FK506 binding proteins 12 and 12.6 (FKBP12 and FKBP12.6) are intracellular receptors for the immunosuppressant drug FK506 (ref. 1). The skeletal muscle ryanodine receptor (RyR1) is isolated as a hetero-oligomer with FKBP12 (ref. 2), whereas the cardiac ryanodine receptor (RyR2) more selectively associates with FKBP12.6 (refs 3, 4, 5). FKBP12 modulates Ca2+ release from the sarcoplasmic reticulum in skeletal muscle and developmental cardiac defects have been reported in FKBP12-deficient mice, but the role of FKBP12.6 in cardiac excitation-contraction coupling remains unclear. Here we show that disruption of the FKBP12.6 gene in mice results in cardiac hypertrophy in male mice, but not in females. Female hearts are normal, despite the fact that male and female knockout mice display similar dysregulation of Ca2+ release, seen as increases in the amplitude and duration of Ca2+ sparks and calcium-induced calcium release gain. Female FKBP12.6-null mice treated with tamoxifen, an oestrogen receptor antagonist, develop cardiac hypertrophy similar to that of male mice. We conclude that FKBP12.6 modulates cardiac excitation-contraction coupling and that oestrogen plays a protective role in the hypertrophic response of the heart to Ca2+ dysregulation.

SummaryIn arterial myocytes the Ca 2+ mobilizing messenger NAADP evokes spatially restricted Ca 2+ bursts from a lysosome-related store that are subsequently amplified into global Ca 2+ waves by Ca 2+ -induced Ca 2+ -release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs). Lysosomes facilitate this process by forming clusters that co-localize with a subpopulation of RyRs on the SR. We determine here whether RyR subtypes 1, 2 or 3 selectively co-localize with lysosomal clusters in pulmonary arterial myocytes using affinity purified specific antibodies. The density of: (1) αlgP120 labelling, a lysosome-specific protein, in the perinuclear region of the cell (within 1.5 μm of the nucleus) was ~4-fold greater than in the sub-plasmalemmal (within 1.5 μm of the plasma membrane) and ~2-fold greater than in the extra-perinuclear (remainder) regions; (2) RyR3 labelling within the perinuclear region was ~4-and ~14-fold greater than that in the extraperinuclear and sub-plasmalemmal regions, and ~2-fold greater than that for either RyR1 or RyR2; (3) despite there being no difference in the overall densities of fluorescent labelling of lysosomes and RyR subtypes between cells, co-localization with αlgp120 labelling within the perinuclear region was ~2-fold greater for RyR3 than for RyR2 or RyR1; (4) co-localization between αlgp120 and each RyR subtype declined markedly outside the perinuclear region. Furthermore, selective block of RyR3 and RyR1 with dantrolene (30μM) abolished global Ca 2+ waves but not Ca 2+ bursts in response to intracellular dialysis of NAADP (10nM). We conclude that a subpopulation of lysosomes cluster in the perinuclear region of the cell and form junctions with SR containing a high density of RyR3 to comprise a trigger zone for Ca 2+ signalling by NAADP.

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The ryanodine receptors are intracellular Ca 2؉ release channels that play a key role in cell signaling via Ca 2؉ . There are three isoforms. Isoform 1 from skeletal muscle and isoform 2 from heart have been characterized. Isoform 3 is widely distributed in many mammalian tissues although in minuscule amounts. Its low abundance has hampered its study. We now describe methodology to isolate mammalian isoform 3 in amounts sufficient for biochemical and biophysical characterization. Bovine diaphragm sarcoplasmic reticulum fractions enriched in terminal cisternae containing both isoforms 1 (>95%) and 3 (<5% of the ryanodine binding) served as starting source. Isoform 3 was selectively immunoprecipitated from the 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid (CHAPS)-solubilized fraction and eluted with peptide epitope. Isoform 3 thus prepared is highly purified as characterized by SDS-polyacryamide gel electrophoresis, Coomassie Blue staining, and by high affinity ryanodine binding. The purified isoform 3 was incorporated into planar lipid bilayers, and its channel properties were studied. Channel characteristics in common with the other two isoforms are slope conductance, higher selectivity to Ca 2؉ versus K ؉ (P Ca/K ϳ6), and response to drugs and ligands. In its response to Ca 2؉ and ATP, it more closely resembles isoform 2. The first two-dimensional structure of isoform 3 was obtained by cryoelectron microscopy and image enhancement techniques.

Recent studies show that three subtypes of RyRs are coexpressed and RyR-gated Ca 2ϩ stores are distributed heterogeneously in systemic vascular myocytes. However, the molecular identity and subcellular distribution of RyRs have not been examined in PASMCs. In this study we detected mRNA and proteins of all three subtypes in rat intralobar PASMCs using RT-PCR and Western blot. Quantitative real-time RT-PCR showed that RyR2 mRNA was most abundant, ϳ15-20 times more than the other two subtypes. Confocal fluorescence microscopy revealed that RyRs labeled with BODIPY TR-X ryanodine were localized in the peripheral and perinuclear regions and were colocalized with sarcoplasmic reticulum labeled with Fluo-5N. Immunostaining showed that the subsarcolemmal regions exhibited clear signals of RyR1 and RyR2, whereas the perinuclear compartments contained mainly RyR1 and RyR3. Ca 2ϩ sparks were recorded in both regions, and their activities were enhanced by a subthreshold concentration of caffeine or by endothelin-1, indicating functional RyR-gated Ca 2ϩ stores. Moreover, 18% of the perinuclear sparks were prolonged [full duration/half-maximum (FDHM) ϭ 193.3 Ϯ 22.6 ms] with noninactivating kinetics, in sharp contrast to the typical fast inactivating Ca 2ϩ sparks (FDHM ϭ 44.6 Ϯ 3.2 ms) recorded in the same PASMCs. In conclusion, multiple RyR subtypes are expressed differentially in peripheral and perinuclear RyR-gated Ca 2ϩ