SIMULTANEOUS RECORDING OF THE CELL-MEMBRANE POTENTIAL AND PROPERTIES OF THE CELL ATTACHED MEMBRANE OF HT29 COLON-CARCINOMA AND CF-PAC CELLS

Reconciliation of the properties of excised single channels and whole cell conductances is one of the major problems in the interpretation of patch clamp data. To combine cell attached and whole cell recordings we have modified the nystatin technique. Low concentrations of nystatin (less-than-or-equal-to 3 * 10(-5) mol/l) were added to the filling solution of the patch pipettes. This permeabilized the cell attached membrane partially and made it possible to measure the potential difference (PD) of the cell in current clamp mode. The input resistance (R(I)) of the cell attached patch was only slightly decreased by nystatin and stayed in the G-OMEGA range, allowing for the simultaneous recording of single channel activity and the input conductance of the cell attached membrane. This technique was examined in HT29 colon carcinoma and CF-PAC cells. In both cells it was shown that this method provides reliable PD measurements. The method was used then to test which type of Cl- channel is activated by carbachol. The PD of HT29 cells was depolarized by carbachol. The depolarization was mainly due to an increase in the Cl- conductance of the cell membrane and was followed by a slight and transient hyperpolarization. No detectable Cl- channels (conductance > 4-8 pS, 300 Hz) were activated in the cell attached membrane, but the input conductance (G0) increased concomitantly with cell depolarization. These results suggest that carbachol induces the opening of very small conductance or very rapidly opening and closing Cl- channels in these cells. The cell attached nystatin (CAN) technique, as described here, may serve as a powerful modification of the single channel patch clamp technique.