METHOD FOR MANIPULATION OF CYTOSOLIC PH IN CELLS CLAMPED IN THE WHOLE-CELL OR PERFORATED-PATCH CONFIGURATIONS
METHOD FOR MANIPULATION OF CYTOSOLIC PH IN CELLS CLAMPED IN THE WHOLE-CELL OR PERFORATED-PATCH CONFIGURATIONS
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DOI：
10.1152/ajpcell.1994.267.4.c1152
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发表时间：
1994-10-01
影响因子：
5.5
通讯作者：
ROTSTEIN, OD
中科院分区：
文献类型：
--
作者：
GRINSTEIN, S;ROMANEK, R;ROTSTEIN, OD
已经开发了许多方法来控制完整细胞的细胞内pH(pH(I))。然而，当使用膜片钳技术研究细胞时，由于细胞内部与记录吸管的连续性，这种方法不适用。灌流移液管法可以用来改变整个细胞构型的pH(I)，但这种方法速度慢，技术要求高，并且在穿孔贴片构型的情况下没有用处。在这份报告中，我们介绍了一个简单的程序，使研究人员能够预测和可逆地改变在整个细胞或穿孔补片模式下夹住的细胞的pH(I)。该方法的基础是通过在吸管溶液中包含大量可渗透的弱电解质，提供几乎无限的细胞内H+(等量)供体/受体系统的储存库。该系统不仅提供了一种施加和维持选定pH(I)的方法，而且通过改变弱电解液的外部浓度，使研究人员能够在实验过程中快速且可逆地改变pH(I)或跨膜Delta pH。通过两种方法在腹膜巨噬细胞中验证了该方法的有效性：1)通过荧光比率测定直接测量单个细胞的pH(I)；2)估计H+选择性电流的反转电位。PH(I)钳制过程在整个电池构型中使用有机或无机弱碱都被证明是有效的。此外，由于NH4+/NH3可以很容易地渗透到由制霉菌素或两性霉素形成的孔中，因此该方法也适用于穿孔贴片结构。
A number of methods have been developed to manipulate the intracellular pH (pH(i)) of intact cells. However, such methods are not applicable when cells are studied using the patch-clamp technique, due to the continuity of the cell interior with the recording pipette. The perfused-pipette method can be used to modify pH(i) in the whole cell configuration, but this approach is slow, technically demanding, and not useful in the case of the perforated-patch configuration. In this report, we introduce a simple procedure that enables the investigator to predictably and reversibly alter pH(i) in cells clamped in either the whole cell or perforated-patch modes. The method is based on the provision of a virtually unlimited reservoir of an intracellular H+ (equivalent) donor/acceptor system, by inclusion of large concentrations of permeable weak electrolytes in the pipette solution. This system not only provides a means for the imposition and maintenance of a chosen pH(i) but, by changing the external concentration of the weak electrolyte, enables the investigator to rapidly and reversibly change pH(i) or the transmembrane Delta pH during the course of an experiment. The effectiveness of the procedure was validated in peritoneal macrophages by two methods: 1) direct measurement of pH(i) in single cells by fluorescence ratio determinations and 2) estimation of the reversal potential of H+-selective currents. The pH(i) clamping procedure is shown to be effective using either organic or inorganic weak bases in the whole cell configuration. In addition, because NH4+/NH3 can readily permeate the pores formed by nystatin or amphotericin, the method is also shown to apply to the perforated-patch configuration.