Single channel analysis of conductance and rectification in cation-selective, mutant glycine receptor channels
Single channel analysis of conductance and rectification in cation-selective, mutant glycine receptor channels
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DOI：
10.1085/jgp.20028553
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发表时间：
2002-05-01
影响因子：
3.8
通讯作者：
Barry, PH
中科院分区：
文献类型：
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作者：
Moorhouse, AJ;Keramidas, A;Barry, PH
配体门控离子通道超家族的成员介导神经系统中的快速突触传递。在本研究中，我们通过表征包含赋予阳离子选择性的孔突变的 α1 同聚人甘氨酸受体通道 (GlyR) 的单通道电导和整流，研究了该通道家族中离子渗透和离子电荷选择性的分子决定因素和机制。 A-1'E 突变体 GlyR 和选择性双突变体（[SDM]、A-1'E、P-2'Delta）GlyR 的平均向内弦电导（-60 mV）分别为 7 pS，平均向外电导分别为 11 和 12 pS（60 mV）。这表明突变并非简单地降低了阴离子渗透性，而是用阳离子电导取代了之前的阴离子电导。额外的突变可以中和环上的正电荷。通道的细胞外月 (SDM+R19'A GlyR) 使电导-电压关系呈线性（60 和 -60 mV 时均为 14 pS）。当该外部带电环变为负电（SDM+R19'E GlyR）时，向内电导进一步增加（至 22 pS），并且现在对外部二价阳离子变得敏感（不存在时为 32 pS）。外部电荷环的突变对电导和整流的影响可以适合仅改变渗透的主要外部能垒高度的模型。当内部二价阳离子不存在时，SDM+R19'A 和 SDM+R19'E GlyR 的平均向外电导增加，这与孔的细胞内端两侧有固定负电荷一致。这支持了我们的假设，即离子电荷选择性引发通过在推定的选择性过滤器处引入带负电的环而反转了孔的静电分布。这些结果还进一步证实了外部孔前庭静电在确定配体门控离子通道的电导和整流特性中的作用。
Members of the ligand-gated ion channel superfamily mediate fast synaptic transmission in the nervous system. In this study, we investigate the molecular determinants and mechanisms of ion permeation and ion charge selectivity in this family of channels by characterizing the single channel conductance and rectification of alpha1 homomeric human glycine receptor channels (GlyRs) containing pore mutations that impart cation selectivity. The A-1'E mutant GlyR and the selectivity double mutant ([SDM], A-1'E, P-2'Delta) GlyR, had mean inward chord conductances (at -60 mV) of 7 pS and mean outward conductances of 11 and 12 pS (60 mV), respectively This indicates that the Mutations have not simply reduced anion permeability, but have replaced the previous anion conductance with a cation one. An additional mutation to neutralize the ring of positive charge at. the extracellular month of the channel (SDM+R19'A GlyR) made the conductance-voltage relationship linear (14 pS at both 60 and -60 mV). When this external charged ring was made negative (SDM+R19'E GlyR), the inward conductance was further increased (to 22 pS) and now became sensitive to external divalent cations (being 32 pS in their absence). The effects of the mutations to the external ring of charge on conductance and rectification could be fit to a model where only the main external energy barrier height for permeation was changed. Mean outward conductances in the SDM+R19'A and SDM+R19'E GlyRs were increased when internal divalent cations were absent, consistent with the intracellular end of the pore being flanked by fixed negative charges. This supports our hypothesis that the ion charge selectivity initiations have inverted the electrostatic profile of the pore by introducing a negatively charged ring at the putative selectivity filter. These results also further confirm the role of external pore vestibule electrostatics in determining the conductance and rectification properties of the ligand-gated ion channels.