Whole-cell currents were investigated in the model salt-secreting epithelium, human T84 cell line, by means of the perforated patch-clamp technique. In the control extracellular medium containing Cl-, depolarizing voltage ramps evoked current responses which peaked at 5.43 +/- 0.81 pA pF-1 at +60 mV and had a reversal potential (Erev) of -38.4 +/- 2.5 mV (n = 23). 2. Activation of the cAMP pathway with forskolin increased the current at +60 mV from 3.81 +/- 0.61 to 20.79 +/- 5.08 pA pF-1 (n = 18). In thirteen cells, Erev was initially shifted towards positive potentials (Erev of the cAMP-activated initial current was -18.2 +/- 1.2 mV) and subsequently shifted towards more negative potentials, consistent with the activation of both Cl- and K+ currents during cAMP stimulation. 3. Increasing the intracellular Ca2+ concentration, [Ca2+]i, with ionomycin (1 microM) or with acetylcholine (1 microM), increased the current at +60 mV from 7.79 +/- 1.57 to 57.50 +/- 12.10 pA pF-1 (n = 6) and from 6.36 to 34.13 pA pF-1 (n = 4), respectively. With both agonists, Erev was shifted either towards the reversal potential for potassium, EK, or towards the reversal potential for chloride, ECl, depending on the cell. 4. In the absence of chloride ions (gluconate substituted), stimulation of the Ca2+ pathway activated a time-independent outward current of large amplitude. This current exhibited inward rectification at positive voltages, reverted at -89.5 +/- 0.2 mV and was markedly reduced by charybdotoxin (10 nM), a specific blocker of Ca(2+)-activated K+ channels. When a voltage step protocol was used, increased [Ca2+]i also activated an outward current at potentials more positive than -40 mV which slowly relaxed during depolarizing steps. 5. The activation of both (i) a time-dependent inwardly rectifying charybdotoxin-sensitive K+ current, and (ii) a time-dependent slowly inactivating current was also produced by cAMP stimulation. 6. We concluded that (i) in the T84 epithelial cells, both Cl- and K+ currents are concomitantly increased by secretagogue stimulation, and (ii) two different types of K+ conductances are activated by either the cAMP or the intracellular Ca2+ secreting pathways.
摘要
采用穿孔膜片钳技术，在模型泌盐上皮细胞系人T84细胞中研究全细胞电流。在含有Cl-的对照细胞外培养基中，去极化电压斜坡诱发电流响应，在+60 mV时峰值为5.43±0.81 pA pF-1，反转电位（Erev）为-38.4±2.5 mV（n = 23）。2. 用福斯可林激活cAMP途径，使+60 mV时的电流从3.81±0.61增加到20.79±5.08 pA pF-1（n = 18）。在13个细胞中，Erev最初向正电位移动（cAMP激活的初始电流的Erev为-18.2±1.2 mV），随后向更负的电位移动，这与cAMP刺激期间Cl-和K+电流的激活一致。3. 用离子霉素（1 μM）或乙酰胆碱（1 μM）增加细胞内Ca2+浓度[Ca2+]i，分别使+60 mV时的电流从7.79±1.57增加到57.50±12.10 pA pF-1（n = 6）和从6.36增加到34.13 pA pF-1（n = 4）。使用这两种激动剂时，Erev根据细胞情况要么向钾的反转电位EK移动，要么向氯的反转电位ECl移动。4. 在无氯离子（用葡萄糖酸盐替代）的情况下，Ca2+途径的刺激激活了一种时间不依赖的大幅度外向电流。该电流在正电压下表现出内向整流，在-89.5±0.2 mV时反转，并且被Ca(2+)激活的K+通道的特异性阻滞剂蝎毒素（10 nM）显著降低。当使用电压阶跃方案时，增加的[Ca2+]i也在比-40 mV更正的电位下激活外向电流，该电流在去极化阶跃期间缓慢松弛。5. cAMP刺激还产生了（i）一种时间依赖的内向整流蝎毒素敏感的K+电流和（ii）一种时间依赖的缓慢失活电流的激活。6. 我们得出结论：（i）在T84上皮细胞中，促分泌剂刺激同时增加Cl-和K+电流；（ii）cAMP或细胞内Ca2+分泌途径激活两种不同类型的K+电导。
