کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
5530491 | 1549309 | 2017 | 9 صفحه PDF | دانلود رایگان |

- Elementary Ca2+ release events imaged through TIRF differs from data driven models.
- Simulate TIRF signals from single channel and clusters to determine discrepancy's.
- Experimental TIRF signals possibly miss fast single-channel events.
High resolution total internal reflection (TIRF) microscopy (TIRFM) together with detailed computational modeling provides a powerful approach towards the understanding of a wide range of Ca2+ signals mediated by the ubiquitous inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) channel. Exploiting this fruitful collaboration further requires close agreement between the models and observations. However, elementary Ca2+ release events, puffs, imaged through TIRFM do not show the rapid single-channel openings and closings during and between puffs as are present in simulated puffs using data-driven single channel models. TIRFM also shows a rapid equilibration of 10Â ms after a channel opens or closes which is not achievable in simulation using standard Ca2+ diffusion coefficients and reaction rates between indicator dye and Ca2+. Furthermore, TIRFM imaging cannot decipher the depth of the channel with respect to the microscope, which will affect the change in fluorescence that the microscope detects, thereby affecting its sensitivity to fast single-channel activity. Using the widely used Ca2+ diffusion coefficients and reaction rates, our simulations show equilibration rates that are eight times slower than TIRFM imaging. We show that to get equilibrium rates consistent with observed values, the diffusion coefficients and reaction rates have to be significantly higher than the values reported in the literature, and predict the channel depth to be 200-250Â nm. Finally, we show that with the addition of noise, short events due to 1-2Â ms opening and closing of channels that are observed in computational models can be missed in TIRFM.
Journal: Cell Calcium - Volume 67, November 2017, Pages 65-73