期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 282, 期 33, 页码 24294-24301出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M703618200
关键词
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资金
- NIGMS NIH HHS [GM047856, GM054867, R01 GM047856, R01 GM054867, R01 GM074821, GM74821] Funding Source: Medline
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.
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