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First published online 21 September 2005
doi: 10.1242/jcs.02582

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Ryanodine receptor binding to FKBP12 is modulated by channel activation state

Wales Heart Research Institute, Department of Cardiology, School of Medicine, Cardiff University, Heath Park, Cardiff, CF14 4XN, UK

View larger version (29K): [in a new window]   Fig. 1. Depletion of endogenous FKBP12 from RyR1 preparations and ELISA binding assays. (A) Western blot of the RyR1 preparation (see Materials and Methods). Lane A, 10 µg recombinant FKBP; lane B, 10 µg RyR1 preparation; lanes C-G, recombinant GST-FKBP12 at 26 nM, 52 nM, 130 nM, 260 nM and 520 nM (20 µl loaded) to test the detection limit of the anti-FKBP12 Ab. Binding of GST-FKBP12 (in solution) to RyR1 coated onto ELISA plate was measured using different agents (see Materials and Methods). (B) Binding dependence on free Ca2+ concentration. Data is normalised by subtracting the binding value for EGTA (<0.01 µM free Ca2+). Open squares indicate Ca2+ buffer alone and the filled squares denote Ca2+ + 1 mM ATP. (C) Dose response to caffeine (open circles) or ruthenium red (filled triangles). Data is normalised by subtracting the binding in buffer alone. n = 5 ± s.e.m., *P<0.05, Student's paired t test. (D) [3H]Ryanodine binding to RyR1 preparations using the various free Ca2+ concentrations ± ATP, caffeine or ruthenium red. White bars indicate conditions favouring high open probability of the channel and grey bars indicate conditions promoting channel closing (Meissner et al., 1988). Data was normalised by dividing the binding values with that in the presence of EGTA. n = 5 ± s.e.m., *P<0.05 compared with the caffeine value, Student's paired t test.

View larger version (21K): [in a new window]   Fig. 2. Surface plasmon resonance sensorgrams and specificity of RyR1 binding to GST-FKPB12. (A) Four representative sensorgrams (in 1 µM Ca2+) of four doubling dilutions of RyR (highest concentration 250 µg/ml). GST-FKBP12 was bound to a Biacore CM3 sensor chip (to 100 RU, not shown) and RyR1 flowed over the surface (30 µl/min, kinetic inject mode). The first arrow indicates when RyR1 contacts the chip followed by the binding curve. The second arrow shows a switch to binding buffer alone and dissociation begins. The vertical RU shift at the arrows results from refractive index change due to the presence of RyR1. BIA Evaluation software analyses the data set and calculates the kinetic constants (see Fig. 3). (B) Either GST-FKBP12 or GST (100 RU) was bound to the sensor chip (not shown). RyR1, 125 µg/ml, was flowed over the surface (30 µl/min). There was binding to GST-FKBP12, but not to GST. (C) A series of RyR1 concentrations was exposed to GST-FKBP12 (as in A) in the presence (open circles) and absence (filled circles) of the FKBP12 inhibitor FK506 (1 µM). Maximum binding was calculated for each concentration. (D) The effect of premixing RyR1 with recombinant FKBP12. The molar ratio of FKBP12:RyR1 was calculated from the protein values and plotted as a percentage of binding in the absence of FKBP12.

View larger version (32K): [in a new window]   Fig. 3. Equilibrium kinetics of the GST-FKBP and RyR1 interaction. (A) The Langmuir equation for equilibrium kinetics and definition of the constants, where [A] and [B] are the molar concentrations of interactants and [AB] the molar concentration of the product. Constants KA, ka and kd were calculated by BIA Evaluation software for kinetic experiments, as illustrated in Fig. 2. For each data set (B-D), the white and grey bars denote conditions where the RyR1 channel was open or closed, respectively (cf ryanodine binding, Fig. 1). (B) The equilibrium constant, KA. The data shows that affinity is greatest when the channel is closed. (C) The association constant, ka, which overall shows no substantial variation nor segregation with channel open and closed states. (D) The dissociation constant, kd, which was greatest when the RyR1 channel was open. n = 4 ± s.e.m., *P<0.05 compared with the EGTA value, Student's unpaired t test.

View larger version (26K): [in a new window]   Fig. 4. Surface plasmon resonance measurements – proportion of RyR1 binding to GST-FKBP12. RyR1 binding was normalised for GST-FKBP binding to the chip surface in each binding cycle. (A) Relationship between RyR1 bound (RU of RyR1 bound/RU of GST-FKBP12 bound to the chip) and RyR1 added. RyR1 was flowed over the chip in binding buffer containing EGTA (open circles), 1 mM Ca2+ (open squares), 1 µM Ca2+ (filled diamonds) and 1 µM Ca2+ + ATP (filled triangles). (B) The results for 1 µM ruthenium red (open circles) and 10 mM caffeine (filled diamonds). (C,D) Average of data from four experiments, with white and grey columns denoting agents that open or close the channel, respectively. C compares 1 µM Ca2+, 1 µM Ca2+ + ATP, and 1 mM Ca2+ with the value for 1 mM EGTA. D compares the binding with 10 mM caffeine with that for 1 µM ruthenium red. n = 4 ± s.e.m., *P<0.05, unpaired Student's t test.