VOLTAGE-GATED CALCIUM CHANNELS - DIRECT OBSERVATION OF THE ANOMALOUS MOLE FRACTION EFFECT AT THE SINGLE-CHANNEL LEVEL
VOLTAGE-GATED CALCIUM CHANNELS - DIRECT OBSERVATION OF THE ANOMALOUS MOLE FRACTION EFFECT AT THE SINGLE-CHANNEL LEVEL
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DOI：
10.1073/pnas.86.13.5207
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发表时间：
1989-07-01
影响因子：
11.1
通讯作者：
TSIEN, RW
中科院分区：
文献类型：
--
作者：
FRIEL, DD;TSIEN, RW
电压门控Ca通道是非常有效的孔：即使在表现出强离子选择性的同时，它们对二价阳离子具有高度渗透性。选择性和离子渗透机制的研究表明，在钙和钡离子的混合物单独的全细胞钙通道电流。这种反常摩尔分数效应（AMFE）为提出离子选择性和渗透机制提供了重要的推动力，这些机制调用多个离子结合位点。然而，单一L型Ca电流的记录并没有证明AMFE [Marban，E.和Yue，D. T.（1988）Biophys. J. 55，594 a（摘要）]，这引起了对它是否是通过开放的Ca通道的离子渗透的表达的怀疑。我们用膜片钳技术记录了PC-12嗜铬细胞瘤细胞L型钙通道。我们的研究结果表明，一个显着的AMFE在单通道水平，但也表明，AMFE只能被发现在限制性条件下的渗透离子浓度和膜电位。虽然当渗透离子以10 mM存在时AMFE在0 mV下是透明的，但当二价阳离子浓度增加至110 mM或膜电位超极化至-40 mV时，这并不明显。我们比较了我们的实验观察与预测的一个单一的文件，两个结合位点模型的钙通道。该模型解释了我们的实验结果。它预测AMFE的条件下，有利于离子-离子相互作用，只要外部结合位点不饱和，由于高渗透离子浓度或负膜电位。
Voltage-gated Ca channels are very efficient pores: even while exhibiting strong ionic selectivity, they are highly permeant to divalent cations. Studies of the mechanism of selectivity and ion permeation have demonstrated that whole-cell Ca channel current in mixtures of Ca and Ba ions alone. This anomalous mole fraction effect (AMFE) has provided an important impetus for proposed mechanisms of ion selectivity and permeation that invoke multiple ion binding sites. However, recordings of unitary L-type Ca currents did not demonstrate that AMFE [Marban, E. and Yue, D. T. (1988) Biophys. J. 55, 594a (abstr.)], raising doubts about whether it is an expression of ion permeation through open Ca channels. We have made patch-clamp recordings from single L-type Ca channels in PC-12 pheochromocytoma cells. Our results demonstrate a significant AMFE at the single-channel level but also indicate that the AMFE can only be found under restrictive conditions of permeant ion concentration and membrane potential. While the AMFE is clear at 0 mV when permeant ions are present at 10 mM, it is not evident when the divalent cation concentration is increased to 110 mM or the membrane potential is hyperpolarized to -40 mV. We compared our experimental observations with predictions of a single-file, two-binding-site model of the Ca channel. The model accounts for our experimental results. It predicts an AMFE under conditions that favor ion-ion interactions, as long as the outer binding site is not saturated due to high permeant ion concentration or negative membrane potential.