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Category: Bacterial Pathogenesis; Microbial Genetics and Molecular Biology
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Bacterial Ion Channels and Their Eukaryotic Homologs is a succinct summarization of the past ten years of research in the field. Emphasizing a multidisciplinary approach, this book will serve as an important reference for ion channel specialists and as a useful introduction to the topic for non-specialists in such fields as microbiology, structural and developmental biology, neuroscience, and biophysics who wish to acquaint themselves with these molecules.
Written by acknowledged experts, this comprehensive volume examines the accumulated knowledge of channel structures and considers how it has advanced the understanding of basic bacterial ion channel properties. The first compendium of its kind, Bacterial Ion Channels provides a historical background and presents an analysis of the structure and function of several types of channels, including potassium, CIC chloride, and sodium ion channels. Chapters delve into such topics as diversity of potassium channels in prokaryotic and eukaryotic cells, selectivity and permeability of bacterial ion channels, voltage- and mechano-sensing, simulation studies of ion channels using molecular modeling, and the role of bacterial ion channels in cell physiology.
Electronic Only, 321 pages, full-color insert, illustrations, index.
K+ is apparently crucial in dealing with biological water on Earth, even today. Dehydration, i.e., external osmotic upshift, demands an adjustment of cellular osmolarity, lest the pressure difference break the membrane. In cases more thoroughly studied, uptake of K+ constitutes the first line of defense against de hydration. Cells from all animal tissues express K+ channels but not necessarily the Na+ and Ca2+ channels. Thus, K2+ channels seem to underlie some basic functions required by all cells, except a few extreme parasitic bacteria, and their variations far outnumber those of Na2+ and Ca22+ channels, which seem to have evolved by gene duplication and filter mutations of ancestral K2+ channels. The cell physiology of Escherichia coli is better documented than that of any other cells, and it is amenable to genetic or molecular biological manipulation. Flowering plants and frolicking animals are the minority, even among eukaryotes. The plant-like Chara sp. and Nitella sp., for example, exhibit a depolarization-activated K+ current. It is therefore reasonable to focus attention on Kch in E. coli in the hope of gaining insights into the functions of prokaryotic K+ channels, even though the electric activities of Kch have not yet been reported. The topic of prokaryotic channels will likely not attract many microbiologists’ attention either, unless their biological roles are illustrated. That nearly every bacterial or archeal genome includes at least one K+ channel gene, however, leaves little doubt that these channels provide selective advantages.
In this chapter, the author describes the Ktn domain and its role as a channel and transporter regulator, briefly considers the other bacterial systems that possess Ktn and Ktn-related domains, and, finally presents a more detailed analysis of one's understanding of the structure and operation of the KefC system. The domain can be found as an integral part of the main channel-forming protein (e.g., MthK, Kch, and KefC) as an ancillary extrinsic membrane protein (KtrA, TrkA) and as a protein attached to the membrane by a single transmembrane span (AmhM in Bacillus pseudofirmus). The protein is one of the shortest members of the family at 434 residues, truncation of the carboxy-terminal domain being responsible for the shorter length and leading to loss of the Ktn domain. Potassium uptake systems in bacteria essentially fall into three categories: K+-transporting primary ATPases, such as Kdp; secondary transporters, such as Kup; and more complex systems, e.g., TrkAEH in Escherichia coli and KtrAB in B. subtilis. The cation-proton antiports 2 (CPA2) family of proteins was originally defined around the monovalent cation-proton antiports but is now thought to contain channels as well as transporters. The analysis of the crystal structures of K+ channels has generated great insights into the mechanism of ion selectivity. It seems almost certain that the analysis of the biochemistry of channels such as MthK and KirBac will provide new insights into the gating mechanism.
As a member of the family Streptomycetaceae, Streptomyces lividans is a gram-positive soil bacterium with a complex growth cycle. In the presence of nutrients this growth is initiated by the germination of spores. Giant liposome-protoplast vesicles derived from the S. lividans mutant strain carrying the plasmid pKCS1 (with the K channel of streptomycetes A (KcsA) gene) showed ion channel activity under neutral pH (7.2) and asymmetric conditions. The negatively charged lipid phosphatidylglycerol (PG) and the nonbilayer lipid PE support tetramerization and membrane association of KcsA better than the zwitterionic bilayer lipid phosphatidylcholine (PC). In vitro studies using a transcription-translation system revealed that no tetramer is formed in a membrane-free reaction but only after the addition of Escherichia coli inner membrane vesicles. With a coupled in vitro transcription-translation system, highest tetramerization was recorded in the presence of pure lipid vesicles, demonstrating that a phospholipid bilayer is the minimal requirement to form the KcsA tetramer. Polyhydroxybutyrate (PHB) and inorganic phosphate (polyP) are widely distributed among prokaryotic and eukaryotic organisms. The results of experimental and comparative structural data support the conclusion that the crystal structure of the tetrameric KcsA does not present the open state. The majority of the virus progeny is released about 8 h after infection. The current model assumes that Kcv is located in the internal membrane of the virus and will be inserted into the plasma membrane of the host cell. The hydration state of K+ ions and their permeation need to be reinvestigated.
Until recently, electrophysiology served as an indirect window into one's understanding of channel gating and structure. A clearer picture of protein movements involved in gating has recently emerged from the merger of crystallographic and spectroscopic studies with functional analysis. These and other emerging results are discussed from the perspective that understanding the molecular process in details of gating helps explain how a wide variety of effectors can function to open or close a target channel, allowing for the large diversity of channels. A large helix opening may not be a requirement for channel gating as a small helix bend can allow K+ ions an adequate path for flow. The cytoplasmic gating ring of the channel is formed by an octamer of RCK domains. The mechanism of channel gating lies at the core of one's understanding of how channels respond to specific stimuli. Information extracted from functional, crystallographic, and spectroscopic studies of prokaryotic channels has revealed molecular details of how the inner helices and the selectivity filter are involved in channel gating. Focusing the gating forces at a consistent position along the ion conduction pathway allows channels to exist with a large diversity of regulatory domains but maintain a conserved core architecture necessary for efficient function.
Ligand-gated ion channels (LGICs) are a major class of ion channels. Postsynaptic LGICs generate electrical signals in response to specific chemical neurotransmitters such as acetylcholine, glutamate, glycine, or γ-aminobutyric acid. Understanding the polymorphism of the genes encoding the GluRs in particular will increase one's understanding of the role of these receptors in neurogenetic variations. Animal glutamate receptors possess both metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors (iGluRs). The iGluRs and mGluRs are classified into subgroups based on their sequence homology, agonist pharmacology, and intracellular transduction mechanisms. The structural and sequence similarity of the different domains of GluRs to proteins from different types of organisms gives rise to some interesting implications in the evolutionary relationship between prokaryotes and eukaryotes. The iGluRs are classified as nmethyl-D-aspartic acid (NMDA) receptors or non-NMDA receptors, which include kainate (KAI) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The first prokaryotic glutamate receptors to be discovered was GluR0 from Synechocystis sp. strain PCC 6803. An interesting observation was made based on scores of the similarity of profiles of the P-loop and M2 helix. Plant glutamate receptors (GLRs) were first identified in Arabidopsis thaliana. The sequencing of the complete genome of A. thaliana revealed the existence of not 1 but 20 genes that encoded putative GLRs. A famous quote of Theodosius Dobzhansky, “Nothing in biology makes sense, except in the light of evolution,” emphasizes the importance of evolutionary studies in biology. Evolutionary inferences essentially rely on diversity among organisms, where the differences are accumulated randomly from some common ancestor.
This chapter gives an overview on voltage-gated K+ channels. The introduction of molecular biology into the channel field, coupled with the ability to use patch-clamping techniques to precisely analyze properties of channels from many different types of cells, revitalized ion channel biophysics. The structure of the pore-forming domain formed by segments S5-P-S6 of the crystal structure of the full-length voltage-gated K+ channel Aeropyrum pernix (KvAP) protein was similar to that of the MthK crystal, suggesting that it was in an open conformation. The chapter discusses experiments performed on Shaker that are most informative about the structure and voltage-sensing domain. Three types of crevasse models can be envisioned. The first model implies that the transmembrane movement of S4 is relatively large and that the rest of the protein remains relatively static. The second model depicts the opposite extreme in which there is little transmembrane movement of S4, but the location of the barrier and the structures of the crevasses change. The third model is a hybrid of the other two; S4 charges move outward during activation, but this movement is accompanied by an inward movement of the transition barrier and a change in the structures of the crevasses. The first and largest category of 6-TM KK+ channel sequences has the same length of S2-S3 linkers as most eukaryotic Kv channels; this category is the most similar to eukaryotic Kv channels. According to the authors, the most closely related sequence to that of KvAP is the KvCA sequence from Clostridium acetobutylicum.
Inward rectifier K+ channels (Kirs) act as a valve or a diode, allowing an inward current upon hyperpolarization but not allowing exit of K+ ions upon depolarization. The degree of rectification in the Kir channels is correlated with the binding affinity of the channel for blocking cations. Kir channels thus serve diverse and important roles throughout the human body and pose major challenges in the recognition of the molecular basis of Kir-mediated channelopathies. The genes for this family of potassium channels encode proteins ranging from ~360 to 500 amino acids. A comprehensive sequence analysis of K+ channels in Caenorhabditis elegans (CE), Drosophila melanogaster (DM), and mammalian genomes has been performed. Although the fundamental pore structure is the same in all members of the K+ channel family, other parts of the sequence indicate significant structural diversity. Phylogenetic analysis of the Kir genes grouped CE together, excluding the genes from the other two species, indicating that gene duplication occurred after the divergence of CE from the lineage-leading mammals and DM. Plants possess K+ channels that conduct primarily at negative voltages. The inward rectification of these K+ channels in plants is independent of intracellular magnesium, thus differing mechanistically from gating of Kir channels in the animal kingdom. Moreover, chimeras of plant and animal Kirs that contain the S1 to S4 segments of plants are activated by hyperpolarization, suggesting that plant Kirs have a membrane topology similar to that of eukaryotic Kv channels.
This chapter focuses on homology modeling and molecular dynamics (MD) simulations studies of ion channels for the range of single cell organisms from prokaryotes to eukaryotes. The glutamate receptor channels (GluRs) share some distant homology in their transmembrane (TM) domains with K channels but possess distinct extracellular ligand-binding domains for which several structures, of both bacterial and mammalian homologs, are known. It can be seen that molecular modeling and simulations can contribute to studies of ion channels in two respects. Modeling studies enable extrapolation from experimental structures of prokaryotic ion channels to molecular models of eukaryotic homologs, thus aiding design and interpretation of, for example, mutation experiments for dissecting structure-function relationships. Ion channel structures and ion channel models may also be used as the basis of multinanosecond MD simulations. Finally, it will become increasingly important to run multiple simulations on multiple channels to allow comparative analysis of simulation results, which in turn will enable the formulation of more general hypotheses concerning the relationship between the conformational dynamics of channel proteins and their physiological functions.
This chapter presents exemplary studies of the structure-function relationship of four membrane channels of diverse function that illustrate the recent advance in membrane protein modeling: aquaporin water channels, the chloride channel, hemolysin, and the mechanosensitive channel of small conductance. Two obstacles stand in the way of the application of molecular dynamics (MD) simulations to membrane channels: the large size of systems to be simulated and the short timescale to which the method traditionally applies. The authors' first case study focused on aquaporin (AQP) water channels. These channels are particularly amenable to MD investigations due to their rather simple function, their great structural rigidity, and the short timescale of the elementary conduction process. The authors characterize the way in which Cl- passes through the ClC channel under extremely favorable conditions: open gates and no proton-coupling to slow the dynamics. Two sections of the chapter closely follow the reports by Aksimentiev and Schulten and by Sotomayor and Schulten. The chapter also presents four case studies that demonstrate the power of MD simulations in unraveling the mechanisms underlying the function of membrane channels. The study of ion and water permeation through hemolysin exemplifies how accurately one can simulate today even very large membrane channel systems. A detailed energetic analysis of ion permeation through chloride channels proposes a two-ion permeation mechanism that can reconcile naturally structural and physiological data. The chapter concludes by suggesting that one can reach more quickly to the goal of understanding membrane channels with computational modeling.
Eukaryotic voltage-sensitive sodium and calcium channels have a major structural subunit that consists of four linked, homologous domains, which contain six putative transmembrane (TM) segments for an overall total of 24. In the putative pore-lining S5-S6 linker, a glutamate residue appears in the position homologous to that acknowledged as the selectivity filter in eukaryotic sodium and calcium channels. Members of the sodium channel family typically have a selectivity ring consisting of the aspartate, glutamate, lysine, and alanine residues contributed from domains I through IV, respectively. The possibility of bacterial sodium channels being involved in rapid flagellar movement has been raised by Clapham and collaborators. Voltage-gated ion channels (VICs) are a subset of the larger P-loop ion channel family. Most voltage-activated channels exhibit two competing responses to membrane depolarization, which initiates both an activation process that results in channel opening and an inactivation process that ultimately results in channel closing. Analyses of the crystal structures of two 2-TM bacterial potassium channels led to the conclusion that the structure of KcsA represented a closed state and that of the related calcium-gated channel, MthK, crystallized in the presence of bound calcium, represented an open state. The 6-TM channels such as Shaker offer easier genetic manipulation, including the possibility of changing four residues in a functional channel for the price of a single mutation. A strong argument has been made that the bacterial sodium channels represent, or at least are closely related to, progenitors of eukaryotic channels.
Several members of the CLC family of proteins are voltage-gated, and this entire family is sometimes termed the voltage-gated family of Cl- channels (chloride channels). Bioinformatics screening of CLC channels suggests the existence of regions in the cytoplasmic carboxyl tail of these proteins that have the propensity to bind actin and possibly other cytoskeletal proteins. While the ClC-0, ClC-1, ClC-2, and ClC-Ka and -Kb branch of the CLC family are generally believed to be plasma membrane channels, the location of the others is controversial, being totally or partially confined to intracellular membranes under normal circumstances. The authors' recent analysis of the human CLCN7 promoter has identified several interesting consensus transcription-factor-binding sites. By mutation, their importance in the transcriptional regulation of this gene has been demonstrated. Transcription factor binding has been demonstrated, and the identification of these factors is under way. Mutations in CLC channels have now been associated with a number of diseases in both humans and other species. Myotonia, the best understood of the CLC diseases, is characterized by a peculiar muscle stiffness that is normally painless, an inability of the muscle to relax after a voluntary contraction. This is purely a muscle phenomenon and does not involve nerve dysfunction. It is sometimes accompanied by weakness, and the stiffness may improve after exercise. Genetic or pharmacological manipulation of the relevant Cl1 channels could treat CLC diseases or, conversely, mimicking some aspect of these diseases could point the way to therapies for other diseases.
Early studies of channels within Escherichia coli native membranes defined many of the fundamental properties of bacterial mechanosensitive channel (MS) channel activities. The observed activity was reported to be modulated, but not gated, by voltage, and a subsequent study demonstrated that amphipaths that intercalate into the membrane asymmetrically can modulate the sensitivity of the channel. The mscS gene family was discovered by classical genetics. The structure of E. coli MscS was solved to 3.9-Å resolution by X-ray crystallography, residues 27 to 280 were resolved. On the other hand, this study used only molecular simulation to model the permeation of the pore, an approach that inherently entertains many assumptions. As implied by some studies, the MscS extended family is not confined to E. coli. A survey of some of the MscS family members from archaea has shown researchers just how diverse the activities encoded by family members can be in conductance, sensitivity, and ionic preference; some homologs even demonstrate cationic rather than anionic preferences. The MscS-like family of channels is extremely large and diverse. In its most streamlined form, e.g., MscS in E. coli, it has many similarities with its counterpart, MscL. It appears to directly sense membrane tension, and it appears to utilize modification of transmembrane (TM) domains tilt and rotation in its gating sequence. Because the MscS and MscL families are so far removed from each other, these preserved features may reflect conserved mechanisms found in many MS channel families.
This chapter discusses properties, structure, and the mechanism of gating of the large mechanosensitive (MS) channel MscL, which is probably the best understood tension-gated channel to date. The progress has been rapid, and within 10 years of MscL cloning we have a reasonably supported structural model of gating. The membrane topology determined with the PhoA fusion approach indicated that the short N-terminal (S1, ~15 residues) and the larger C-terminal (S3, ~40 residues) segments are cytoplasmic, whereas the loop connecting M1 and M2 segments (S2, ~25 residues) resides on the extracellular side (periplasm). MscL is activated directly by tension in the lipid bilayer in which the protein is embedded. Upon a strong osmotic downshift, hydrostatic pressure building up inside the cell causes a distension of the elastic cell wall and eventually stresses the inner membrane. Analysis of occupancies of substates and rates of subtransitions as functions of tension provided valuable information about the positions of intermediate states and major barriers on the reaction coordinate. MscL remains stable and functional in liposomes made of exogenous lipids. Initial characterization of MscL using scanning cysteine mutagenesis, site-specific spin labeling, and electron paramagnetic resonance (EPR) spectroscopy demonstrated that the transmembrane region of EcoMscL has essentially the same organization as TbMscL, validating the correctness of the homology-based alignment of the EcoMscL model. The hypothetical S1 bundle was proposed to act as the second gate because a poreoccluding element was needed to explain the postulated expanded low-conducting substate.
This chapter provides an overview on the role of bacterial channels in cell physiology. The proposed role of mechanosensitive (MS) channels is the rapid and nonspecific release of solutes from the cell in response to the generation of excessive turgor pressure. This model has now been tested and verified for E. coli. In most bacterial cells Cl- ions have not been predicted to have significant roles in cell physiology. In some halophilic bacteria Cl- is accumulated to provide salt balance during growth in very high salt concentrations. The aquaglyceroporins are pores that allow the permeation of water and small linear polyhydric molecules through the bacterial membrane. The cell faces two types of problems that relate to ammonium ions. First, there is the need to scavenge for ammonium when the concentration in the environment is low. Second, cells growing on broth may encounter a surfeit of cytoplasmic ammonium ions arising from the deamination of amino acids. In a wide range of bacterial genera the genes for AmtB and GlnK exist as an operon, and recent work suggests that the former is a pore that is used by cells to accelerate the passage of ammonium ions across the membrane and that GlnK is a specific component of the nitrogen-regulatory circuit that controls its activity. The increased understanding of mechanistic aspects of the channel selectivity and gating has advanced rapidly, but an appreciation of the role of channels in cell physiology lags well behind.
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At A Glance
Bacterial Ion Channels and Their Eukaryotic Homologs is a succinct summarization of the past ten years of research in the field. Emphasizing a multidisciplinary approach, this book will serve as an important reference for ion channel specialists and as a useful introduction to the topic for nonspecialists in such fields as microbiology, structural and developmental biology, neuroscience, and biophysics who wish to acquaint themselves with these molecules. Written by acknowledged experts, this comprehensive volume examines the accumulated knowledge of channel structures and considers how it has advanced the understanding of basic bacterial ion channel properties. The first compendium of its kind, Bacterial Ion Channels provides a historical background and presents an analysis of the structure and function of several types of channels, including potassium, CIC chloride, and sodium ion channels. Chapters delve into such topics as diversity of potassium channels in prokaryotic and eukaryotic cells, selectivity and permeability of bacterial ion channels, voltage- and mechano-sensing, simulation studies of ion channels using molecular modeling, and the role of bacterial ion channels in cell physiology.
Description
This is a series of reviews focused on bacterial ion channels. Included are potassium, sodium, chloride and water. Structural information obtained from these channels is related to corresponding activities in eukaryotic cells.
Purpose
The goal is to provide an up-to-date review of work in the area of bacterial ion channels. This worthwhile goal is well met.
Audience
The intended audience is investigators working in the ion channel field (both pro- and eukaryotic) and other scientists with a general interest in the topic. Those likely to profit from reading this book range from post-doctoral fellows to senior investigators. The editors have assembled a diverse group of well qualified contributors.
Features
A major section is devoted to the various types of potassium channels (ligand, voltage, etc.) with subsequent sections covering sodium, chloride, and water transport mechanisms. The chapters are up-to-date and cover the diverse approaches used in studying these transmembrane molecules. Each chapter is accompanied by a thorough bibliography that allows easy access to primary sources. Of particular value are discussions of the relationships between the bacterial channels and their eukaryotic counterparts. Thus, crystal structures are available for some of the prokaryotic channels providing data that should help our understanding of these processes in higher animals. Two of the chapters review modeling and molecular dynamic approaches, areas of growing interest. Similarly, the discussion of a glutamate receptor potassium channel will be of particular interest to investigators working in neuronal systems. One minor concern is that illustrations are sparse within the text, with a set of color plates in the center
of the book. This is always difficult for the reader. Nonetheless, the book is a strong contribution and of interest to investigators in fields other than the ion channels themselves.
Assessment
This is a very interesting book. Although removed from my own interest, I enjoyed reading it and learned a good deal. I expect that others will have a similar experience.
Doody Enterprises
Reviewer: Eugene Davidson, PhD (Georgetown University School of Medicine)
Review Date: Unknown
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