Kullman Lisen, Winterhalter Mathias, Bezrukov Sergey M
Laboratory of Physical and Structural Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-0924 USA.
Biophys J. 2002 Feb;82(2):803-12. doi: 10.1016/S0006-3495(02)75442-8.
Transport of sugars through maltoporin channels reconstituted into planar lipid membranes has traditionally been addressed using multichannel preparations. Here we show that single-channel experiments offer new possibilities to reveal molecular details of the interaction between the sugar and the channel. We analyze time-resolved transient interruptions in the maltoporin ionic current in the presence of differently sized maltodextrins. We find for all studied sugars, from maltotriose to maltoheptaose, that only one sugar molecule is required to completely block one of the pores in the maltoporin trimer. The probability of simultaneous blockage of different pores increases with sugar concentration in a manner that demonstrates their mutual independence. The maltoporin channel is asymmetric and, added from one side only, predominantly inserts in an oriented manner. The asymmetry of the channel structure manifests itself in two ways. First, it is seen as an asymmetrical response to applied voltage at otherwise symmetrical conditions; second, as asymmetrical rates of sugar entry into the channel with asymmetrical (one-sided) sugar addition. Importantly, we find that the sugar residence time in the pore does not depend on which side the sugar is added. This voltage-dependent time is the same for symmetrical, cis, or trans sugar addition. This observation suggests that once a sugar molecule is captured by the "greasy slide" of the channel, it spends enough time there to "forget" from what entrance it was captured. This also means that the blockage events studied here represent sugar translocation events, and not just binding at and release from the same entrance of the channel.
传统上，通过重构到平面脂质膜中的麦芽糖孔蛋白通道运输糖类一直是使用多通道制剂来研究的。在这里，我们表明单通道实验为揭示糖类与通道之间相互作用的分子细节提供了新的可能性。我们分析了在存在不同大小的麦芽糊精时麦芽糖孔蛋白离子电流中的时间分辨瞬态中断情况。我们发现，对于所有研究的糖类，从麦芽三糖到麦芽七糖，只需一个糖分子就能完全阻断麦芽糖孔蛋白三聚体中的一个孔。不同孔同时被阻断的概率随糖浓度增加，这表明它们相互独立。麦芽糖孔蛋白通道是不对称的，并且仅从一侧添加时，主要以定向方式插入。通道结构的不对称性以两种方式表现出来。首先，在其他条件对称的情况下，它表现为对施加电压的不对称响应；其次，在不对称（单侧）添加糖时，糖进入通道的速率不对称。重要的是，我们发现糖在孔中的停留时间并不取决于糖是从哪一侧添加的。对于对称、顺式或反式添加糖，这种电压依赖性时间是相同的。这一观察结果表明，一旦糖分子被通道的“油腻滑道”捕获，它会在那里停留足够长的时间以“忘记”它是从哪个入口被捕获的。这也意味着这里研究的阻断事件代表糖的转运事件，而不仅仅是在通道同一入口处的结合和释放。
