To explore the mechanisms of inhibition of insulin secretion in pancreatic β-cells by oxygen free radicals, we studied the effects of H2O2 on membrane currents using the patch-clamp technique. Exposure of β-cells to H2O2 (≥30 (μmol/1) increased the activity of ATP-sensitive potassium (K+ATP) channels without changing the single channel conductance in cell-attached membrane patches. Action currents observed during superfusion of 11.1 mmol/1 glucose were suppressed. In inside-out membrane patches, the activity of K+ATP channels was not influenced by H2O2. In conventional whole-cell clamp experiments using a pipette solution containing 3 mmol/1 ATP, H2O2 did not influence the membrane currents. However, H2O2 did activate the K+ATP channel current in perforated whole-cell clamp configurations. The increased K+ATP channel current was reversed by subsequent exposure to 11.1 mmol/12-ketoisocaproic acid. In cell-attached membrane patches, the K+ATP channel current evoked by exposure to 30 μmol/l H2O2 was inhibited by exposure to 11.1 mmol/l glyceraldehyde, whereas the channel was again activated by exposure to 0.3 mmol/l H2O2. Subsequent superfusion of 11.1 mmol/l 2-ketoisocaproic acid inhibited the channel; this effect was counteracted by exposure to 10 mmol/l H2O2. Transient inhibition of K+ATP channels with provocation of action potentials was observed after washout of 100 μmol/l H2O2 during superfusion of 2.8 or 11.1 mmol/l glucose. We conclude that H2O2 has no direct effect on the K+ATP channels but that it indirectly activates the channels when it is exposed to β-cells under conditions in which the cellular metabolism is physiologically regulated.

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