ATP-SENSITIVE K-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( CHANNELS OF SKELETAL)

In the present work, we have investigated whether thiol-dependent redo x mechanisms play a role in the regulation of ATP-sensitive K+ (K-ATP) channels present on the surface membrane of skeletal muscle fibers fr om 5-7-month-old (''young adult'') and 24-26-month-old (''aged'') rats . The K-ATP channels were surveyed by using patch-damp techniques. Con tinuous recordings of channel activity were performed in the inside-ou t configuration at a constant voltage at 20 degrees, in the presence o f 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 so lution, dramatically increased K-ATP channel activity. In contrast, wh en patches were excised from aged rat fibers no increase of channel ac tivity was detected. Open probability (P-open) analysis in the range o f potentials from -70 mV to -t60 mV revealed that the P-open of the ch annels 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 ch annels present in the patches of aged rat fitters was also observed. N o change with aging was found in the single-channel conductance, which was 60 pS. The application of increasing concentrations of the sulfhy dryl group-reducing agents L-cysteine (5 mu M to 5 mM) and N-acetyl-L- cysteine (0.5-5 mM) restored the P-open of the channels of aged rat fi bers without increasing the number of functional channels. Thimerosal, a sulfhydryl group-oxidizing agent, and glybenclamide applied to the cytoplasmic face of K-ATP channels from fibers of either young adult o r aged rats dramatically abolished channel openings. However, the K-AT P channels of aged rat fibers were 30-200 times more sensitive to the inhibitory effects of these chemicals. In both young adult and aged ra t fibers the effect of thimerosal was reversed only by addition of L-c ysteine. In contrast, the effect of glybenclamide was fully reversible . Moreover, after preincubation of aged rat channels with 1 mM L-cyste ine, 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 K-ATP channel proteins contain thiol groups essential for channel activity. Oxidation of thes e groups occurs during aging and prolonged channel closure. This modif ication may explain the altered pharmacological response to both thime rosal and glybenclamide observed in aged rat skeletal muscle fibers.