Grygorczyk R, Tabcharani J A, Hanrahan J W
Department of Physiology, McGill University, 3655 Drummond Street, Montréal, Québec, Canada H3G 1Y6.
J Membr Biol. 1996 May;151(2):139-48. doi: 10.1007/s002329900065.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated, ATP-dependent chloride channel which may have additional functions. Recent reports that CFTR mediates substantial electrodiffusion of ATP from epithelial cells have led to the proposal that CFTR regulates other ion channels through an autocrine mechanism involving ATP. The aim of this study was to determine the ATP conductance of wild-type CFTR channels stably expressed in Chinese hamster ovary cells using patch clamp techniques. In the cell-attached configuration with 100 mM Mg middle dot ATP or Tris middle dot ATP solution in the pipette and 140 mM NaCl in the bath, exposing cells to forskolin caused the activation of a low-conductance channel having kinetics resembling those of CFTR. Single channel currents were negative at the resting membrane potential (Vm), consistent with net diffusion of Cl from the cell into the pipette. The transitions decreased in amplitude, but did not reverse direction, as Vm was clamped at increasingly positive potentials to enhance the driving force for inward ATP flow (>+80 mV). In excised patches, single channel currents did not reverse under essentially biionic conditions (Clin/ATPout or ATPin/Clout), although PKA-activated currents were clearly visible in the same patches at voltages where they would be carried by chloride ions. Moreover, with NaCl solution in the bath and a mixture of ATP and Cl in the pipette, the single channel I/V curve reversed at the predicted equilibrium potential for chloride. CFTR channel currents disappeared when patches were exposed to symmetrical ATP solutions and were restored by reexposure to Cl solution. Finally, in the whole-cell configuration with NaCl in the bath and 100 mM MgATP or TrisATP in the pipette, cAMP-stimulated cells had time-independent, outwardly rectifying currents consistent with CFTR selectivity for external Cl over internal ATP. Whole-cell currents reversed near Vm = -55 mV under these conditions, however the whole cell resistance measured at -100 mV was comparable to that of the gigaohm seal between the plasma membrane and glass pipette (7 Gomega). We conclude that CFTR does not mediate detectable electrodiffusion of ATP.
囊性纤维化跨膜传导调节因子（CFTR）是一种由环磷酸腺苷（cAMP）激活、依赖三磷酸腺苷（ATP）的氯离子通道，可能还具有其他功能。最近有报道称，CFTR介导上皮细胞中ATP的大量电扩散，这导致有人提出CFTR通过涉及ATP的自分泌机制调节其他离子通道。本研究的目的是使用膜片钳技术测定稳定表达于中国仓鼠卵巢细胞中的野生型CFTR通道的ATP传导性。在细胞贴附模式下，移液管中含有100 mM Mg·ATP或Tris·ATP溶液，浴槽中含有140 mM NaCl，将细胞暴露于福斯可林会激活一个低电导通道，其动力学特性与CFTR相似。在静息膜电位（Vm）下，单通道电流为负，这与氯离子从细胞净扩散到移液管中一致。随着Vm被钳制在越来越正的电位以增强ATP内向流动的驱动力（>+80 mV），电流跃迁幅度减小，但方向未反转。在切除的膜片中，在基本双离子条件下（细胞内氯离子/细胞外ATP或细胞内ATP/细胞外氯离子），单通道电流未反转，尽管在相同膜片中，蛋白激酶A（PKA）激活的电流在氯离子携带电流的电压下清晰可见。此外，当浴槽中为NaCl溶液，移液管中为ATP和氯离子的混合物时，单通道电流-电压（I/V）曲线在预测的氯离子平衡电位处反转。当膜片暴露于对称的ATP溶液时，CFTR通道电流消失，重新暴露于氯离子溶液后恢复。最后，在全细胞模式下，浴槽中为NaCl，移液管中为100 mM MgATP或TrisATP，cAMP刺激的细胞具有与CFTR对细胞外氯离子相对于细胞内ATP的选择性一致的时间无关的外向整流电流。在这些条件下，全细胞电流在Vm = -55 mV附近反转，然而在-100 mV处测量的全细胞电阻与质膜和玻璃移液管之间的千兆欧封接电阻相当（7 GΩ）。我们得出结论，CFTR不介导可检测到的ATP电扩散。
