Horn R, Marty A
Laboratoire de Neurobiologie, Ecole Normale Supèrieure, Paris, France.
J Gen Physiol. 1988 Aug;92(2):145-59. doi: 10.1085/jgp.92.2.145.
A new method is described as an alternative to whole-cell recording in order to prevent "wash-out" of the muscarinic response to acetylcholine (ACh) in rat lacrimal gland cells. The membrane of a cell-attached patch is permeabilized by nystatin in the patch pipette, thus providing electrical continuity between the pipette and the cytoplasm of the cell without the loss or alteration of cytoplasmic compounds necessary for the maintenance of the response to ACh. With normal whole-cell recording in these cells, the response to ACh, seen as the activation of Ca-activated K and Cl currents, lasts for approximately 5 min. With the nystatin method, the response is not diminished after 1 h. Nystatin, applied extracellularly, is shown to cause a rapid and reversible increase of membrane conductance to cations. In the absence of wash-out, we were able to obtain dose-response curves for the effect of ACh on Ca-activated K currents. An increase of [ACh] caused an increase in the K current, with apparent saturation at concentrations above approximately 1 microM ACh. The delay between ACh application and the activation of K current was inversely related to [ACh] and reached a minimum value of 0.7-1.0 s at high [ACh].
本文描述了一种新方法，作为全细胞记录的替代方法，以防止大鼠泪腺细胞对乙酰胆碱（ACh）的毒蕈碱反应出现“洗脱”现象。通过在膜片吸管中加入制霉菌素使细胞贴附膜片的膜通透化，从而在吸管与细胞胞质之间提供电连续性，而不会丢失或改变维持对ACh反应所需的胞质化合物。在这些细胞中进行正常全细胞记录时，对ACh的反应（表现为钙激活钾电流和氯电流的激活）持续约5分钟。使用制霉菌素方法时，1小时后反应不会减弱。研究表明，细胞外应用制霉菌素会导致膜对阳离子的电导迅速且可逆地增加。在不存在“洗脱”的情况下，我们能够获得ACh对钙激活钾电流影响的剂量反应曲线。[ACh]的增加会导致钾电流增加，在ACh浓度高于约1 microM时出现明显的饱和现象。施加ACh与激活钾电流之间的延迟与[ACh]呈负相关，在高[ACh]时达到最小值0.7 - 1.0秒。
