Thorn P, Petersen O H
Medical Research Council Secretory Control Research Group, University of Liverpool, United Kingdom.
J Gen Physiol. 1992 Jul;100(1):11-25. doi: 10.1085/jgp.100.1.11.
The activation of the nonselective cation channels in mouse pancreatic acinar cells has been assessed at low agonist concentrations using patch-clamp whole cell, cell-attached patch, and isolated inside-out patch recordings. Application of acetylcholine (ACh) (25-1,000 nM) and cholecystokinin (CCK) (2-10 pM) evoked oscillatory responses in both cation and chloride currents measured in whole cell experiments. In cell-attached patch experiments we demonstrate CCK and ACh evoked opening of single 25-pS cation channels in the basolateral membrane. Therefore, at least a component of the whole cell cation current is due to activation of cation channels in the basolateral acinar cell membrane. To further investigate the reported sensitivity of the cation channel to intracellular ATP and calcium we used excised inside-out patches. Micromolar Ca2+ concentrations were required for significant channel activation. Application of ATP and ADP to the intracellular surface of the patch blocked channel opening at concentrations between 0.2 and 4 mM. The nonmetabolizable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP, 0.2-2 mM), also effectively blocked channel opening. The subsequent removal of ATP caused a transient increase in channel activity not seen with the removal of ADP or AMP-PNP. Patches isolated into solutions containing 2 mM ATP showed channel activation at micromolar Ca2+ concentrations. Our results show that ATP has two separate effects. The continuous presence of the nucleotide is required for operation of the cation channels and this action seems to depend on ATP hydrolysis. ATP can also close the channel and this effect can be demonstrated in excised inside-out patches when ATP is added to the bath after a period of exposure to an ATP-free solution. This action does not require ATP hydrolysis. Under physiological conditions hormonal stimulation can open the nonselective cation channels and this can be explained by the rise in the intracellular free Ca2+ concentration.
利用膜片钳全细胞、细胞贴附式膜片和分离的内向外膜片记录技术，在低激动剂浓度下评估了小鼠胰腺腺泡细胞中非选择性阳离子通道的激活情况。在全细胞实验中，应用乙酰胆碱（ACh）（25 - 1000 nM）和胆囊收缩素（CCK）（2 - 10 pM）可诱发阳离子电流和氯离子电流的振荡反应。在细胞贴附式膜片实验中，我们证明CCK和ACh可诱发基底外侧膜中单个25 - pS阳离子通道的开放。因此，全细胞阳离子电流的至少一部分是由于腺泡细胞基底外侧膜中阳离子通道的激活所致。为了进一步研究报道的阳离子通道对细胞内ATP和钙的敏感性，我们使用了分离的内向外膜片。需要微摩尔浓度的Ca2+才能显著激活通道。将ATP和ADP应用于膜片的细胞内表面，在0.2至4 mM的浓度下可阻断通道开放。不可代谢的ATP类似物5'-腺苷酰亚胺二磷酸（AMP-PNP，0.2 - 2 mM）也能有效阻断通道开放。随后去除ATP会导致通道活性短暂增加，而去除ADP或AMP-PNP则不会出现这种情况。分离到含有2 mM ATP溶液中的膜片在微摩尔浓度的Ca2+下显示出通道激活。我们的结果表明ATP有两种不同的作用。阳离子通道的运作需要核苷酸的持续存在，这种作用似乎依赖于ATP水解。ATP也可以关闭通道，当在无ATP溶液中暴露一段时间后将ATP添加到浴液中时，这种效应可以在分离的内向外膜片中得到证明。这种作用不需要ATP水解。在生理条件下，激素刺激可打开非选择性阳离子通道，这可以用细胞内游离Ca2+浓度的升高来解释。
