The 12 kDa FK506-binding protein, FKBP12, modulates the Ca2+-flux properties of the type-3 ryanodine receptor
Author:
Van Acker Kristel1, Bultynck Geert1, Rossi Daniela2, Sorrentino Vincenzo2, Boens Noel3, Missiaen Ludwig1, De Smedt Humbert1, Parys Jan B.1, Callewaert Geert1
Affiliation:
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
Abstract
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.
Publisher
The Company of Biologists
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