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.
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.
复制标题
年轻成年和老年大鼠骨骼肌纤维的 ATP 敏感 K 通道：硫醇依赖性氧化还原机制可能参与其生物物理和药理学特性的年龄相关修饰。
作者：
D. Tricarico;D. Camerino
在本工作中，我们研究了5-7月龄(“青年”)和24-26月龄(“老年”)大鼠骨骼肌纤维表面膜上存在的ATP敏感K+(KATP)通道的调节中，硫醇依赖的氧化还原机制是否起作用。用膜片钳技术检测KATP通道。在膜两侧存在150 mMKCl的情况下，在20度恒定电压下，以内向外配置进行通道活动的连续记录。正如预期的那样，将幼年成年大鼠纤维上附着的细胞斑块切除到不含ATP的溶液中，显著增加了KATP通道的活性。相反，当从老化的大鼠纤维上切除斑块时，没有检测到通道活动的增加。在-70 mV~+60 mV电位范围内的开放概率(Popen)分析表明，老年大鼠纤维通道的开放概率比年轻成年大鼠的低7.5倍。此外，还观察到老年大鼠纤维斑块中的功能通道数量减少。单通道电导没有随时间变化，为60ps。在不增加功能通道数目的情况下，增加巯基还原剂L-半胱氨酸(5微米~5 mM)和N-乙酰基-L-半胱氨酸(0.5~5 mM)的浓度，可使衰老大鼠纤维的通道恢复开放。硫柳汞和格列本脲作用于KATP通道的细胞质表面，无论是从幼年大鼠还是老年大鼠的纤维上，都显著地消除了通道的开放。然而，老年大鼠纤维的KATP通道对这些化学物质的抑制作用的敏感性是后者的30-200倍。在幼年和老年大鼠的纤维中，硫柳汞的作用仅被L-半胱氨酸逆转。相比之下，格列本脲的作用是完全可逆的。此外，用1 mM L-半胱氨酸预先孵育老龄大鼠的通道后，格列本脲的阻断作用减弱，与年轻成年大鼠的作用相似。这些观察使我们得出结论，在大鼠骨骼肌中，KATP通道蛋白含有通道活动所必需的硫醇基团。这些基团的氧化发生在老化和长时间的通道关闭期间。这一修饰可能解释了在老年大鼠骨骼肌纤维中观察到的硫柳汞和格列本脲的药理反应改变。
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 fibers.