Kyrozis A, Reichling D B
Department of Physiology and Cellular Biophysics, Columbia University, New York, NY 10032, USA.
J Neurosci Methods. 1995 Mar;57(1):27-35. doi: 10.1016/0165-0270(94)00116-x.
The antibiotic gramicidin, when incorporated into lipid membranes, forms pores that are exclusively permeable to monovalent cations and small unchanged molecules. We report the use of gramicidin for perforated patch-clamp recordings in the whole-cell mode. Recordings were performed in cultured rat spinal cord dorsal horn neurons. Cells had stable resting potentials and series resistances for times routinely exceeding 60 min. To test if intracellular chloride concentration ([Cl]i) remains stable with this technique, we measured responses to agonists of glycine and GABAA receptors, both of which gate chloride conductances. The driving force for these responses remained stable at values that differed significantly from values that would be expected if [Cl-]i were biased towards pipette [Cl-]. We conclude that gramicidin perforated-patch recording, in addition to other properties of the perforated-patch recording technique, has the advantage of not altering [Cl-]i. It is, therefore, an electrophysiological method particularly suitable for studies of anionic channels when [Cl-]i is a variable of interest, as well as for studies of homeostatic [Cl-]i regulation.
抗生素短杆菌肽掺入脂质膜后会形成仅对单价阳离子和不带电荷的小分子具有通透性的孔道。我们报道了短杆菌肽在全细胞模式下用于穿孔膜片钳记录的应用。记录在培养的大鼠脊髓背角神经元中进行。细胞具有稳定的静息电位和串联电阻，常规情况下可保持超过60分钟。为了测试采用该技术时细胞内氯离子浓度（[Cl]i）是否保持稳定，我们测量了对甘氨酸和GABAA受体激动剂的反应，这两种受体均可控制氯离子电导。这些反应的驱动力保持稳定，其值与假设[Cl-]i偏向移液管内[Cl-]时预期的值有显著差异。我们得出结论，除了穿孔膜片钳记录技术的其他特性外，短杆菌肽穿孔膜片钳记录具有不改变[Cl-]i的优点。因此，当[Cl-]i是一个重要变量时，它是一种特别适合研究阴离子通道以及研究[Cl-]i稳态调节的电生理方法。
