During the course of experiments involving the oxidative stress of ion channels, interesting results with anti-oxidants were obtained. Inside-out patches were used from HEK 293 cells expressing both α and β subunits of BK (maxi-K) channels cloned from human cells (Ahring et al. 1997). Recordings were carried out at 23 ± 2 °C in symmetrical solutions containing (mM): KC1 144, MgCl2 1, EGTA 10 and Hepes 10; pH 7.2. A VH of -40 to -65 mV was selected for a given patch and applied for 20 s at intervals. The probability that patches manifested open channels (P) was approximately 0.03 and variable over time, but unit current was constant.
The commonly used food antioxidant BHT (2, 6-di-tert-butyl-4-methylphenol), dissolved in methanol, was added to the recording bath. At a bath concentration of 100 µM it reduced channel activity (i.e. P) to a negligible level within 3 min (8 experiments) whilst 10 µM BHT had a similar but sometimes slower effect (6 experiments). 1 µM BHT had no effect in 23 min (2 experiments) and reduced P over 12 min in one experiment. Methanol was without any effect. The inhibition in 100 µM BHT was partially reversed on removing the BHT. In contrast, the lipophilic antioxidant α-tocopherol had no effect over 10-80 min exposure (50 µM, 2 experiments and 100 µM, 2 experiments). Trolox (6-hydroxy-2, 5, 7, 8-tetramethyl-chroman-2-carboxylic acid), a water-soluble antioxidant analogue of vitamin E, was also without effect on channel activity (50 µM, 4 experiments over 30 min). However, the non-antioxidant 3, 5-di-tert-butyltoluene, in which the OH group in BHT is replaced by H, promptly inhibited channel activity (100 µM, 3 experiments; 10 µM, 3 experiments). 1 µM had no effect (2 experiments).
Thus the two inhibitory compounds act by other than a redox mechanism.
This work was supported by The Wellcome Trust.