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

Abstract

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 a...

Family of prokaryote cyclic nucleotide-modulated ion channels

“…4A). A similar ion selectivity was described for other bacterial CNG channels, such as MloK1 (13) and MmaK (22).…”

“…Bacterial CNG channels could only be successfully studied as reconstituted proteins in liposomes (13,42) or in giant E. coli spheroplasts (22). In the present study, fluorescence imaging and electrophysiological patch-clamp recording demonstrated that SthK-GFP can be expressed as a functional ion channel protein in the plasma membrane of Xenopus oocytes.…”

“…We could experimentally demonstrate that SthK channels are highly selective for K + over Na + ions. Importantly, SthK has several sequence features that closely resemble eukaryote cyclic nucleotidemodulated channels, including a C-linker region, which is missing in previously studied prokaryote homologs, such as MloK1 (10,12,13) and MmaK (22). Together, these data make the SthK channel a promising candidate for future structural analysis to learn more about how mammalian CNG and HCN channels work.…”

Gating of the TrkH ion channel by its associated RCK protein TrkA

“…All mutations were made with the QuikChange kit (Agilent Technologies) and verified by sequencing through the entire coding regions. Giant spheroplasts were prepared from E. coli following a protocol described previously 29-31 , with minor modifications. In brief, a single colony co-transformed with TrkH and TrkA was incubated in 5 ml of modified LB medium (0.5% NaCl, 1% tryptone and 0.5% yeast extract) by shaking at 250 rpm, 37 °C.…”

“…All solutions of perfused nucleotides were prepared from sodium salts. No pressure was applied to the membrane after the GΩ seal formation was achieved, which excluded the endogenous mechanosensitive channel activities 31 . The analog signals were amplified by an Axon-200B patch clamp amplifier (Molecular Devices Inc), and filtered at 1 kHz using the built-in Bessel filter, digitized at 100 μs by Digidata 1322a (Molecular Devices Inc), and recorded to a computer hard disk.…”