Molecular template for a voltage sensor in a novel K+ channel. III. Functional reconstitution of a sensorless pore module from a prokaryotic Kv channel.
Molecular template for a voltage sensor in a novel K+ channel. III. Functional reconstitution of a sensorless pore module from a prokaryotic Kv channel.
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DOI：
10.1085/jgp.200810077
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发表时间：
2008-12
期刊：
影响因子：
--
通讯作者：
Montal M
中科院分区：
文献类型：
--
作者：
Santos JS;Grigoriev SM;Montal M
KvLm是单核细胞增多性李斯特氏菌的原核电压门控性K+(Kv)通道。KvLm的电压敏感模块(跨膜片段S1-S4)的序列是非典型的，因为它只包含真核生物Kv的8个已知的电压敏感决定的保守带电残基中的3个。相反，KvLm的孔道模块(PM)，包括S4-S5接头和细胞质尾部(接头-S5-P-S6-C末端)是高度保守的。在这里，全长(FL)-KvLm和KvLm-PM蛋白被表达、纯化并重组为巨大的脂质体。重组的FL-KvLm的性质很好地反映了在大肠杆菌球体中异源表达的通道的特征：激活电压右移，微摩尔四丁基铵阻断亲和力，以及与真核细胞Kv相当的单通道电导。相反，通过PM的离子电流概括了FL-KvLm的电导和阻断特性，而KvLm-PM只表现出基本的电压依赖性。鉴于KvLm-PM显示了FL-KvLm和其他真核Kv的许多导电性质，包括严格的离子选择性，我们得出结论，在没有电压感应模块的情况下，PM亚基在脂质双层中的自组装产生了类似于天然KvLm的导电低聚物，并且真核Kv通道中观察到的电压传感和PM的结构独立性最初是在原核Kv通道的设计中自然实现的。总而言之，结果表明，这一强大的功能模块将被证明是一个有价值的分子模板，用于耦合新的传感器，并阐明PM残基对Kv传导特性的特定贡献。
KvLm is a prokaryotic voltage-gated K+ (Kv) channel from Listeria monocytogenes. The sequence of the voltage-sensing module (transmembrane segments S1-S4) of KvLm is atypical in that it contains only three of the eight conserved charged residues known to be deterministic for voltage sensing in eukaryotic Kv's. In contrast, the pore module (PM), including the S4-S5 linker and cytoplasmic tail (linker-S5-P-S6-C-terminus) of KvLm, is highly conserved. Here, the full-length (FL)-KvLm and the KvLm-PM only proteins were expressed, purified, and reconstituted into giant liposomes. The properties of the reconstituted FL-KvLm mirror well the characteristics of the heterologously expressed channel in Escherichia coli spheroplasts: a right-shifted voltage of activation, micromolar tetrabutylammonium-blocking affinity, and a single-channel conductance comparable to that of eukaryotic Kv's. Conversely, ionic currents through the PM recapitulate both the conductance and blocking properties of the FL-KvLm, yet the KvLm-PM exhibits only rudimentary voltage dependence. Given that the KvLm-PM displays many of the conduction properties of FL-KvLm and of other eukaryotic Kv's, including strict ion selectivity, we conclude that self-assembly of the PM subunits in lipid bilayers, in the absence of the voltage-sensing module, generates a conductive oligomer akin to that of the native KvLm, and that the structural independence of voltage sensing and PMs observed in eukaryotic Kv channels was initially implemented by nature in the design of prokaryotic Kv channels. Collectively, the results indicate that this robust functional module will prove valuable as a molecular template for coupling new sensors and to elucidate PM residue–specific contributions to Kv conduction properties.
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