MECHANISM OF ANION PERMEATION THROUGH CHANNELS GATED BY GLYCINE AND GAMMA-AMINOBUTYRIC-ACID IN MOUSE CULTURED SPINAL NEURONS
MECHANISM OF ANION PERMEATION THROUGH CHANNELS GATED BY GLYCINE AND GAMMA-AMINOBUTYRIC-ACID IN MOUSE CULTURED SPINAL NEURONS
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DOI：
10.1113/jphysiol.1987.sp016493
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发表时间：
1987-04-01
影响因子：
5.5
通讯作者：
SAKMANN, B
中科院分区：
文献类型：
--
作者：
BORMANN, J;HAMILL, OP;SAKMANN, B
1.甘氨酸受体（GlyR）和γ-甘氨酸受体的离子选择性和离子转运性质用膜片钳技术研究了小鼠脊髓神经元索马膜上的氨基丁酸受体（GABAR）通道。2.电流-电压（I-V）关系的发射机激活的电流从全细胞测量与145 mM-Cl-细胞内和细胞外，表现出外向整流。在电压跃变实验中，GlyR和GABAR通道的瞬时I-V关系为线性，稳态I-V关系为外向整流，表明GlyR和GABAR通道的门控具有电压敏感性。3.全细胞电流的逆转电位移动56 mV每10倍的变化，在内部Cl-活性表明激活Cl-选择性通道。两个通道的K+/Cl-渗透率比值（PK/PCl）均小于0.05。4.对于GABAR通道，大的多原子阴离子的渗透性顺序为甲酸根>碳酸氢根>乙酸根>磷酸根>丙酸根;磷酸根和丙酸根在GlyR通道中不可测量地渗透。这表明开放的GlyR和GABAR通道具有5.2和5.6埃的有效孔径，分别两种通道对小阴离子的相对渗透率顺序为SCN- > I- > Br- > Cl- > F-。5. GlyR和GABAR通道是多电导态通道。在细胞贴附的补丁的单通道斜率电导接近0 mV的膜电位为29，18和10 pS的甘氨酸，和28，17和10 pS的GABA激活的通道。GlyR和GABAR通道最常观察到的（主要）电导状态分别为29和17 pS。6.在细胞外和细胞内浓度均为145 mM-Cl-的外向斑中，GlyR通道的电导状态为46、30、20和12 pS，GABAR通道的电导状态为44、30、19和12 pS。最常见的主要状态是46 pS的GlyR和30 pS的GABAR通道。7.在两个膜面上相等的140 mM浓度的小阴离子中测量的单通道电导揭示了两个通道的Cl- > Br- > I- > SCN- > F-的电导序列。这几乎是相反的顺序，发现这些离子的premeability表明存在的离子在通道中的结合位点。8. GlyR和GABAR通道对Cl-的电导-活性关系均为双曲线。最大电导（γ max）为92和72 pS; GlyR和GABAR通道的半饱和活性（Km）分别为108和155 mM-Cl-。9. SCN-的电导-活性关系的特征在于最大电导为35 pS（GlyR）和32 pS（GABAR）。半饱和活性分别为46和57 mM。在早期的SCN-和Cl-的混合物在两个膜两侧的通道电导表现出异常的摩尔分数依赖性。这表明通道含有至少两个阴离子结合位点。10. GlyR和GABAR通道在它们的主态Cl-电导中的差异可能是由小的能量差（约1 kJ mol ~（-1））在Cl ~-离子与通道壁相互作用中的作用。GlyR和GABAR通道显示可比的电导状态的观察，显示类似的渗透性和电导序列的小阴离子，一个类似的电导饱和曲线的Cl-和SCN-和类似的摩尔分数依赖性，其电导在Cl-SCN-混合物可以表明，这两个受体通道具有类似的分子结构，在这些地区的亚基，形成衬里的通道。
1. The ion-selective and ion transport properties of glycine receptor (GlyR) and .gamma.-aminobutyric acid receptor (GABAR) channels in the soma membrane of mouse spinal cord neurones were investigated using the whole-cell, cell-attached and outside-out patch versions of the patch-clamp technique. 2. Current-voltage (I-V) relations of transmitter-activated currents obtained from whole-cell measurements with 145 mM-Cl- intracellularly and extracellularly, showed outward rectification. In voltage-jump experiments, the instantaneous I-V relations were linear, and the steady-state I-V relations were rectifying outwardly indicating that the gating of GlyR and GABAR channels is voltage sensitive. 3. The reversal potential of whole-cell currents shifted 56 mV per tenfold change in internal Cl- activity indicating activation of Cl--selective channels. The permeability ratio of K+ to Cl- (PK/PCl) was smaller than 0.05 for both channels. 4. The permeability sequence for large polyatomic anions was formate > bicarbonate > acetate > phosphate > proprionate for GABAR channels; phosphate and propionate were not measurably permeant in GlyR channels. This indicates that open GlyR and GABAR channels have effective pore diameters of 5.2 and 5.6 .ANG., respectively. The sequence of relative permeabilities for small anions was SCN- > I- > Br- > Cl- > F- for both channels. 5. GlyR and GABAR channels are multi-conductance-state channels. In cell-attached patches the single-channel slope conductances close to 0 mV membrane potential were 29, 18 and 10 pS for glycine, and 28, 17 and 10 pS for GABA-activated channels. The most frequently observed (main) conductance states were 29 and 17 pS for the GlyR and GABAR channel, respectively. 6. In outside-out patches with equal extracellular and intracellular concentrations of 145 mM-Cl-, the conductance states were 46, 30, 20 and 12 pS for GlyR channels and 44, 30, 19 and 12 pS for GABAR channels. The most frequently occurring main state was 46 pS for the GlyR and 30 pS for the GABAR channel. 7. Single-channel conductances measured in equal 140 mM concentrations of small anions on both membrane faces revealed a conductance sequence of Cl- > Br- > I- > SCN- > F- for both channels. This is nearly the inverse sequence of that found for the premeability of these ions indicating the presence of binding sites for ions in the channel. 8. The conductance-activity relation of GlyR and GABAR channels for Cl- was described by hyperbolic curves. The maximal conductances (.gamma.max) were 92 and 72 pS; the half-saturation activities (Km) were 108 and 155 mM-Cl- for GlyR and GABAR channels, respectively. 9. The conductance-activity relation for SCN- was characterized by maximal conductances of 35 pS (GlyR) and 32 pS (GABAR). The half-saturation activities were 46 and 57 mM, respectively. In early mixtures of SCN- and Cl- on both membranes sides the channel conductance showed anomalous mole-fraction dependence. This indicates that the channels contain at least two binding sites for anions. 10. The difference between GlyR and GABAR channels in their main-state Cl- conductance could be caused by a small energy difference (.apprx. 1 kJ mol-1) in the interaction of Cl- ions with the channel wall. The observation that both GlyR and GABAR channels display comparable conductance states, show similar permeability and conductance sequences for small anions, a similar conductance saturation curve for Cl- and SCN- and similar mole-fraction dependence of their conductance in Cl--SCN- mixtures could indicate that both receptor channels have a similar molecular structure in those regions of their subunits that form the lining of the channel.