Single-channel measurements of an N-acetylneuraminic acid-inducible outer membrane channel in Escherichia coli.
Single-channel measurements of an N-acetylneuraminic acid-inducible outer membrane channel in Escherichia coli.
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DOI：
10.1007/s00249-011-0781-5
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发表时间：
2012-03
影响因子：
2
通讯作者：
Eisenberg, Bob
中科院分区：
文献类型：
--
作者：
Giri, Janhavi;Tang, John M.;Wirth, Christophe;Peneff, Caroline M.;Eisenberg, Bob
NanC是一种参与唾液酸（Neu5Ac，即n -乙酰神经氨酸）摄取的大肠杆菌外膜蛋白。NanC基因的表达由Neu5Ac诱导和控制。Neu5Ac的转运机制尚不清楚。NanC的结构最近得到了解决（PDB代码：2WJQ），包括一个独特的正电荷（碱性）侧链排列，与酸性糖运输的作用一致。然而，最初的NanC功能测量未能发现其在唾液酸运输中的作用，可能是因为实验中使用的离子条件。我们在这里表明，通常用于测量外膜孔功能的离子条件不适用于纳米碳纳米管。pH 7.0下的NanC单通道具有：(1)电导在100 mM KCl至3 M KCl中为100 pS至800 pS，(2)阴离子对阳离子的选择性（在250 mM KCl中为+16 mV || 1m KCl），以及(3)两种形式的电压依赖性门控（通道闭合高于±200 mV）。当HEPES浓度在pH 7.4、250 mM KCl中从100 μM增加到100 mM时，单通道电导降低了50%，这与晶体结构中观察到的两个HEPES结合位点一致。研究替代缓冲液，我们发现磷酸盐干扰通道电导。当磷酸盐浓度在250 mM KCl中从0 mM增加到5 mM， pH为8.0时，单通道电导降低19%。令人惊讶的是，溶液中的TRIS与ag| AgCl电极发生反应，即使电极位于琼脂- kcl桥的远端也会产生伪影。合适的NanC基线溶液为250 mM KCl，调整至pH 7.0，无缓冲液。
NanC is an Escherichia coli outer membrane protein involved in sialic acid (Neu5Ac, i.e., N-acetylneuraminic acid) uptake. Expression of the NanC gene is induced and controlled by Neu5Ac. The transport mechanism of Neu5Ac is not known. The structure of NanC was recently solved (PDB code: 2WJQ) and includes a unique arrangement of positively charged (basic) side chains consistent with a role in acidic sugar transport. However, initial functional measurements of NanC failed to find its role in the transport of sialic acids, perhaps because of the ionic conditions used in the experiments. We show here that the ionic conditions generally preferred for measuring the function of outer-membrane porins are not appropriate for NanC. Single channels of NanC at pH 7.0 have: (1) conductance 100 pS to 800 pS in 100 mM KCl to 3 M KCl), (2) anion over cation selectivity (Vreversal = +16 mV in 250 mM KCl || 1 M KCl), and (3) two forms of voltage-dependent gating (channel closures above ±200 mV). Single-channel conductance decreases by 50% when HEPES concentration is increased from 100 μM to 100 mM in 250 mM KCl at pH 7.4, consistent with the two HEPES binding sites observed in the crystal structure. Studying alternative buffers, we find that phosphate interferes with the channel conductance. Single-channel conductance decreases by 19% when phosphate concentration is increased from 0 mM to 5 mM in 250 mM KCl at pH 8.0. Surprisingly, TRIS in the baths reacts with Ag|AgCl electrodes, producing artifacts even when the electrodes are on the far side of agar–KCl bridges. A suitable baseline solution for NanC is 250 mM KCl adjusted to pH 7.0 without buffer.
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