Figure 1.

PMA has no effect on human neutrophils in conventional whole-cell configuration but produces dramatic effects in permeabilized patches. (A) Lack of effect of PMA in a human neutrophil studied in conventional whole-cell recording configuration. After 16 min in whole-cell configuration, 60 nM PMA was applied. Superimposed are currents during test pulses to +60 mV applied from a holding potential (V_hold) of −40 mV just before PMA was added and 23 min later. The bath and pipette solutions were identical to those used to study PMA effects in permeabilized-patch recordings (see Methods). (B) Creating an NH₄⁺ gradient has been shown to establish pH_i in whole-cell configuration (13, 17). To validate this approach for amphotericin-permeabilized patch recording, we determined the reversal potential, V_rev, of H⁺ currents in human neutrophils. Average V_rev data are plotted against the Nernst potential for NH₄⁺, E_NH4⁺, which ideally is the same as E_H if the NH₄⁺ gradient successfully establishes pH_i. Mean ±SD values of V_rev are plotted for 5, 12, 15, and 51 neutrophils (Left to Right) in 1-, 3-, 15-, and 50-mM NH₄⁺ bath solutions, respectively. These data confirm that NH₄⁺ can permeate amphotericin pores, because otherwise pH_i would be uncontrolled, and V_rev would have been the same in all solutions. The dashed line shows the Nernst potential for NH₄⁺ that would obtain if the NH₄⁺ gradient controlled pH_i perfectly and if the H⁺ channel were perfectly selective for H⁺, which appears the case (18). The control of pH_i is excellent with gradients ≤10. Because we are interested in physiological responses, we operated in the range of a 50//50 or 15//50 gradient (pH_i = pH_o or pH_i = pH_o −0.5), where the control is excellent. V_rev was similar before and after PMA treatment, with an average change of <4 mV in 12 cells where V_rev was measured in the same solutions before and after PMA. When the permeabilized-patch configuration is used, PMA has dramatic effects on H⁺ currents. Identical families of voltage pulses were applied to a human neutrophil before (C) and after (D) 10-min exposure to 60 nM PMA. From a holding potential of −60 mV, 8-s pulses were applied to −20 mV through +20 mV in 10-mV increments. The inward tail currents after repolarization to −60 mV are labeled with the test pulse voltage. The presence of a distinct inward tail current after the pulse to −10 mV reveals activation of the H⁺ conductance negative to the tail current reversal potential, V_rev, measured in this cell, −3 mV. Before PMA was added, depolarizing pulses larger than shown in C elicited “normal” H⁺ currents.