labeled B1 to B4. (B–H) Co-IP of RyR1 and FKBP12 in SR preparations. Co-IP samples were loaded at protein concentrations of approximately 3, 6, and 9 μg. (B) RyR1 and FKBP12 in homogenate (H) and B4 vesicles (n = 5). (C) RyR1 and FKBP12 in P2 and B4 vesicles (n = 6). (D) RyR1 and FKBP12 in control-incubated homogenate and homogenate incubated with GST-cleaved FKBP12 (cFKBP12) (n = 3). (E) RyR1 and FKBP12 in B4 and solubilized B4 (n = 5). (F) RyR1, GST-FKBP12, and FKBP12 in incubated B4 without GST-FKBP12 (control) and B4 incubated with GST-FKBP12 (n = 4). (G) Supernatants following Co-IP control-incubated B4 and B4 incubated with GST-FKBP12 (n = 5). (H) B4 and B4 incubated with GST-cleaved FKBP12 (cFKBP12) (n = 3). (B–H) The images in each figure show immune-stained bands from the same SDS-PAGE gel. The upper image in each panel shows the RyR1 band, and the lower image shows the…

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  Figure 2. S107 prevents FKBP12’s dissociation from RyR1 during processing. Results for the Co-IP of RyR1 and FKBP12 in SR preparations in the following preparation in the presence of 20 µM of S107. Co-IP samples were loaded at protein concentrations of approximately 3, 6, and 9 μg. (A) RyR1 and FKBP12 in homogenate (H) and B4 vesicles in the presence of S107, as labeled. (B) RyR1 and FKBP12 in P2 and B4 vesicles in the presence of S107, as labeled. The images in each panel show bands from the same SDS-PAGE gel, and images in (A,B) are from the same gel as the images shown in Figure 1B,C, respectively. The upper image in each panel shows the RyR1 band, and the lower image shows the FKBP12 band, with vertically aligned images obtained from the same lane. Positions of MW markers are provided in (A) with corresponding arrows indicating marker positions in (A,B). The graphs (A) show the average relative FKBP12/RyR1 in the homogenate (left) and in B4 (right) processed with S107 (n = 5). The FKBP12/RyR1 ratios were first calculated for the RyR1 and FKBP12 bands in the same lanes; then, the H + S107 ratio was expressed relative to the H ratio (left), and the B4 + S107 ratio was expressed relative…

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  Figure 3. Biphasic FKBP12-induced changes in the activity of native RyR1 channels from B3 and B4 sucrose gradient fractions. RyR1 channel activity recorded in artificial lipid bilayers. (A) Current recordings obtained from native RyR1 channels during typical experiments over 4 to 8 min of control recording before FKBP12 was added to the cytoplasmic (cis) solution containing different [Ca2+] and [ATP] specified above the records. Upper “control” recordings were obtained before FKBP12 addition, and the lower recordings at the times below each record. The [FKBP12]s are provided at the top of each panel. Currents were at +40 mV, with upward deflections indicating channel opening from the baseline (indicated by the arrow on the left-hand side of each row) to the maximum open channel level (broken lines in each record). (i) 10 µM cis Ca2+: No change in activity after 12 min exposure to 5 nM FKBP12. (ii) 1 µM cis Ca2+: increased activity after 23 min with 5 nM FKBP12. (iii) 1 µM cis Ca2+: increased activity after 5 min with 0.2 µM FKBP12. (iv) 1 µM cis Ca2+: increased activity after 9 min with 1.0 µM FKBP12. (v) 1 µM cis Ca2+: increased activity after 5 min with 5.0 µM FKBP12. (B–D) Analysis of…

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  Figure 4. Changes in native RyR1 single-channel gating parameters during exposure to FKBP12. Gating parameters, mean open time (To, left column), mean closed time (Tc, middle column), and event frequency (Fo, right column) were measured from the single-channel recording at the same time as the Po values in Figure 3. Refer to Figure 3 for general details that are not repeated here and for the number of experiments. Average data are plotted as a function of time and [FKBP12]. (A) 1–5 nM FKBP12; (B) 0.2 µM FKBP12; (C) 1 and 5 µM FKBP12. The data for the one channel recorded with 5 µM FKBP12 are included because they provide additional support for the trends seen with 1 µM FKBP12. Filled symbols indicate the average values, with the s.e.m. indicated by vertical capped bars. The broken red lines surround data sets that show trends in the gating parameter that are most clearly associated with the rapid onset of activation (To in (A)). Blue asterisks * in indicate significant differences from the control after the addition of FKBP12.

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  Figure 5. Effects of FBP12 on partially purified solubilized RyR1 channels. Ion channel activity recorded in artificial lipid bilayers. Current recordings obtained from solubilized RyR1 channels during typical experiments during 4 to 8 min control recording before FKBP12 was added to the cytoplasmic solution containing 1 µM free Ca2+ and for periods of ~20–38 min in the presence of FKBP12. (A–D) Column (i) shows examples of channel currents recorded before (upper record) and at the indicated times after FKBP12 addition (lower records). Currents are shown at +40 mV, with upward deflections indicating channel opening. In each recording, the solid red line marks the baseline (zero current level), and the broken red line marks the maximum open level for a single channel. The open level for a second full conductance opening is not specifically marked, as it is seldom clearly seen and cannot be separated from multiple sub-conductance openings. Column (ii): Plots of individual measurements of relative I′F at +40 mV (filled circles) and at −40 mV (open circles). Column (iii): average relative I′F (including independent values at +40 and −40 mV). Column (iv): Average I′F, shown to illustrate the…

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  Figure 6. (A,B) FKBP12 inhibition of RyR1 is enhanced by S107. Ion channel activity recorded from native RyR1 channels (A(i)) and from solubilized RyR1 channels (B(i)), before FKBP12 addition to the cytoplasmic solution containing 1 µM free Ca2+ and then at various times over ~20–38 min exposures to 0.2 µM FKBP12 alone or 0.2 µM FKBP12 with 20 µM S107 added 5–10 min after FKBP12. Currents are shown at +40 mV, with upward deflections indicating channel opening. Relative Po for native RyR1 channels and I′F for solubilized RyR1 exposed to 0.2 µM FKBP12 in the absence of S107 in Figure 3 and Figure 5 are included in (A(ii),B(ii)), respectively, for comparison with the S107 data sets. Measurements of relative Po or relative I′F at +40 mV and −40 mV are included in the average relative Po and relative I′F, as described in the legends of Figure 3 and Figure 5. (A(ii),B(ii)) Individual measurements of relative Po or relative I′F at +40 mV (filled circles) and −40 mV (open circles) with 0.2 µM FKBP12 alone (royal blue symbols) or with 0.2 µM FKBP12 + S107 (cyan symbols). (A(iii),(Biii)) Graphs of average relative Po for native RyR1 channels (n = 6) and average I′F for solubilized RyR1 channels (n =…

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  Figure 7. Normalization highlights the dominance of increases in Imean in the effects of FKBP12 on solubilized RyR1 channel activity and I′F. The relative Imean and Imax were calculated for the Imean and Imax data in Figure 5 and Figure 6. Both Imean and Imax in the presence of FKBP12 were normalized to their control value for each individual bilayer before obtaining the average relative values. Details of the experiments, including the number of observations, are the same as those in the legend in Figure 5. In (A–E), panel (i) shows the relative Imax in individual bilayers at +40 mV (filled circles) and −40 mV (open circles). Panel (ii) shows the average relative Imax. Panel (iii) shows relative Imean from individual bilayers at +40 mV (filled circles) and −40 mV (open circles). Panel (iv) shows the average relative Imean. For average data in (ii,iv), symbols show average relative values, and the s.e.m. is indicated by the vertical capped bars. Data are shown in (A) for 1–10 nM FKBP12, in (B) for 0.2 µM FKBP12, in (C) for 1.0 µM FKBP12, and in (D) for 5.0 µM FKBP12, and in (E), data for 0.2 µM FKBP12 are repeated (filled royal blue circles) for comparison with 0.2 µM FKBP12 + S107 (cyan…

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  Figure 8. The negative co-operativity model suggests that changes in subunit occupation by FKBP12 within each tetramer might explain the changes in RyR1 channel activity associated with exogenous FKBP12 binding. (A,B) illustrate patterns of FKBP12 occupation of RyR1 subunits in RyR1 tetramers from B4 vesicles (A) and solubilized RyR1 (B). The control horizontal panel for B4 vesicles shows examples of four control RyR1 tetramers before adding exogenous FKBP12.From left to right, 1 tertamer with no FkBP12 bound, 2 tetramers with 1 FKBP12 bound, and 1 tetramer with 2 FKBP12 molecules bound, approximating the mean of 1.25 (Results). The control horizontal panel for four solubilized RyR1 tetramers is shown with 3 tetramers having 0 FKBP12 molecules bound, and 1 RyR1 tetramer with 1 FKBP bound. This is an overestimate of the 0.19 tetramers with FKBP12 bound (Discussion) but is shown to indicate that a small but finite number of solubilized RyR1 channels contain 1 subunit with FKBP12 bound. 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…