Anoxic decrease in potassium outward currents of hippocampal cultured neurons in absence and presence of dithionite

The effect of brief anoxia on voltage dependent K(+)-currents of hippocampal cultured neurons was studied. The oxygen scavenger dithionite (hydrosulphite) was previously used for creating zero oxygen pressure. However, dithionite consumes O(2) in parallel with generation of superoxide radicals and is a strongly reducing agent. In this study anoxia was produced by perfusion of the neurons with a solution bubbled with nitrogen for 1 h using a chamber with an argon layer isolating the anoxic bath flow from atmospheric oxygen in presence and absence of dithionite. Oxygen partial pressure of dithionite-free solution was determined by oxygen dependent quenching of the phosphorescence of Pd-coproporphyrin to be 0.15+/-0. 02 Torr (values are given as mean+/-S.D., n=6). Slow (I(K))- and fast (I(A))-inactivating K(+)-currents were measured with the patch clamp technique in the whole cell configuration. Exposure of the neurons to anoxia reversibly decreased the amplitude of I(K) at a test pulse of 0 mV to 77+/-12% (n=7) in absence and to 83+/-7% (n=6) in presence of 2 mM dithionite; the amplitude of I(A) decreased to 78+/-11% in absence and to 82+/-9% in presence of 2 mM dithionite. Voltage dependence of activation and inactivation shifted 5 min after exposure to anoxia reversibly by about 6 mV in depolarizing direction. The decay times of inactivation were insensitive to anoxia. Dithionite had no significant effects on K(+)-currents. In 15 of 21 neurons not employed for analysis on K(+)-currents, a reversible increase in holding current under dithionite was observed. In absence of dithionite in 4 of 19 neurons the holding current reversibly increased during anoxia. Although dithionite does not affect K(+)-currents, changes in holding current show that the dithionite may affect neurons independently of oxygen deprivation.