title: Dysregulated ryanodine receptors mediate cellular toxicity: restoration of normal phenotype by FKBP12.6.
abstract: Ca2+ homeostasis is a vital cellular control mechanism in which Ca2+ release from intracellular stores plays a central role. Ryanodine receptor (RyR)-mediated Ca2+ release is a key modulator of Ca2+ homeostasis, and the defective regulation of RyR is pathogenic. However, the molecular events underlying RyR-mediated pathology remain undefined. Cells stably expressing recombinant human RyR2 (Chinese hamster ovary cells, CHOhRyR2) had similar resting cytoplasmic Ca2+ levels ([Ca2+]c) to wild-type CHO cells (CHOWT) but exhibited increased cytoplasmic Ca2+ flux associated with decreased cell viability and proliferation. Intracellular Ca2+ flux increased with human RyR2 (hRyR2) expression levels and determined the extent of phenotypic modulation. Co-expression of FKBP12.6, but not FKBP12, or incubation of cells with ryanodine suppressed intracellular Ca2+ flux and restored normal cell viability and proliferation. Restoration of normal phenotype was independent of the status of resting [Ca2+]c or ER Ca2+ load. Heparin inhibition of endogenous inositol trisphosphate receptors (IP3R) had little effect on intracellular Ca2+ handling or viability. However, purinergic stimulation of endogenous IP3R resulted in apoptotic cell death mediated by hRyR2 suggesting functional interaction occurred between IP3R and hRyR2 Ca2+ release channels. These data demonstrate that defective regulation of RyR causes altered cellular phenotype via profound perturbations in intracellular Ca2+ signaling and highlight a key modulatory role of FKBP12.6 in hRyR2 Ca2+ channel function.
authors.affiliation: Department of Cardiology, Wales Heart Research Institute, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom. georgech@cf.ac.uk
authors.forename: Christopher H
authors.initials: CH
authors.lastname: George
authors.forename: Gemma V
authors.initials: GV
authors.lastname: Higgs
authors.forename: John J
authors.initials: JJ
authors.lastname: Mackrill
authors.forename: F Anthony
authors.initials: FA
authors.lastname: Lai
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000187:drug effects; D016923:Cell Death; D002455:Cell Division; D003408:Cresols / Q000494:pharmacology; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D015870:Gene Expression; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors; D010641:Phenotype; D018160:Receptors, Cytoplasmic and Nuclear / Q000187:drug effects; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000502:physiology; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction; D022021:Tacrolimus Binding Proteins / Q000235:genetics / Q000502:physiology*; D014162:Transfection
chemical_list: D015220:Calcium Channels; D003408:Cresols; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D018048:Receptors, Purinergic P2; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C006984:chlorocresol; D000255:Adenosine Triphosphate; D006493:Heparin; D022021:Tacrolimus Binding Proteins; C403484:tacrolimus binding protein 1B; D002118:Calcium