The Donnan potential between cytoplasm and pipette in the whole-cell mode can account for the difference in cell-attached and current-clamp V_m estimates.A, Three superimposed cell-attached current traces from a cell with spontaneous single-channel activity (K_“ATP”). Two independent estimates forV_m can be obtained from these records. With the single-channel amplitude at V_h = −60 mV and the single K_“ATP” channel conductance calculated from the slope of the open levels during the voltage ramp stimulation, the driving force for K⁺ flux (V_patch −E_K; withV_patch = V_h +V_m and E_K ≈ 0 mV) amounts to 10.8 pA/84 pS = 128.6 mV atV_h = −60 mV, yielding an estimatedV_m of −68.6 mV. The value determined forV_m from reversal of the macroscopic K(V) current was −69.2 mV. The close agreement between these two values indicates that the ramp stimulation does not significantly affectV_m. B, Subsequent whole-cell current-clamp measurement in the same cell gave a value of −53 ± 3 mV for V_m. C1, Evidence for a change in Donnan junction potential with time after the establishment of whole-cell recordings in the same cell as A andB. Whole-cell K(V) currents activated by ramp stimuli showed a gradual hyperpolarizing shift in voltage dependence.C2, Both the activation threshold (open circles) and the voltage corresponding to the half-maximal current (filled circles) shifted by approximately −15 mV during the first 4–8 min after break-in. Current traces were normalized to compensate for a partial rundown of the K(V) current (C1, inset). The slice was from a 15-d-old rat.