铜对大鼠海马CAl区神经元A-电流和延迟整流钾电流的影响

【作者】 牛志电；

【导师】 阮迪云；

【作者基本信息】 中国科学技术大学 ， 生物物理学， 2006， 博士

【摘要】 铜是一种重要的过渡金属，在中枢神经系统中起着重要的作用。但是，铜代谢的紊乱对中枢神经系统是有害的，尤其是铜浓度过度升高时。中枢神经元中含铜量病理性的升高会引起严重的神经学紊乱，如神经退行性病变和智力障碍，人们推测这与威尔逊病（WD）和阿尔茨海默病（AD）的发病机制有关。近来的研究表明，电压依赖性钾通道的功能异常与学习和记忆的损伤、共济失调、癫痫、神经性耳聋等一些神经性疾病的产生有关，其中电压依赖性瞬间外向钾通道和延迟整流钾通道的功能还与学习和记忆有关。由于WD患者和AD的早期患者的脑部海马均含有高水平的铜，故在本课题中，我们应用全细胞膜片钳技术，在急性分离的大鼠海马CA1区锥体神经元上比较详细地研究生理和病理浓度的Cu²⁺对电压依赖性瞬间外向钾电流（L_A）和延迟整流钾电流（I_K）的效应。主要研究结果如下： 1．急性铜暴露对大鼠海马CA1区神经元A-型钾电流的影响 胞外1，10，30，100和1000μM Cu²⁺以剂量依赖性的方式可逆地抑制I_A的幅度，其IC₅₀值为130μM。较高浓度的铜，即100和300μM Cu²⁺能显著地使I_A的激活曲线向去极化方向移动(V_1/2:对照，1.0±0.2mV；100μM Cu²⁺，8.2±1.1mV；300μM Cu²⁺，18.2±1.1mV)，提示Cu²⁺抑制I_A的激活。对于稳态失活曲线，100和300μM Cu²⁺也能显著地使其向去极化方向移动(V_1/2:对照，-87.7±2.0mV；100μM Cu²⁺，-82.5±0.5mV；300μM Cu²⁺，-78.7±1.5mV)，提示在任何一个给定的膜电位处，通道的失活受到Cu²⁺的抑制。此外，100和300μM Cu²⁺还能明显促进A-型钾通道的失活，并减缓通道从失活状态的恢复。结果提示：生理和病理浓度的Cu²⁺可能通过对A-型钾通道的抑制影响大鼠海马CA1区神经元的兴奋性。而这也可能涉及到WD和早期阶段AD的病理过程，并可能与WD病人的智力障碍以及AD患者早期记忆丧失和认知功能下降有关。 2．铜对急性分离的大鼠海马CA1区神经元延迟整流钾电流的抑制 胞外1，10，30，100和1000μM Cu²⁺以剂量依赖性的方式可逆地抑制I_K的幅度，且高浓度的Cu²⁺（1000μM）对I_K的抑制也仅仅为64％。其IC₅₀值为100更多还原

【Abstract】 Copper (Cu²⁺) is an essential transition metal that plays a critical role in the central nervous system （CNS）. However, aberrant copper homeostasis is harmful to CNS and excess copper is extremely toxic in the CNS. Pathologically increased accumulation of copper in central neurons induce severe neurological disorders, such as neurological degeneration, mental retardation, which has been postulated to play a role in the pathogenesis of Wilson’s disease （WD） and Alzheimer’s disease （AD）. Recently, many human neurological disease pathgenesis has been shown to be related to abnormalities of voltage-gated K⁺ channel function, such as learning and memory impairing, ataxia, epilepsy and deafness. Recent evidence demonstrated that voltage-dependent transient outward and delayed rectifier K⁺ channel function have been implicated in learning and memory. In addition, high concentrations of Cu²⁺ has been measured in the brain for patients with WD or AD. Thus, in this study, we investigated in detail the effects of physiologically and pathologically relevant copper on I_a and I_k by using the whole-cell patch-clamp technique in the acutely dissociated rat hippocampal CA1 pyramid neurons. The main results were summarized as following:1. Effects of copper on A-type potassium currents （I_A） in acutely dissociated rathippocampal CA1 neuronsExtracellular application of various concentrations of Cu²⁺（1-1000 μM） reversibly reduced the amplitude of I_a in a dose-dependent manner with an IC₅₀ value of 130 μM. 100 and 300 μM Cu²⁺significantly shifted the V_1/2 of steady-state activation curve to the depolarizing direction by 7.2 and 17.2 mV, respectively, indicating that Cu²⁺ decreased the activation of I_a. For state-inactivation curves, 100 and 300 μM Cu²⁺ markedly shifted the V_1/2 to the depolarizing direction by 5.2 and 9.0 mV, respectively, indicating that channels were less likely to be inactivated at higher concentrations of Cu²⁺ at any given potential. In addition, higher concentrations Cu²⁺ markedly increased the decay at a prepulse potential of -110 mV and significantly slowed the recovery of I_k from inactivation. These results suggest it is possible for更多还原