Prokaryotic K + channels: From crystal structures to diversity (original) (raw)
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The molecular biology of K+ channels
Current Opinion in Cell Biology, 1991
It is now clear that voltage-gated K+ channels are encoded by a set of multigene subfamilies. Expression of different members of these subfamilies, coupled with mutational analysis, has advanced our knowledge of the structure and function of voltage-dependent K+ channels.
Journal of Biological Chemistry, 2001
Sequence similarity among and electrophysiological studies of known potassium channels, along with the three-dimensional structure of the Streptomyces lividans K ؉ channel (KcsA), support the tenet that voltagegated K ؉ channels (Kv channels) consist of two distinct modules: the "voltage sensor" module comprising the N-terminal portion of the channel up to and including the S4 transmembrane segment and the "pore" module encompassing the C-terminal portion from the S5 transmembrane segment onward. To substantiate this modular design, we investigated whether the pore module of Kv channels may be replaced with the pore module of the prokaryotic KcsA channel. Biochemical and immunocytochemical studies showed that chimeric channels were expressed on the cell surface of Xenopus oocytes, demonstrating that they were properly synthesized, glycosylated, folded, assembled, and delivered to the plasma membrane. Unexpectedly, surface-expressed homomeric chimeras did not exhibit detectable voltage-dependent channel activity upon both hyperpolarization and depolarization regardless of the expression system used. Chimeras were, however, strongly dominant-negative when coexpressed with wild-type Kv channels, as evidenced by the complete suppression of wild-type channel activity. Notably, the dominant-negative phenotype correlated well with the formation of stable, glycosylated, nonfunctional, heteromeric channels. Collectively, these findings imply a structural compatibility between the prokaryotic pore module and the eukaryotic voltage sensor domain that leads to the biogenesis of non-responsive channels. Our results lend support to the notion that voltage-dependent channel gating depends on the precise coupling between both protein domains, probably through a localized interaction surface.
Functional Characterization of a Prokaryotic Kir Channel
Journal of Biological Chemistry, 2004
The Kir gene family encodes inward rectifying K ؉ (Kir) channels that are widespread and critical regulators of excitability in eukaryotic cells. A related gene family (KirBac) has recently been identified in prokaryotes. While a crystal structure of one member, Kir-Bac1.1, has been solved, there has been no functional characterization of any KirBac gene products. Here we present functional characterization of KirBac1.1 reconstituted in liposomes. Utilizing a 86 Rb ؉ uptake assay, we demonstrate that KirBac1.1 generates a K ؉-selective permeation path that is inhibited by extraliposomal Ba 2؉ and Ca 2؉ ions. In contrast to KcsA (an acid-activated bacterial potassium channel), KirBac1.1 is inhibited by extraliposomal acid (pK a ϳ 6). This characterization of KirBac1.1 activity now paves the way for further correlation of structure and function in this model Kir channel.
K(+) channels: function-structural overview
Comprehensive Physiology, 2012
Potassium channels are particularly important in determining the shape and duration of the action potential, controlling the membrane potential, modulating hormone secretion, epithelial function and, in the case of those K(+) channels activated by Ca(2+), damping excitatory signals. The multiplicity of roles played by K(+) channels is only possible to their mammoth diversity that includes at present 70 K(+) channels encoding genes in mammals. Today, thanks to the use of cloning, mutagenesis, and the more recent structural studies using x-ray crystallography, we are in a unique position to understand the origins of the enormous diversity of this superfamily of ion channels, the roles they play in different cell types, and the relations that exist between structure and function. With the exception of two-pore K(+) channels that are dimers, voltage-dependent K(+) channels are tetrameric assemblies and share an extremely well conserved pore region, in which the ion-selectivity filter re...
Journal of Biological Chemistry, 2007
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. . 2 The abbreviations used are: Mmak, cyclic nucleotide-gated potassium channel from M. magnetotacticum; MloK1, M. loti cyclic nucleotidegated potassium channel; CNG, cyclic nucleotide-gated ion channel; HCN, hyperpolarization-activated cyclic nucleotide-gated ion channel; CNBD, cyclic nucleotide binding domain; MES, 4-morpholineethanesulfonic acid; TM, transmembrane.
VKCDB: voltage-gated K+ channel database updated and upgraded
Nucleic acids research, 2011
The Voltage-gated K(+) Channel DataBase (VKCDB) (http://vkcdb.biology.ualberta.ca) makes a comprehensive set of sequence data readily available for phylogenetic and comparative analysis. The current update contains 2063 entries for full-length or nearly full-length unique channel sequences from Bacteria (477), Archaea (18) and Eukaryotes (1568), an increase from 346 solely eukaryotic entries in the original release. In addition to protein sequences for channels, corresponding nucleotide sequences of the open reading frames corresponding to the amino acid sequences are now available and can be extracted in parallel with sets of protein sequences. Channels are categorized into subfamilies by phylogenetic analysis and by using hidden Markov model analyses. Although the raw database contains a number of fragmentary, duplicated, obsolete and non-channel sequences that were collected in early steps of data collection, the web interface will only return entries that have been validated as ...
Intra-membrane molecular interactions of K+ channel proteins
2013
Ion channel proteins regulate complex patterns of cellular electrical activity and ionic signaling. Certain K + channels play an important role in immunological biodefense mechanisms of adaptive and innate immunity. Most ion channel proteins are oligomeric complexes with the conductive pore located at the central subunit interface. The long-term activity of many K + channel proteins is dependent on the concentration of extracellular K + ; however, the mechanism is unclear. Thus, this project focused on mechanisms underlying structural stability of tetrameric K + channels. Using KcsA of Streptomyces lividans as a model K + channel of known structure, the molecular basis of tetramer stability was investigated by: 1. Bioinformatic analysis of the tetramer interface. 2. Effect of two local anesthetics (lidocaine, tetracaine) on tetramer stability. 3. Molecular simulation of drug docking to the ion conduction pore. The results provide new insights regarding the structural stability of K + channels and its possible role in cell physiology.