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First published online 17 February 2004
doi: 10.1242/jcs.00948

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The 12 kDa FK506-binding protein, FKBP12, modulates the Ca²⁺-flux properties of the type-3 ryanodine receptor

Kristel Van Acker^1,*, Geert Bultynck^1,*, Daniela Rossi², Vincenzo Sorrentino², Noel Boens³, Ludwig Missiaen¹, Humbert De Smedt¹, Jan B. Parys¹ and Geert Callewaert^1,

¹ Laboratorium voor Fysiologie, Campus Gasthuisberg O/N, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium
² Section of Molecular Medicine, Department of Neuroscience, University of Siena, via Aldo Moro 5, I-53100 Siena, Italy
³ Afdeling Fotochemie en Spectroscopie, Departement Chemie, Celestijnenlaan 200F, B-3001 Heverlee, Belgium

View larger version (39K): [in a new window]   Fig. 1. Expression and FKBP12-binding properties of wtRyR3 and V2322DRyR3 channels. (A) RyR3-expression levels (upper blot) and FKBP12-expression levels (lower blot) were determined in the microsomal fraction of control, wtRyR3- and V2322DRyR3-overexpressing HEK293 cells (2 µg protein/lane and 25 µg protein/lane, respectively) by western blot analysis, using the monoclonal m34C RyR antibody (1/4000) and the polyclonal FKBP12 antibody (1/1000), respectively. (B) FKBP12 expression was detected in the microsomal fraction of wtRyR3-overexpressing cells (Micros) by western blot analysis, using the polyclonal anti-FKBP12 antibody (1/1000). Microsomes were treated with 25 µM FK506 and collected by centrifugation. The supernatants (Sn) of this fraction and the FK506-treated microsomes (Pellet) were analysed for FKBP12 expression. All lanes contained 25 µg protein. (C) Immunoblot (m34C RyR antibody; 1/3000) showing RyR3 in the microsomes of HEK293 cells (Micros; 2 µg) expressing either wtRyR3 or V2322DRyR3, the solubilised fraction (Sol; 8 µg) and the fraction retained after incubation with GST or GST-FKBP12 immobilised on glutathione-Sepharose 4B (GST or GST-FKBP12; 16 µg). Localization of RyR3 and of molecular mass markers are indicated.

View larger version (20K): [in a new window]   Fig. 2. Effect of caffeine and A23187 on Ca2+ efflux from non-permeabilised wtRyR- and V2322DRyR3-expressing HEK293 cells. (A) 45Ca2+ efflux was measured in wtRyR3 (squares) and V2322DRyR3-expressing cells (circles) in Ca2+-free medium. After 8 minutes, cells were challenged for 2 minutes with 10 mM caffeine. (B) The total-releasable Ca2+ was measured by challenging the cells with 10 µM A23187 in wtRyR3- (squares) and V2322DRyR3-expressing cells (circles) in the same medium. The results are plotted as fractional loss as a function of time. Representative traces from five independent experiments are shown. Data points are given as mean±s.d.

View larger version (18K): [in a new window]   Fig. 3. Cytosolic Ca2+ signals evoked by 1 mM caffeine in wtRyR3- and V2322DRyR3-expressing HEK293 cells. (A,B) Ca2+ responses and the corresponding first differentiate dA/dt of wtRyR3- (continuous line) and V2322DRyR3-expressing (dotted line) HEK293 cells stimulated with 1 mM caffeine as indicated by the black horizontal bar. (C,D) Ca2+ transients in V2322DRyR3-expressing HEK293 cells were characterised by a larger amplitude and a faster rate of rise than in wtRyR3-expressing cells. Amplitude (A) expressed as % F/F: 77.6±8.8, n=62 versus 140.0±15.2, n=48 in the wtRyR3- and V2322DRyR3-expressing cells (P<0.001), respectively. Rate of rise (dA/dt): 71.4±8.1, n=62 versus 107.6±11.9, n=48 in the wtRyR3- and V2322DRyR3-expressing HEK293 cells (P<0.01), respectively.

View larger version (14K): [in a new window]   Fig. 4. Dose-response curve for the caffeine-induced cytosolic Ca2+ signals. wtRyR3- (filled squares) and V2322DRyR3- (open squares) expressing HEK293 cells were stimulated with increasing caffeine concentrations. Each data point is the averaged maximal amplitude of two to three independent experiments. EC50 values amounted to 0.3 mM and 5.2 mM for the wtRyR3 and the V2322DRyR3 group, respectively. Sigmoidal fitting was performed using MicrocalTM Origin (version 6.0) software.

View larger version (33K): [in a new window]   Fig. 5. Spontaneous Ca2+ sparks in wtRyR3- and V2322DRyR3-expressing HEK293 cells. (A,B) Individual traces show time courses of spontaneous Ca2+ sparks in wtRyR3- and V2322DRyR3-expressing cells, respectively. The contour images show the corresponding regions over which the relative changes in fluorescence were integrated. (C) The bar chart shows that the percentage sparking was significantly larger in the V2322DRyR3- than in the wtRyR3-expressing cells (68.3±3.8 versus 44.4±2.7, n=18, P<0.001). (D-F) The Ca2+-spark properties, mean amplitude and duration were significantly smaller in the V2322DRyR3-expressing than in the wtRyR3-expressing cells whereas the spark frequency was not affected. Amplitude (A) expressed as % F/F: 40.2±1.3, n=109 versus 48.6±2.0, n=82 in the V2322DRyR3- and wtRyR3-expressing cells (P<0.001), respectively. Duration (seconds): 0.37±0.02, n=109 versus 0.56±0.06, n=82 in the V2322DRyR3- and wtRyR3-expressing cells (P=0.001), respectively. Frequency (per second): 1.62±0.23, n=12 versus 1.29±0.16, n=12 in the V2322DRyR3- and wtRyR3-expressing cells, respectively (P=0.24).

View larger version (17K): [in a new window]   Fig. 6. FKBP12 overexpression in wtRyR3- and V2322DRyR3-expressing HEK293 cells. (A) FKBP12 expression levels were determined in the homogenate of wtRyR3- and V2322DRyR3-expressing HEK293 cells after exogenous overexpression of FKBP12 by western blot analysis, using the polyclonal FKBP12 antibody (1/1000). FKBP12 expression was increased by 2.07±0.27 (n=4) in wtRyR3-expressing cells and 1.63±0.22 (n=4) in V2322DRyR3-expressing cells. All lanes contained 30 µg protein. (B) The bar chart shows that the percentage sparking was significantly reduced in the wtRyR3 cells overexpressing FKBP12 (44.4±2.7, n=18 versus 5.3±2.7, n=7, P<0.001) while FKBP12 overexpression did not significantly affect the percentage sparking in V2322DRyR3-expressing cells (68.3±3.8, n=18 versus 57.7±3.2, n=10, P<0.068).

View larger version (15K): [in a new window]   Fig. 7. Model for the generation of Ca2+ sparks by RyR isoforms and the regulation by FKBP12. The first line shows a schematic representation of wtRyR1, wtRyR3 and V2322DRyR3 tetramers with their putative degree of FKBP12 saturation. At resting Ca2+ levels, the affinity of FKBP12 for wtRyR1 is high (4 FKBPs/RyR), for wtRyR3 is intermediate (2 FKBPs/RyR) and for V2322DRyR3 is low (0 FKBPs/RyR). In this model, we propose that the relative FKBP12 saturation degree determines the stability of the closed state of the RyR channel and the sensitivity to channel activators, such as caffeine. This model explains the absence of spontaneous Ca2+ sparks in wtRyR1-(FKBP12)4-expressing cells and the increased occurrence of Ca2+ sparks in V2322DRyR3-expressing cells (68% of cells) as compared to wtRyR3-(FKBP12)2 expressing cells (44% of cells).

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