Patch clamp and phenotypic analyses of a prokaryotic cyclic nucleotide-gated K+ channel using Escherichia coli as a host
Patch clamp and phenotypic analyses of a prokaryotic cyclic nucleotide-gated K+ channel using Escherichia coli as a host
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DOI：
10.1074/jbc.m703618200
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发表时间：
2007-08-17
影响因子：
4.8
通讯作者：
Choe, Senyon
中科院分区：
文献类型：
--
作者：
Kuo, Mario Meng-Chiang;Saimi, Yoshiro;Choe, Senyon
原核离子通道在为理解离子过滤和通道门控机制提供结构模型方面具有重要价值。然而，它们的功能检测仍然很少见，通常通过将纯化的通道蛋白掺入人工脂膜来进行。在这里，我们通过检测从磁螺杆菌MmaK克隆的一个假定的环核苷酸门控K+通道来证明利用大肠杆菌来进行功能分析。当在野生型大肠杆菌细胞中表达时，MmaK使宿主对外部施加的毫摩尔浓度的K+敏感，表明MmaK在体内形成了一个功能正常的K+管道。在将这些细胞扩增成巨球形质体后，通过膜片钳可以很容易地在切除的E.col膜上检测到宏观和微观的MmaK电流。我们发现MmaK确实受到亚微摩尔cAMP的门控，类似于10倍高的cGMP浓度，表现为一种内向整流的K+特异性电流，在-100 mV处的单位电导为10.8ps。此外，MmaK仅在细胞质一侧被弱酸性的pH灭活。MmaK的体外生物物理特性与其在大肠杆菌中的体内表型相关，暗示其作为细胞内cAMP和调节细菌膜电位的pH传感器的关键作用。以MmaK功能研究为例，我们建立了一个方便和通用的系统，为生物物理分析提供了一个方便和通用的系统，可以进一步与微生物遗传学相吻合。
Prokaryotic ion channels have been valuable in providing structural models for understanding ion filtration and channel-gating mechanisms. However, their functional examinations have remained rare and usually been carried out by incorporating purified channel protein into artificial lipid membranes. Here we demonstrate the utilization of Escherichia coli to host the functional analyses by examining a putative cyclic nucleotide-gated K+ channel cloned from Magnetospirillum magnetotacticum, MmaK. When expressed in wild-type E. coli cells, MmaK renders the host sensitive to millimolar concentrations of externally applied K+, indicating MmaK forms a functional K+ conduit in the E. coli membrane in vivo. After enlarging these cells into giant sphero-plasts, macro- and microscopic MmaK currents are readily detected in excised E. col imembrane patches by a patch clamp. We show that MmaK is indeed gated by submicromolar cAMP and similar to 10-fold higher concentration of cGMP and manifests as an inwardly rectified, K+ - specific current with a 10.8 pS unitary conductance at -100 mV. Additionally, MmaK is inactivated by slightly acidic pH only from the cytoplasmic side. Our in vitro biophysical characterizations of MmaK correlate with its in vivo phenotype in E. coli, implicating its critical role as an intracellular cAMP and pH sensor for modulating bacterial membrane potential. Exemplified by MmaK functional studies, we establish that E. coli and its giant spheroplast provide a convenient and versatile system to express foreign channels for biophysical analyses that can be further dovetailed with microbial genetics.