Determination of gap junctional intercellular communication by capacitance measurements

Abstract

Electrical coupling between cells is usually measured using the double-patch-clamp technique with cell pairs. Here, a single patch-clamp technique that is not limited to cell pairs is described to determine electrical coupling between cells. Capacitance measurements in clusters of normal rat kidney (NRK) fibroblasts were used to study intercellular communication. In the whole-cell patch-clamp configuration capacitive transients were evoked by applying small voltage pulses. Total membrane capacitance was calculated from these capacitive transients after determination of access resistance, membrane conductance, and the decay constant of the transients, or alternatively by integrating the current transient. We found that in clusters of one to ten cells, membrane capacitance increased linearly with cell number, showing that the cells are electrically coupled. Membrane conductance of the cluster of cells also increased, as expected for cells that are well coupled. In subconfluent and confluent cultures, high membrane conductances together with large capacitive transients were observed, indicative of electrical coupling. Capacitance could only be determined qualitatively under these conditions, due to space clamp problems. In the presence of the gap junctional inhibitors halothane, heptanol or octanol, capacitance of all clusters of cells fell to single-cell levels, showing a complete uncoupling of the cells. The tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also uncoupled the cells completely, with 10 min. We conclude that capacitance measurements can provide a useful tool to study changes in intercellular communication in clusters of cells.