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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

Author for correspondence (e-mail: geert.callewaert{at}med.kuleuven.ac.be)

Accepted 20 October 2003

We have characterised the functional regulation of the type-3 ryanodine receptor by the 12 kDa FK506-binding protein. Wild-type type-3 ryanodine receptor and mutant type-3 ryanodine receptor in which the critical valine at position 2322 in the central 12 kDa FK506-binding protein binding site was substituted by aspartate, were stably expressed in human embryonic kidney cells. In contrast to the wild-type receptor, the mutant receptor was strongly impaired in binding to immobilised glutathione S-transferase 12 kDa FK506-binding protein. Caffeine-induced ⁴⁵Ca²⁺-efflux was markedly increased in cells expressing mutant type-3 ryanodine receptor whereas the maximal-releasable Ca²⁺ was not affected. Confocal Ca²⁺ imaging provided clear evidence for a much higher sensitivity of the mutant receptor, which showed global Ca²⁺ release at about 20-fold lower caffeine concentrations than the wild-type receptor. Spontaneous Ca²⁺ sparks were observed in both wild-type- and mutant-expressing cells but the number of sparking cells was about 1.5-fold higher in the mutant group, suggesting that the degree of FK506 binding controls the stability of the closed state of ryanodine receptor channels. Furthermore, overexpression of 12 kDa FK506-binding protein decreased the number of sparking cells in the wild-type-expressing cells whereas it did not affect the number of sparking cells in cells expressing the mutant receptor. Concerning spark properties, the amplitude and duration of Ca²⁺ sparks mediated by mutant channels were significantly reduced in comparison to wild-type channels. This suggests that functional coupling between different mutant type-3 ryanodine receptor channels in a cluster is impaired. Our findings show for the first time that the central binding site for the 12 kDa FK506-binding protein of type-3 ryanodine receptor, encompassing the critical valine proline motif, plays a crucial role in the modulation of the Ca²⁺ release properties of the type-3 ryanodine receptor channel, including the regulation of both global Ca²⁺ responses and spontaneous Ca²⁺ sparks.

Key words: 12 kDa FK506-binding protein, Ryanodine receptor type 3, Ca²⁺ signalling

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