Tabcharani J A, Boucher A, Eng J W, Hanrahan J W
Department of Physiology, McGill University, Montreal, Quebec, Canada.
J Membr Biol. 1994 Nov;142(2):255-66. doi: 10.1007/BF00234947.
Agonists that elevate calcium in T84 cells stimulate chloride secretion by activating KBIC, an inwardly rectifying K channel in the basolateral membrane. We have studied the regulation of this channel by calcium, nucleotides and phosphorylation using patch clamp and short-circuit current (ISC) techniques. Open probability (Po) was independent of voltage but declined spontaneously with time after excision. Rundown was slower if patches were excised into a bath solution containing ATP (10 microM-5 mM), ATP (0.1 mM)+protein kinase A (PKA; 180 nM), or isobutylmethylxanthine (IBMX; 1 mM). Analysis of event durations suggested that the channel has at least two open and two closed states, and that rundown under control conditions is mainly due to prolongation of the long closed time. Channel activity was restimulated after rundown by exposure to ATP, the poorly hydrolyzable ATP analogue AMP-PNP, or ADP. Activity was further enhanced when PKA was added in the presence of MgATP, but only if free calcium concentration was elevated (400 nM). Nucleotide stimulation and inward rectification were both observed in nominally Mg-free solutions. cAMP modulation of basolateral potassium conductance in situ was confirmed by measuring currents generated by a transepithelial K gradient after permeabilization of the apical membrane using alpha-toxin. Finally, protein kinase C (PKC) inhibited single KBIC channels when it was added directly to excised patches. These results suggest that nonhydrolytic binding of nucleotides and phosphorylation by PKA and PKC modulate the responsiveness of the inwardly rectifying K channel to Ca-mediated secretagogues.
可提高T84细胞内钙离子水平的激动剂通过激活KBIC（一种位于基底外侧膜的内向整流钾通道）来刺激氯离子分泌。我们使用膜片钳和短路电流（ISC）技术研究了钙离子、核苷酸和磷酸化对该通道的调节作用。开放概率（Po）与电压无关，但在膜片切除后会随时间自发下降。如果将膜片切除到含有ATP（10微摩尔/升 - 5毫摩尔/升）、ATP（0.1毫摩尔/升）+蛋白激酶A（PKA；180纳摩尔/升）或异丁基甲基黄嘌呤（IBMX；1毫摩尔/升）的浴液中，通道电流衰减会更慢。对事件持续时间的分析表明，该通道至少有两个开放状态和两个关闭状态，在对照条件下的电流衰减主要是由于长关闭时间的延长。在电流衰减后，通过暴露于ATP、难以水解的ATP类似物AMP - PNP或ADP可重新刺激通道活性。当在MgATP存在的情况下加入PKA时，活性会进一步增强，但前提是游离钙离子浓度升高（400纳摩尔/升）。在名义上无镁的溶液中也观察到了核苷酸刺激和内向整流现象。使用α - 毒素使顶端膜通透后，通过测量跨上皮钾梯度产生的电流，证实了原位cAMP对基底外侧钾电导的调节作用。最后，当蛋白激酶C（PKC）直接添加到切除的膜片中时，它会抑制单个KBIC通道。这些结果表明，核苷酸的非水解性结合以及PKA和PKC的磷酸化作用可调节内向整流钾通道对钙介导的促分泌剂的反应性。
