【作者】 李伟；

【导师】 徐涛； 丁久平；

【作者基本信息】 华中科技大学 ， 生物物理， 2007， 博士

【摘要】 大电导的电压和Ca2+激活的K+通道(BK或MaxiK通道)在哺乳动物的组织中广泛的表达,起着各种各样重要的生理作用,如连系胞内钙信号通路和膜兴奋性,调节神经递质的释放等。BK通道由α亚基和β亚基构成,β2和β3亚基能使通道的电流失活。其中hβ3亚基在组织中的定位和功能还不清楚。尽管失活的电流在重组的BK通道中已经被广泛的研究,但是在原代细胞尤其是神经元中研究的还很少。在本文的工作中,我们在大鼠腰椎L4-L6背根神经节(DRG)的小神经元上发现了一种失活的Ca2+依赖的K+电流,它能够被大电导的BK通道特异的阻断剂Charybdotoxin (CTX)和Iberiotoxin (IbTX)所阻断,证实是失活的BKi电流。木瓜蛋白酶(papain)可以从胞外作用,改变通道的失活性质。我们在全细胞和单通道(inside-out)记录中均发现96%的神经元上存在着快失活的成分和/或慢失活的成分。用单指数或/和双指数拟合失活的平均单通道电流得到失活时间常数(τ)。这些失活时间常数的分布可以用两相的高斯函数拟合,分别得到快的失活时间常数为τi,fast=4.6 ms、慢的失活时间常数为τi,slow=31.8 ms。根据这两种电流成分的失活动力学和单通道电流的特征,我们证明了大鼠小DRG神经元上存在着β2和β3b亚基。单通道记录(inside-out)中,我们发现DRG神经元上存在失活的中等电导的BKi通道。在K+平衡溶液中,单通道电导为100-200 pS不等,指出大鼠DRG神经元上中等电导的BK通道是以slo2/slo1/β2/β3b组合的形式存在。在电流钳的模式下,我们用100 nM ChTX阻断BK通道后,小DRG神经元动作电位的爆发频率增加,动作电位的时程增宽。说明BK电流对复极化相和兴奋性起作用。我们认为小DRG神经元上的BKi电流可能在调制痛觉信息从外周向中枢传递的过程中起着重要的作用。更多还原

【Abstract】 Large conductance voltage- and Ca2+-activated K+ channels (BK or MaxiK channels) are almost ubiquitously expressed among mammalian tissues, which play a crucial role in coupling changes in submembrane calcium concentration with changes in membrane potential and excitability. BK channels are formed fromα(Slo1) gene with itsβfamily, two of which,β2 andβ3, show ability to inactivate currents. It remains to be demonstrated where the hβ3- subunit is expressed in native tissues and what are the functional roles. Even though the inactivation of BK channels has been extensively studied mostly in expression experiments, rarely do they exhibit inactivation in native cells, especially in neurons. It is unclear what kind of native BK channels exists in Dorsal root ganglia (DRG) neurons and what are their properties and functional roles.In the current study, we have identified and characterized an inactivating Ca2+-dependent potassium (KCa) current in small neurons from rat lumbar L4-L6 dorsal root ganglia (DRG). The KCa currents are verified as inactivating BK currents (BKi) due to their sensitivity to the specific BK channel blockers, Charybdotoxin (CTX) and Iberiotoxin (IbTX). We demonstrated that the inactivation of BKi could be removed by extracellular treatment with papain.Using whole-cell recordings and inside-out patches we found that BK channels in DRG neurons exhibited a rapid inactivation and/or a slow inactivation in 96% cells. Ensemble inactivating currents from single-channel recordings were best fitted to single- or double-exponential function, which yield inactivation time constant (τ). Two gaussian fits to distribution of inactivation time constant indicate that the inactivation time constants are 4.6 ms (fast) and 31.8 ms (slow). Based on the kinetics of inactivation and the properties of single channel currents, our results indicate the presence of bothβ2 andβ3b in small DRG neurons. In inside-out patches, we found inactivating medium conductance BK currents, and the single-channel conductance varied from 100 to 200 pS in symmetrical K+ solution, indicated that there might be a combination of slo2/slo1/β2/β3 in rat dorsal root ganglion neurons.At current-clamp, blockade of the BKi channels by applying 100 nM ChTX results in increased firing and broadened action potentials. The BK currents contribute to repolarization phase and excitability and we propose BKi in small DRG neurons might play an important role in modulating nociceptive input from the peripheral to the CNS.更多还原