ATP-sensitive K+ channels of skeletal muscle fibers from young adult and aged rats: possible involvement of thiol-dependent redox mechanisms in the age-related modifications of their biophysical and pharmacological properties

In the present work, we have investigated whether thiol-dependent redox mechanisms play a role in the regulation of ATP-sensitive K+ (KATP) channels present on the surface membrane of skeletal muscle fibers from 5-7-month-old ("young adult") and 24-26-month-old ("aged") rats. The KATP channels were surveyed by using patch-clamp techniques. Continuous recordings of channel activity were performed in the inside-out configuration at a constant voltage at 20 degrees, in the presence of 150 mM KCl on both sides of the membrane. As expected, the excision of cell-attached patches from young adult rat fibers, into ATP-free solution, dramatically increased KATP channel activity. In contrast, when patches were excised from aged rat fibers no increase of channel activity was detected. Open probability (Popen) analysis in the range of potentials from -70 mV to +60 mV revealed that the Popen of the channels of aged rat fibers was about 7.5 times lower than that of young adult rat fibers. Moreover, a decrease in the number of functional channels present in the patches of aged rat fibers was also observed. No change with aging was found in the single-channel conductance, which was 60 pS. The application of increasing concentrations of the sulfhydryl group-reducing agents L-cysteine (5 microM to 5 mM) and N-acetyl-L-cysteine (0.5-5 mM) restored the Popen of the channels of aged rat fibers without increasing the number of functional channels. Thimerosal, a sulfhydryl group-oxidizing agent, and glybenclamide applied to the cytoplasmic face of KATP channels from fibers of either young adult or aged rats dramatically abolished channel openings. However, the KATP channels of aged rat fibers were 30-200 times more sensitive to the inhibitory effects of these chemicals. In both young adult and aged rat fibers the effect of thimerosal was reversed only by addition of L-cysteine. In contrast, the effect of glybenclamide was fully reversible. Moreover, after preincubation of aged rat channels with 1 mM L-cysteine, the blocking effect of glybenclamide was reduced and was similar to that observed in young adult rat fibers. These observations lead us to conclude that, in rat skeletal muscle, the KATP channel proteins contain thiol groups essential for channel activity. Oxidation of these groups occurs during aging and prolonged channel closure. This modification may explain the altered pharmacological response to both thimerosal and glybenclamide observed in aged rat skeletal muscle f