McKinney L C, Gallin E K
Department of Physiology, Armed Forces Radiobiology Research Institute, Bethesda, Maryland 20889-5145.
J Membr Biol. 1992 Dec;130(3):265-76. doi: 10.1007/BF00240483.
The whole-cell patch clamp technique was used to test whether intracellular application of G-protein activators affect ionic currents in murine macrophages. Both the J774.1 macrophage-like cell line and primary bone marrow derived macrophages were used. Cells were bathed in Na Hanks' solution and intracellularly dialyzed (via the patch pipette) with K Hanks (145 mM KCl, < 100 nM Ca) plus or minus the G-protein activators GTP gamma S (10 microM), GppNHp (10 microM), or AIF4- (200 microM AlCl3 + 5 mM KF). In the absence of G-protein activators, only two K currents, an inwardly rectifying K current (Kir) and an outward, inactivating K current (Ko) were observed. In the presence of protein activators, two effects were observed: (i) the Kir conductance, which is stable for up to 30 min under control conditions, decayed twice as fast and (ii) an outwardly rectifying, noninactivating current appeared. The induced outward current appeared < 2 min after attaining the whole-cell patch clamp configuration. The current could be distinguished from the Kir and Ko currents on the basis of its direction of rectification (outward), barium sensitivity (> 1 mM), and kinetics (no time-dependent inactivation). Intracellular application of GTP (500 microM), GDP (500 microM), cAMP (100 microM + 0.5 mM ATP), or IP3 (20 microM) did not induce the current; 100 microM ATP gamma S activated a half-maximal amount of current. Induction of outward current by 10 microM GTP gamma S could be prevented by pre-exposing cells to pertussis toxin but not cholera toxin. This current is K selective since (i) its induction was accompanied by hyperpolarization of the cell toward EK, even after Kir had "washed out", (ii) it was present after > 90% of both intracellular and extracellular Cl were replaced by isethionate, and (iii) the induced outward conductance was absent when Ki was completely replaced by Cs, and was reduced by approximately 1/3 when [K]i was reduced by 1/3. Quinidine (1 mM) and 4-aminopyridine (10 mM) inhibited the current, but apamin (1 microM) and charybdotoxin (1 microM) did not.
采用全细胞膜片钳技术来检测向小鼠巨噬细胞内施加G蛋白激活剂是否会影响离子电流。使用了J774.1巨噬细胞样细胞系和原代骨髓来源的巨噬细胞。细胞浸浴在Na Hanks溶液中，并通过膜片电极向细胞内透析（通过膜片移液管）K Hanks溶液（145 mM KCl，<100 nM Ca），并添加或不添加G蛋白激活剂GTPγS（10 μM）、GppNHp（10 μM）或AlF4-（200 μM AlCl3 + 5 mM KF）。在没有G蛋白激活剂的情况下，仅观察到两种钾电流，一种内向整流钾电流（Kir）和一种外向、失活的钾电流（Ko）。在存在蛋白激活剂的情况下，观察到两种效应：（i）在对照条件下稳定达30分钟的Kir电导衰减速度加快了两倍；（ii）出现了一种外向整流、非失活电流。诱导的外向电流在达到全细胞膜片钳配置后不到2分钟出现。根据其整流方向（外向）、钡敏感性（>1 mM）和动力学（无时间依赖性失活），该电流可与Kir和Ko电流区分开来。向细胞内施加GTP（500 μM）、GDP（500 μM）、cAMP（100 μM + 0.5 mM ATP）或IP3（20 μM）均未诱导出该电流；100 μM ATPγS激活了半数最大电流。预先将细胞暴露于百日咳毒素可阻止10 μM GTPγS诱导外向电流，但霍乱毒素则不能。该电流具有钾选择性，因为（i）即使在Kir“洗脱”后，其诱导仍伴随着细胞向EK方向的超极化；（ii）在细胞内和细胞外的Cl均被羟乙基磺酸替代>90%后该电流仍存在；（iii）当Ki完全被Cs替代时，诱导的外向电导消失，当[K]i降低1/3时，外向电导降低约1/3。奎尼丁（1 mM）和4-氨基吡啶（10 mM）可抑制该电流，但蜂毒明肽（1 μM）和蝎毒素（1 μM）则不能。
