Inwardly permeating Na ions generate the voltage dependence of resurgent Na current in cerebellar Purkinje neurons.
Inwardly permeating Na ions generate the voltage dependence of resurgent Na current in cerebellar Purkinje neurons.
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DOI：
10.1523/jneurosci.0376-10.2010
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发表时间：
2010-04-21
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影响因子：
--
通讯作者：
Raman IM
中科院分区：
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作者：
Aman TK;Raman IM
小脑浦肯野神经元的电压门控钠通道表达内源性开放通道阻断蛋白。这种阻断剂在正电位下结合通道，在负电位下解除结合，产生复苏的Na电流并允许快速放电。复苏电流的宏观电压依赖性提出了一个问题，即阻断剂是否直接感知膜电位或是否间接赋予电压依赖性。因为我们以前发现，向内渗透的Na离子促进解离的阻滞剂，我们测量了电压钳位电流在不同的Na梯度测试渗透离子的作用，在产生的电压依赖性的解除阻滞。在反向梯度，向外复苏电流很小或不存在，这表明解除封锁通常需要“敲除”钠。然而，在强负电位下的向内复苏电流在反向比在控制梯度中更大。此外，在反向梯度和Na浓度降低的对照梯度中，阻断状态的占据时间延长，表明当内向电流较小时阻断更稳定。因此，反向梯度转移了阻断的电压依赖性，使得即使在负电位条件化后也能诱发复苏电流。此外，在控制梯度，峰值复苏电流线性下降的驱动力在调节步骤，这表明块的稳定性直接与内向钠电流幅度。因此，阻断剂解结合的电压依赖性几乎完全来自占据外部孔的Na离子的排斥。阻断蛋白缺乏电压敏感性表明阻断剂的结合位点位于膜场之外，在渗透途径中。
Voltage-gated Na channels of cerebellar Purkinje neurons express an endogenous open-channel blocking protein. This blocker binds channels at positive potentials and unbinds at negative potentials, generating a resurgent Na current and permitting rapid firing. The macroscopic voltage-dependence of resurgent current raises the question of whether the blocker directly senses membrane potential or whether voltage-dependence is conferred indirectly. Because we previously found that inwardly permeating Na ions facilitate dissociation of the blocker, we measured voltage-clamped currents in different Na gradients to test the role of permeating ions in generating the voltage-dependence of unblock. In reverse gradients, outward resurgent currents were tiny or absent, suggesting that unblock normally requires “knockoff” by Na. Inward resurgent currents at strongly negative potentials, however, were larger in reverse than in control gradients. Moreover, occupancy of the blocked state was prolonged both in reverse gradients and in control gradients with reduced Na concentrations, indicating that block is more stable when inward currents are small. Accordingly, reverse gradients shifted the voltage-dependence of block, such that resurgent currents were evoked even after conditioning at negative potentials. Additionally, in control gradients, peak resurgent currents decreased linearly with driving force during the conditioning step, suggesting that the stability of block varies directly with inward Na current amplitude. Thus, the voltage-dependence of blocker unbinding results almost entirely from repulsion by Na ions occupying the external pore. The lack of voltage sensitivity of the blocking protein suggests that the blocker’s binding site lies outside the membrane field, in the permeation pathway.