Solsona C, Innocenti B, Fernández J M
Department of Cell Biology, Medical School, University of Barcelona, Spain.
Biophys J. 1998 Feb;74(2 Pt 1):1061-73. doi: 10.1016/S0006-3495(98)74030-5.
We have inflated patch-clamped mast cells by 3.8 +/- 1.6 times their volume by applying a hydrostatic pressure of 5-15 cm H2O to the interior of the patch pipette. Inflation did not cause changes in the cell membrane conductance and caused only a small reversible change in the cell membrane capacitance (36 +/- 5 fF/cm H2O). The specific cell membrane capacitance of inflated cells was found to be 0.5 microF/cm2. High-resolution capacitance recordings showed that inflation reduced the frequency of exocytotic fusion events by approximately 70-fold, with the remaining fusion events showing an unusual time course. Shortly after the pressure was returned to 0 cm H2O, mast cells regained their normal size and appearance and degranulated completely, even after remaining inflated for up to 60 min. We interpret these observations as an indication that inflated mast cells reversibly disassemble the structures that regulate exocytotic fusion. Upon returning to its normal size, the cell cytosol reassembles the fusion pore scaffolds and allows exocytosis to proceed, suggesting that exocytotic fusion does not require soluble proteins. Reassembly of the fusion pore can be prevented by inflating the cells with solutions containing the protease pronase, which completely blocked exocytosis. We also interpret these results as evidence that the activity of the fusion pore is sensitive to the tension of the plasma membrane.
我们通过向膜片吸管内部施加5 - 15 cm H₂O的静水压力，将肥大细胞的体积膨胀至其原有体积的3.8±1.6倍。膨胀并未引起细胞膜电导的变化，仅使细胞膜电容产生了微小的可逆变化（36±5 fF/cm H₂O）。发现膨胀后细胞的比细胞膜电容为0.5 μF/cm²。高分辨率电容记录显示，膨胀使胞吐融合事件的频率降低了约70倍，其余融合事件呈现出异常的时间进程。压力恢复到0 cm H₂O后不久，肥大细胞恢复正常大小和外观，并完全脱颗粒，即使在膨胀长达60分钟后也是如此。我们将这些观察结果解释为膨胀的肥大细胞可逆地拆解调节胞吐融合的结构的迹象。恢复正常大小后，细胞胞质溶胶重新组装融合孔支架并使胞吐作用得以进行，这表明胞吐融合不需要可溶性蛋白质。用含有蛋白酶链霉蛋白酶的溶液使细胞膨胀可阻止融合孔的重新组装，这完全阻断了胞吐作用。我们还将这些结果解释为融合孔的活性对质膜张力敏感的证据。
