کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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5530539 | 1549310 | 2017 | 13 صفحه PDF | دانلود رایگان |
- We generated FKBP-YCaMP and FKBP-GCaMP6 as genetically engineered RyR2-targeted probes.
- FKBP-YCaMP signals were too small (â¼20%) and too slow (2-3Â s) to detect Ca2+-sparks.
- FKBP-GCaMP6 had sufficient on-rate to monitor dyadic Ca2+ sparks in the Z-lines.
- Isoproterenol did not dissociate FKBP-GCaMP6 from z-lines; enhanced Ca2+ spark frequency.
The details of cardiac Ca2+ signaling within the dyadic junction remain unclear because of limitations in rapid spatial imaging techniques, and availability of Ca2+ probes localized to dyadic junctions. To critically monitor ryanodine receptors' (RyR2) Ca2+ nano-domains, we combined the use of genetically engineered RyR2-targeted pericam probes, (FKBP-YCaMP, Kd = 150 nM, or FKBP-GCaMP6, Kd = 240 nM) with rapid total internal reflectance fluorescence (TIRF) microscopy (resolution, â¼80 nm). The punctate z-line patterns of FKBP,2-targeted probes overlapped those of RyR2 antibodies and sharply contrasted to the images of probes targeted to sarcoplasmic reticulum (SERCA2a/PLB), or cytosolic Fluo-4 images. FKBP-YCaMP signals were too small (â¼20%) and too slow (2-3 s) to detect Ca2+ sparks, but the probe was effective in marking where Fluo-4 Ca2+ sparks developed. FKBP-GCaMP6, on the other hand, produced rapidly decaying Ca2+ signals that: a) had faster kinetics and activated synchronous with ICa3 but were of variable size at different z-lines and b) were accompanied by spatially confined spontaneous Ca2+ sparks, originating from a subset of eager sites. The frequency of spontaneously occurring sparks was lower in FKBP-GCaMP6 infected myocytes as compared to Fluo-4 dialyzed myocytes, but isoproterenol enhanced their frequency more effectively than in Fluo-4 dialyzed cells. Nevertheless, isoproterenol failed to dissociate FKBP-GCaMP6 from the z-lines. The data suggests that FKBP-GCaMP6 binds predominantly to junctional RyR2s and has sufficient on-rate efficiency as to monitor the released Ca2+ in individual dyadic clefts, and supports the idea that β-adrenergic agonists may modulate the stabilizing effects of native FKBP on RyR2.
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Journal: Cell Calcium - Volume 66, September 2017, Pages 98-110