CFTR channels expressed in CHO cells do not have detectable ATP conductance

被引:77
作者
Grygorczyk, R
Tabcharani, JA
Hanrahan, JW
机构
[1] Department of Physiology, McGill University, Montréal, Que. H3G 1Y6
关键词
CFTR; cystic fibrosis; chloride channel; ATP conductance; secretion;
D O I
10.1007/s002329900065
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 . ATP or Tris . 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 (V-m), consistent with net diffusion of Cl from the cell into the pipette. The transitions decreased in amplitude, but did not reverse direction, as V-m 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 (Cl-in/ATP(out) or ATP(in)/Cl-out), 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 V-m = -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 G Omega). We conclude that CFTR does not mediate detectable electrodiffusion of ATP.
引用
收藏
页码:139 / 148
页数:10
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