Deng Pan-Yue, Klyachko Vitaly A
Departments of Cell Biology and Physiology, Biomedical Engineering, CIMED, Washington University, St Louis, MO, USA.
J Physiol. 2016 Jan 1;594(1):83-97. doi: 10.1113/JP271031. Epub 2015 Nov 18.
Single-channel recordings in CA3 pyramidal neurons revealed that large-conductance calcium-activated K(+) (BK) channel open probability was reduced by loss of fragile X mental retardation protein (FMRP) and that FMRP acts on BK channels by modulating the channel's gating kinetics. Fmr1/BKβ4 double knockout mice were generated to genetically upregulate BK channel activity in the absence of FMRP. Deletion of the BKβ4 subunit alleviated reduced BK channel open probability via increasing BK channel open frequency, but not through prolonging its open duration. Genetic upregulation of BK channel activity via deletion of BKβ4 normalized action potential duration, excessive glutamate release and short-term synaptic plasticity during naturalistic stimulus trains in excitatory hippocampal neurons in the absence of FMRP. Genetic upregulation of BK channel activity via deletion of BKβ4 was sufficient to normalize excessive epileptiform activity in an in vitro model of seizure activity in the hippocampal circuit in the absence of FMRP. Loss of fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS), yet the mechanisms underlying the pathophysiology of FXS are incompletely understood. Recent studies identified important new functions of FMRP in regulating neural excitability and synaptic transmission via both translation-dependent mechanisms and direct interactions of FMRP with a number of ion channels in the axons and presynaptic terminals. Among these presynaptic FMRP functions, FMRP interaction with large-conductance calcium-activated K(+) (BK) channels, specifically their auxiliary β4 subunit, regulates action potential waveform and glutamate release in hippocampal and cortical pyramidal neurons. Given the multitude of ion channels and mechanisms that mediate presynaptic FMRP actions, it remains unclear, however, to what extent FMRP-BK channel interactions contribute to synaptic and circuit defects in FXS. To examine this question, we generated Fmr1/β4 double knockout (dKO) mice to genetically upregulate BK channel activity in the absence of FMRP and determine its ability to normalize multilevel defects caused by FMRP loss. Single-channel analyses revealed that FMRP loss reduced BK channel open probability, and this defect was compensated in dKO mice. Furthermore, dKO mice exhibited normalized action potential duration, glutamate release and short-term dynamics during naturalistic stimulus trains in hippocampal pyramidal neurons. BK channel upregulation was also sufficient to correct excessive seizure susceptibility in an in vitro model of seizure activity in hippocampal slices. Our studies thus suggest that upregulation of BK channel activity normalizes multi-level deficits caused by FMRP loss.
对CA3锥体神经元进行的单通道记录显示，脆性X智力低下蛋白（FMRP）缺失会降低大电导钙激活钾（BK）通道的开放概率，且FMRP通过调节通道的门控动力学作用于BK通道。通过基因手段在缺乏FMRP的情况下上调BK通道活性，构建了Fmr1/BKβ4双敲除小鼠。BKβ4亚基的缺失通过增加BK通道的开放频率而非延长其开放持续时间，缓解了BK通道开放概率的降低。在缺乏FMRP的情况下，通过缺失BKβ4对BK通道活性进行基因上调，可使兴奋性海马神经元在自然刺激序列期间的动作电位持续时间、过量谷氨酸释放和短期突触可塑性恢复正常。在缺乏FMRP的情况下，通过缺失BKβ4对BK通道活性进行基因上调足以使海马回路癫痫活动体外模型中过度的癫痫样活动恢复正常。脆性X智力低下蛋白（FMRP）缺失会导致脆性X综合征（FXS），但其病理生理学的潜在机制尚未完全明确。最近的研究发现FMRP在通过翻译依赖性机制以及FMRP与轴突和突触前终末中许多离子通道的直接相互作用来调节神经兴奋性和突触传递方面具有重要的新功能。在这些突触前FMRP功能中，FMRP与大电导钙激活钾（BK）通道，特别是其辅助β4亚基的相互作用，调节海马和皮质锥体神经元的动作电位波形和谷氨酸释放。然而，鉴于介导突触前FMRP作用的离子通道和机制众多，目前尚不清楚FMRP - BK通道相互作用在多大程度上导致了FXS中的突触和回路缺陷。为了研究这个问题，我们构建了Fmr1/β4双敲除（dKO）小鼠，以在缺乏FMRP的情况下通过基因手段上调BK通道活性，并确定其使FMRP缺失导致的多级缺陷恢复正常的能力。单通道分析显示，FMRP缺失降低了BK通道的开放概率，而这种缺陷在dKO小鼠中得到了补偿。此外，dKO小鼠在海马锥体神经元的自然刺激序列期间表现出正常的动作电位持续时间、谷氨酸释放和短期动力学。BK通道上调也足以纠正海马切片癫痫活动体外模型中过度的癫痫易感性。因此，我们的研究表明，BK通道活性上调可使FMRP缺失导致的多级缺陷恢复正常。
