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

Abstract 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.