Patch Clamp and Phenotypic Analyses of a Prokaryotic Cyclic Nucleotide-gated K+ Channel Using Escherichia coli as a Host (original) (raw)

2007, Journal of Biological Chemistry

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 spheroplasts, macro-and microscopic MmaK currents are readily detected in excised E. coli membrane patches by a patch clamp. We show that MmaK is indeed gated by submicromolar cAMP and ϳ10-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. Recent sequencing of bacterial genomes reveals that ion channels evolved as early as three billion years ago. K ϩ channels, for example, are widely spread in all life forms, Bacteria, Archaea, and Eukarya (1, 2). Because prokaryotic channel genes can often be heterologously expressed in Escherichia coli at high yield, the channel proteins so produced have laid an inroad to determine their crystal structures. Beginning with the prelude of MacKinnon and Doyle (3) crystal structures of these channels have raised our understanding of the molecular bases of ion channels as illustrated in several atomic structures of prokaryotic K ϩ channels (4-9). Functional interpretation of prokaryotic channel structures by electrophysiological methods, however, has not been straightforward. The main technical barrier is that the prokaryotic channel activities are often difficult to analyze under the existing methodology. A common strategy has been reconstitution of the purified channel protein into artificial lipids for bilayer lipid membrane measurement (5, 10, 11), a process that relies on the chance survival of the channel during detergent extraction and lipid reconstitution. In some cases, the reconstituted channel activities can only be demonstrated with the low resolution 86 Rb ϩ uptake assay (9, 12-14). An often overlooked opportunity to capture these channels in action is the very membrane of the E. coli cells in which they are commonly overproduced. Although the rod of this bacterium (0.75-m diameter, 2 m in length) is about the dimension of a patch clamp pipette tip, there are genetic and pharmacological ways of generating giant E. coli some ten times its original size. In 1987, Martinac et al. (15) first described the enlargement of E. coli into giant spheroplast for direct patch clamp examination of the native mechanosensitive channels (16). Besides mechanosensitive channels, however, this pioneering method has seldom been extended to study the activities of other foreign channels. We have optimized the methods of functional preparation of giant E. coli spheroplast as well as patch clamp of the enlarged membrane to study MthK, the RCK (regulating the conductance of K ϩ)-containing K ϩ channel from Methanobacterium thermoautotrophicum (5). The success in detecting the ensemble current of MthK in E. coli membrane led us to discover its hitherto unknown properties, including deactivation, desensitization, acidic inactivation, and Cd 2ϩ activation (17). In this report, we illustrate the optimized method of giant spheroplast preparation and gigOhm seal formation in detail by functional expression and biophysical characterization of a bacterial cyclic nucleotide-gated K ϩ channel, MmaK 2 from M. magnetotacticum. Related extension of microbial genetics to ion channel research is also discussed. EXPERIMENTAL PROCEDURES Molecular Biology and Phenotype Analysis-The mmaK gene (NCBI 46202428) from M. magnetotacticum (genomic DNA from Dr. B. Martinac, University of Queensland, Austra-* This work was supported by National Institutes of Health Grants GM74821