Tricarico D, Camerino D C
Department of Pharmacobiology, Faculty of Pharmacy, University of Bari, Italy.
Mol Pharmacol. 1994 Oct;46(4):754-61.
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.
在本研究中，我们探究了硫醇依赖性氧化还原机制是否在调节5至7月龄（“青年成年”）和24至26月龄（“老年”）大鼠骨骼肌纤维表面膜上存在的ATP敏感性钾通道（KATP通道）中发挥作用。通过膜片钳技术对KATP通道进行检测。在膜两侧均存在150 mM KCl的情况下，于20摄氏度恒定电压下以膜外翻式配置对通道活性进行连续记录。正如预期的那样，将青年成年大鼠纤维上的细胞贴附膜片切除并置于无ATP溶液中，会显著增加KATP通道活性。相反，当从老年大鼠纤维上切除膜片时，未检测到通道活性增加。在 -70 mV至 +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Popen比青年成年大鼠纤维的低约7.5倍。此外，还观察到老年大鼠纤维膜片中功能性通道数量减少。单通道电导未随衰老发生变化，为60 pS。应用浓度逐渐增加的巯基还原剂L - 半胱氨酸（5 microM至5 mM）和N - 乙酰 - L - 半胱氨酸（0.5 - 5 mM）可恢复老年大鼠纤维通道的Popen，而不增加功能性通道数量。硫柳汞，一种巯基氧化剂以及格列本脲应用于青年成年或老年大鼠纤维的KATP通道胞质面时，会显著消除通道开放。然而，老年大鼠纤维的KATP通道对这些化学物质的抑制作用敏感度高30至200倍。在青年成年和老年大鼠纤维中，硫柳汞的作用仅通过添加L - 半胱氨酸得以逆转。相反，格列本脲的作用是完全可逆的。此外，用1 mM L - 半胱氨酸对老年大鼠通道进行预孵育后，格列本脲的阻断作用减弱，且与在青年成年大鼠纤维中观察到的情况相似。这些观察结果使我们得出结论，在大鼠骨骼肌中，KATP通道蛋白含有对通道活性至关重要的巯基。这些基团在衰老和通道长时间关闭过程中会发生氧化。这种修饰可能解释了在老年大鼠骨骼肌纤维中观察到的对硫柳汞和格列本脲药理学反应的改变。
