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Pore froming Proteins: Reviews
(59 References)
Montoya, M. and E. Gouaux (2003). "Beta-barrel membrane protein folding and structure viewed through the lens of alpha-hemolysin." Biochim Biophys Acta 1609(1): 19-27.
The beta-barrel is a transmembrane structural motif commonly encountered in bacterial outer membrane proteins and pore-forming toxins (PFTs). Alpha-hemolysin (alphaHL) is a cytotoxin secreted by Staphylococcus aureus that assembles from a water-soluble monomer to form a membrane-bound heptameric beta-barrel on the surface of susceptible cells, perforating the cell membranes, leading to cell death and lysis. The mechanism of heptamer assembly, which has been studied extensively, occurs in a stepwise manner, and the structures of the initial, monomeric form and final, membrane-embedded pore are known. The toxin's ability to assemble from an aqueous, hydrophilic species to a membrane-inserted oligomer is of interest in understanding the assembly of PFTs in particular and the folding and structure of beta-barrel membrane proteins in general. Here we review the structures of the monomeric and heptamer states of LukF and alphaHL, respectively, the mechanism of toxin assembly, and the relationships between alphaHL and nontoxin beta-barrel membrane proteins.
Herson, P. S. and J. P. Adelman (2003). "It takes two to tango, but three to ISA." Neuron 37(3): 370-2.
Rapidly inactivating A-type potassium channels are important determinants of firing frequency in many excitable cells. Nadal et al. (in this issue of Neuron) purified A-type potassium (I(SA)) channels from rat cerebellum and identified a novel beta subunit. This protein, DPPX, associates with the pore-forming subunits and endows previously elusive kinetic properties on A-type channels formed from cloned subunits.
Weiger, T. M., A. Hermann, et al. (2002). "Modulation of calcium-activated potassium channels." J Comp Physiol A Neuroethol Sens Neural Behav Physiol 188(2): 79-87.
Potassium currents play a critical role in action potential repolarization, setting of the resting membrane potential, control of neuronal firing rates, and regulation of neurotransmitter release. The diversity of the potassium channels that generate these currents is nothing less than staggering. This diversity is generated by multiple genes (as many as 100 and perhaps more in some creatures) encoding the pore-forming channel alpha subunits, alternative splicing of channel gene transcripts, formation of heteromultimeric channels, participation of auxiliary (non-pore-forming) beta and other subunits, and modulation of channel properties by post-translational modifications and other mechanisms. Prominent among the potassium channels are several families of calcium activated potassium channels, which are highly selective for potassium ions as their charge carrier, and require intracellular calcium for channel gating. The modulation of one of these families, that of the large conductance calcium activated and voltage-dependent potassium channels, has been especially widely studied. In this review we discuss a few selected examples of the modulation of these channels, to illustrate some of the molecular mechanisms and physiological consequences of ion channel modulation.
Schwanstecher, C. and M. Schwanstecher (2002). "Nucleotide sensitivity of pancreatic ATP-sensitive potassium channels and type 2 diabetes." Diabetes 51 Suppl 3: S358-62.
Type 2 diabetes is generally perceived as a polygenic disorder, with disease development being influenced by both hereditary and environmental factors. However, despite intensive investigations, little progress has been made in identifying the genes that impart susceptibility to the common late-onset forms of the disease. E23K, a common single nucleotide polymorphism in K(IR)6.2, the pore-forming subunit of pancreatic beta-cell ATP-sensitive K(+) (K(ATP)) channels, significantly enhances the spontaneous open probability of these channels, and thus modulates sensitivities toward inhibitory and activatory adenine nucleotides. Based on previous association studies, we present evidence that with an estimated attributable proportion of 15% in Caucasians, E23K in K(IR)6.2 appears to be the most important genetic risk factor for type 2 diabetes yet identified.
Portnoy, D. A., V. Auerbuch, et al. (2002). "The cell biology of Listeria monocytogenes infection: the intersection of bacterial pathogenesis and cell-mediated immunity." J Cell Biol 158(3): 409-14.
Listeria monocytogenes has emerged as a remarkably tractable pathogen to dissect basic aspects of cell biology, intracellular pathogenesis, and innate and acquired immunity. In order to maintain its intracellular lifestyle, L. monocytogenes has evolved a number of mechanisms to exploit host processes to grow and spread cell to cell without damaging the host cell. The pore-forming protein listeriolysin O mediates escape from host vacuoles and utilizes multiple fail-safe mechanisms to avoid causing toxicity to infected cells. Once in the cytosol, the L. monocytogenes ActA protein recruits host cell Arp2/3 complexes and enabled/vasodilator-stimulated phosphoprotein family members to mediate efficient actin-based motility, thereby propelling the bacteria into neighboring cells. Alteration in any of these processes dramatically reduces the ability of the bacteria to establish a productive infection in vivo.
Panchal, R. G., M. L. Smart, et al. (2002). "Pore-forming proteins and their application in biotechnology." Curr Pharm Biotechnol 3(2): 99-115.
Proteins and peptides that form membrane-spanning pores and channels comprise a diverse class of molecules ranging from short peptides that are unregulated and create non-selective pathways to large ion channel proteins that are highly regulated and exhibit exquisite selectivity for particular ions. The diversity of regulation and selectivity, together with recent advances in protein "re-engineering" technology, provide a strong framework on which to build custom molecules with wide-ranging biotechnological application. Here we review a selection of pore-forming peptides and proteins from a number of different species to highlight their structural and functional diversity. The current and potential uses of native and re-engineered molecules are discussed together with a novel strategy to re-engineer alpha-hemolysin to create targeted and regulable cell-killing agents termed proimmunolysins. Numerous pore-forming peptides are currently in development as antimicrobial agents with potential application as anti-tumorigenic agents. In addition to their roles as biotherapeutic agents, pore-forming proteins are also being developed as biosensors for a range of different analytes. Recent examples of this technology include the use of alpha-hemolysin with an adapter molecule to create sensors for organic molecules and gramicidin as a general-purpose sensor for a range of analytes. These approaches promise to deliver a configurable binding site for analytes encoded in a readily measured electrical signal. The number of applications for pore-forming molecules is sure to grow in both quantity and diversity with increased knowledge of the fundamental structure and function of pores.
Noebels, J. L. (2002). "Sodium channel gene expression and epilepsy." Novartis Found Symp 241: 109-20; discussion 120-3, 226-32.
Na+ channelopathies that prolong membrane depolarization lead to neuronal bursting, abnormal network synchronization, and various patterns of episodic neurological disorders, including epilepsy. Two distinct pathways exist for generating epileptic phenotypes based on inherited disorders of voltage-gated Na+ ion channels. The first pathway is direct, involving mutations in genes encoding the pore-forming alpha1 and regulatory beta subunits of the channel that directly alter current amplitude or kinetics. These mutations favour repetitive firing and network hyperexcitability, although often the circuits most vulnerable to functional alterations are not easy to identify and the emergent clinical phenotypes are difficult to predict. The second pathway involves mutation of other genes that lead to downstream modifications in Na+ channel expression. Two clinically relevant examples of localization-related vulnerability in brain are described that illustrate how specific phenotypes arise from both direct and secondary pathways. Selective expression of the cardiac SCN5A channel within limbic regions of brain may explain why mutation of the gene for this tetrodotoxin-insensitive current may be associated with seizures. Ectopic expression of type II Na+ channels along axonal internodes in hypomyelinated brain may reveal why deletion of the myelin basic protein gene leads to subcortical seizure patterns. Analysis of these models offers insight into developmental processes that control the cellular expression and plasticity of Na+ channel genes, and will help to clarify mechanisms of hereditary Na+ channel-based epileptogenesis.
Nerbonne, J. M. and W. Guo (2002). "Heterogeneous expression of voltage-gated potassium channels in the heart: roles in normal excitation and arrhythmias." J Cardiovasc Electrophysiol 13(4): 406-9.
In the mammalian myocardium, there are marked regional differences in action potential waveforms and frequency dependences. This heterogeneity impacts the normal dispersion of ventricular repolarization and appears to reflect the differential expression of voltage-gated K+ channels. Multiple types of voltage-gated K+ currents have been distinguished in mammalian ventricles and, in many cases, the K+ (Kv) channel pore-forming (alpha) and accessory (beta) subunits encoding these channels have been identified. In the diseased myocardium, remodeling of voltage-gated K+ currents occurs, influencing propagation and rhythmicity, effects that can lead to increased dispersion of ventricular repolarization and create substrates for reentrant arrhythmias. Targeting the K+ channels that function to maintain the normal dispersion of ventricular repolarization could be effective in treating cardiac arrhythmias.
Korovkina, V. P. and S. K. England (2002). "Molecular diversity of vascular potassium channel isoforms." Clin Exp Pharmacol Physiol 29(4): 317-23.
1. One essential role for potassium channels in vascular smooth muscle is to buffer cell excitation and counteract vasoconstrictive influences. Several molecular mechanisms regulate potassium channel function. The interaction of these mechanisms may be one method for fine-tuning potassium channel activity in response to various physiological and pathological challenges. 2. The most prevalent K+ channels in vascular smooth muscle are large-conductance calcium- and voltage-sensitive channels (maxi-K channels) and voltage-gated channels (Kv channels). Both channel types are complex molecular structures consisting of a pore-forming alpha-subunit and an ancillary beta-subunit. The maxi-K and Kv channel alpha-subunits assemble as tetramers and have S4 transmembrane domains that represent the putative voltage sensor. While most vascular smooth muscle cells identified to date contain both maxi-K and Kv channels, the expression of individual alpha-subunit isoforms and beta-subunit association occurs in a tissue-specific manner, thereby providing functional specificity. 3. The maxi-K channel alpha-subunit derives its molecular diversity by alternative splicing of a single-gene transcript to yield multiple isoforms that differ in their sensitivity to intracellular Ca2+ and voltage, cell surface expression and post- translational modification. The ability of this channel to assemble as a homo- or heterotetramer allows for fine-tuning control to intracellular regulators. Another level of diversity for this channel is in its association with accessory beta-subunits. Multiple beta-subunits have been identified that can arise either from separate genes or alternative splicing of a beta-subunit gene. The maxi-K channel beta-subunits modulate the channel's Ca2+ and voltage sensitivity and kinetic and pharmacological properties. 4. The Kv channel alpha-subunit derives its diverse nature by the expression of several genes. Similar to the maxi-K channel, this channel has been shown to assemble as a homo- and heterotetramer, which can significantly change the Kv current phenotype in a given cell type. Association with a number of the ancillary beta-subunits affects Kv channel function in several ways. Beta-subunits can induce inactivating properties and act as chaperones, thereby regulating channel cell-surface expression and current kinetics.
Isom, L. L. (2002). "Beta subunits: players in neuronal hyperexcitability?" Novartis Found Symp 241: 124-38; discussion 138-43, 226-32.
Voltage-gated Na+ channels are glycoprotein complexes responsible for initiation and propagation of action potentials in excitable cells such as central and peripheral neurons, cardiac and skeletal muscle myocytes, and neuroendocrine cells. Mammalian Na+ channels are heterotrimers, composed of a central, pore-forming a subunit and two auxiliary beta subunits. The a subunits form a gene family with at least 10 members. Mutations in alpha subunit genes have been linked to paroxysmal disorders such as epilepsy, long QT syndrome, and hyperkalaemic periodic paralysis in humans, and motor endplate disease and cerebellar ataxia in mice. Three genes encode Na + channel beta subunits with at least one alternative splice product. A mutation in the beta1 subunit gene has been linked to generalized epilepsy with febrile seizures plus type 1 (GEFS+1) in a human family with this disease. Na+ channel beta subunits are multifunctional. They modulate channel gating and regulate the level of channel expression at the plasma membrane. More recently, they have been shown to function as cell adhesion molecules in terms of interaction with extracellular matrix, regulation of cell migration, cellular aggregation, and interaction with the cytoskeleton. Structure-function studies have resulted in the preliminary assignment of functional domains in the beta1 subunit. A Na+ channel signalling complex is proposed that involves beta subunits as channel modulators as well as cell adhesion molecules, other cell adhesion molecules such as neurofascin and contactin, RPTPbeta, and extracellular matrix molecules such as tenascin.
Isom, L. L. (2002). "The role of sodium channels in cell adhesion." Front Biosci 7: 12-23.
Voltage-gated sodium channels are unique in that they combine action potential conduction with cell adhesion. Mammalian sodium channels are heterotrimers, composed of a central, pore-forming alpha subunit and two auxiliary beta subunits. The alpha subunits are members of a large gene family containing the voltage-gated sodium, potassium, and calcium channels. Sodium channel alpha subunits form a gene subfamily with at least eleven members. Mutations in sodium channel alpha subunit genes have been linked to paroxysmal disorders such as epilepsy, long QT syndrome (LQT), and hyperkalemic periodic paralysis in humans, and motor endplate disease and cerebellar ataxia in mice. Three genes encode the sodium channel beta subunits with at least one alternative splice product. Unlike the pore-forming alpha subunits, the sodium channel beta subunits are not structurally related to beta subunits of calcium and potassium channels. Sodium channel beta subunits are multifunctional. They modulate channel gating and regulate the level of channel expression at the plasma membrane. We have shown that beta subunits also function as cell adhesion molecules (CAMs) in terms of interaction with extracellular matrix molecules, regulation of cell migration, cellular aggregation, and interaction with the cytoskeleton. A mutation in SCN1B has been shown to cause GEFS+1 epilepsy in human families. We propose that the sodium channel signaling complex at nodes of Ranvier involves beta subunits as channel modulators as well as CAMs, other CAMs such as neurofascin and contactin, RPTPbeta, and extracellular matrix molecules such as tenascin. Finally, we explore other subunits of voltage-gated ion channels as potential CAM candidates.
Hoenderop, J. G., B. Nilius, et al. (2002). "Molecular mechanism of active Ca2+ reabsorption in the distal nephron." Annu Rev Physiol 64: 529-49.
The identification of the epithelial Ca(2+) channel (ECaC) complements the group of Ca(2+) transport proteins including calbindin-D28K, Na(+)/Ca(2+) exchanger and plasma membrane Ca(2+)-ATPase, which are co-expressed in 1,25(OH)2D3- responsive nephron segments. ECaC constitutes the rate-limiting apical entry step in the process of active transcellular Ca(2+) transport and belongs to a superfamily of Ca(2+) channels that includes the vanilloid receptor and transient receptor potential channels. This new Ca(2+) channel consists of six transmembrane-spanning domains, including a pore-forming hydrophobic stretch between domain 5 and 6. The C- and N-terminal tails contain several conserved regulatory sites, implying that the channel function is modulated by regulatory proteins. The distinctive functional properties of ECaC include a constitutively activated Ca(2+) permeability, a high selectivity for Ca(2+), hyperpolarization-stimulated and Ca(2+)-dependent feedback regulation of channel activity, and 1,25(OH)2D3-induced gene activation. This review covers the distinctive properties of this new highly Ca(2+)-selective channel and highlights the implications for active transcellular Ca(2+) reabsorption in health and disease.
Heuck, A. P. and A. E. Johnson (2002). "Pore-forming protein structure analysis in membranes using multiple independent fluorescence techniques." Cell Biochem Biophys 36(1): 89-101.
A large number of transmembrane proteins form aqueous pores or channels in the phospholipid bilayer, but the structural bases of pore formation and assembly have been determined experimentally for only a few of the proteins and protein complexes. The polypeptide segments that form the transmembrane pore and the secondary structure that creates the aqueous-lipid interface can be identified using multiple independent fluorescence techniques (MIFT). The information obtained from several different, but complementary, fluorescence analyses, including measurements of emission intensity, fluorescence lifetime, accessibility to aqueous and to lipophilic quenching agents, and fluorescence resonance energy transfer (FRET) can be combined to characterize the nature of the protein-membrane interaction directly and unambiguously. The assembly pathway can also be determined by measuring the kinetics of the spectral changes that occur upon pore formation. The MIFT approach therefore allows one to obtain structural information that cannot be obtained easily using alternative techniques such as crystallography. This review briefly outlines how MIFT can reveal the identity, location, conformation, and topography of the polypeptide sequences that interact with the membrane.
Hanlon, M. R. and B. A. Wallace (2002). "Structure and function of voltage-dependent ion channel regulatory beta subunits." Biochemistry 41(9): 2886-94.
Voltage-dependent K(+), Ca(2+), and Na(+) channels play vital roles in basic physiological processes, including management of the action potential, signal transduction, and secretion. They share the common function of passively transporting ions across cell membranes; thus, it would not be surprising if they should exhibit similarities of both structure and mechanism. Indeed, the principal pore-forming (alpha) subunits of each show either exact or approximate 4-fold symmetry and share a similar transmembrane topology, and all are gated by changes in membrane potential. Furthermore, these channels all possess an auxiliary polypeptide, designated the beta subunit, which plays an important role in their regulation. Despite considerable functional semblences and abilities to interact with structurally similar alpha subunits, however, there is considerable structural diversity among the beta subunits. In this review, we discuss the similarities and differences in the structures and functions of the beta subunits of the voltage-dependent K(+), Ca(2+), and Na(+) channels.
Gilbert, R. J. (2002). "Pore-forming toxins." Cell Mol Life Sci 59(5): 832-44.
Pore-forming toxins are widely distributed proteins which form lesions in biological membranes. In this review, bacterial pore-forming toxins are treated as a paradigm and discussed in terms of the structural principles on which they work. Then, a large family of bacterial toxins, the cholesterol-binding toxins, are analyzed in depth to provide an overview of the processes involved in pore formation. The ways in which the cholesterol-binding toxins (cholesterol-dependent cytolysins) interact with membranes and form pores, the structure of the monomeric soluble and oligomeric pore-forming states, and the effects of the toxin on membrane structure are discussed. By surveying the range of work which has been done on cholesterol-binding toxins, a working model is elaborated which reconciles two current, apparently diametrically opposed, models for their mechanism.
Gentschev, I., G. Dietrich, et al. (2002). "The E. coli alpha-hemolysin secretion system and its use in vaccine development." Trends Microbiol 10(1): 39-45.
Many Gram-negative bacteria use a type I secretion system to translocate proteins, including pore-forming toxins, proteases, lipases and S-layer proteins, across both the inner and outer membranes into the extracellular surroundings. The Escherichia coli alpha-hemolysin (HlyA) secretion system is the prototypical and best characterized type I secretion system. The structure and function of the components of the HlyA secretion apparatus, HlyB, HlyD and TolC, have been studied in great detail. The functional characteristics of this secretion system enable it to be used in a variety of different applications, including the presentation of heterologous antigens in live-attenuated bacterial vaccines. Such vaccines can be an effective delivery system for heterologous antigens, and the use of a type I secretion system allows the antigens to be actively exported from the cytoplasm of the bacterial carrier rather than only becoming accessible to the host immune system after bacterial disintegration.
Frey, J. and P. Kuhnert (2002). "RTX toxins in Pasteurellaceae." Int J Med Microbiol 292(3-4): 149-58.
RTX toxins (repeats in the structural toxin) are pore-forming protein toxins produced by a broad range of pathogenic Gram-negative bacteria. In vitro, RTX toxins mostly exhibit a cytotoxic and often also a hemolytic activity. They are particularly widespread in species of the family Pasteurellaceae which cause infectious diseases, most frequently in animals but also in humans. Most RTX toxins are proteins with a molecular mass of 100-200 kDa and are post-translationally activated by acylation via a specific activator protein. The repeated structure of RTX toxins, which gave them their name, is composed of iterative glycine-rich nonapeptides binding Ca2+ on the C-terminal half of the protein. Genetic analysis of RTX toxins of various species of Pasteurellaceae and of a few other Gram-negative bacteria gave evidence of horizontal transfer of genes encoding RTX toxins and led to speculations that RTX toxins might have originated from Pasteurellaceae. The toxic activities of RTX toxins in host cells may lead to necrosis and apoptosis and the underlying detailed mechanisms are currently under investigation. The impact of RTX toxins in pathogenicity and the immune responses of the host were described for several species of Pasteurellaceae. Neutralizing antibodies were shown to significantly reduce the cytotoxic activity of RTX toxins. They constitute a valuable strategy in the development of immuno-prophylactics against several animal diseases caused by pathogenic species of Pasteurellaceae. Although many RTX toxins possess cytotoxic and hemolytic activities toward a broad range of cells and erythrocytes, respectively, a few RTX toxins were shown to have cytotoxic activity only against cells of specific hosts and/or show cell-type specificity. Further evidence exists that RTX toxins play a potential role in host specificity of certain pathogens.
Duclohier, H. (2002). "How do channel- and pore-forming helical peptides interact with lipid membranes and how does this account for their antimicrobial activity?" Mini Rev Med Chem 2(4): 331-42.
Animals and plants defend themselves against pathogenic micro-organisms by the rapid mobilization of polycationic helical amphipathic peptides. Interactions with membranes induce optimal orientation and mutual structural changes, allowing for example to form transbilayer ion channels or pores whose properties are compared in this review. Physicochemical studies of peptide-lipid interactions provide attractive approaches for drug design.
Dirksen, R. T. and G. Avila (2002). "Altered ryanodine receptor function in central core disease: leaky or uncoupled Ca(2+) release channels?" Trends Cardiovasc Med 12(5): 189-97.
Central core disease (CCD) is an autosomal-dominant human congenital myopathy that is associated with at least 22 different mutations in the skeletal muscle isoform of ryanodine receptor (RyR1). CCD mutations in RyR1 have been proposed to lead to the formation of sarcoplasmic reticulum (SR) Ca(2+) release channels that are excessively leaky to Ca(2+). Although some of the CCD mutations in RyR1 may indeed result in leaky SR Ca(2+) release channels, the leaky-channel hypothesis may not represent the only mechanism for muscle weakness in this disorder. The presence of an alternate mechanism of muscle weakness in CCD is supported by the observation that muscle cells expressing a CCD mutation in the putative pore-forming segment of RyR1 (I4898T) exhibit a functional uncoupling of SR Ca(2+) release from sarcolemmal depolarization. These observations cannot be explained by the leaky-channel hypothesis and indicate that muscle weakness in some forms of CCD arises from an alternate and completely unexpected mechanism, termed "excitation-contraction uncoupling."
Deutsch, C. (2002). "Potassium channel ontogeny." Annu Rev Physiol 64: 19-46.
Potassium channels are multi-subunit complexes, often composed of several polytopic membrane proteins and cytosolic proteins. The formation of these oligomeric structures, including both biogenesis and trafficking, is the subject of this review. The emphasis is on events in the endoplasmic reticulum (ER), particularly on how, where, and when K(+) channel polypeptides translocate and integrate into the bilayer, oligomerize and fold to form pore-forming units, and associate with auxiliary subunits to create the mature channel complex. Questions are raised with respect to the sequence of these events, when biogenic decisions are made, models for integration of K(+) channel transmembrane segments, crosstalk between the cell surface and ER, and recognition of compatible partner subunits. Also considered are determinants of subunit composition and stoichiometry, their consequence for trafficking, mechanisms for ER retention and export, and sequence motifs that direct channels to the cell surface. It is these mechanistic issues that govern the differential distributions of K(+) conductances at the cell surface, and hence the electrical activity of cells and tissues underlying both the physiology and pathophysiology of an organism.
Delcour, A. H. (2002). "Structure and function of pore-forming beta-barrels from bacteria." J Mol Microbiol Biotechnol 4(1): 1-10.
Crystallographic studies of the past ten years have revealed that many outer membrane proteins and bacterial toxins are constructed on the beta-barrel motif. Two structural classes can be identified. The first class, represented by the porins, includes monomeric or multimeric proteins where each beta-barrel is formed from a single polypeptide. The second class features proteins where the beta-barrel is itself a multimeric assembly, to which each subunit contributes a few beta-strands. In addition to structural investigations, much work has also been devoted to the functional aspects of these proteins, and to the relationships between structure and function. Here we present a review of the structural and the functional properties of some of the best-studied examples of these various classes of proteins, namely the general-diffusion, specific and ligand-gated porins, multidrug efflux proteins and the staphylococcal toxin alpha-hemolysin.
Cossart, P. (2002). "Molecular and cellular basis of the infection by Listeria monocytogenes: an overview." Int J Med Microbiol 291(6-7): 401-9.
This review rather than covering the whole field, intends to highlight the particularly interesting properties of some proteins involved in the infection by Listeria monocytogenes and the general interest in some of the approaches used to analyze the molecular and cellular basis of the infection. After an introduction to the bacterium and to the disease, a description of the infection at the cellular level will be given. The specific features of the pore-forming toxin listeriolysin O, as a protein particularly well adapted to the intracellular lifestyle of L. monocytogenes will be discussed. By describing in detail how the bacterium moves inside cells, particular attention will be given to show how addressing this issue has provided key answers and instrumental tools to cell biologists studying actin-based motility. The analysis of the entry process and in particular the studies derived from the specificity of internalin for its receptor will demonstrate how an apparently reductionist approach can help generating relevant animal models, identification of virulence factors and demonstration of their role in vivo. The virulence gene cluster and its regulation by PrfA will be presented and discussed in the framework of the recently determined genome sequence.
Almeida-Campos, F. R., F. S. Noronha, et al. (2002). "The multitalented pore-forming proteins of intracellular pathogens." Microbes Infect 4(7): 741-50.
Being an intracellular pathogen demands being able to invade a host cell, to circumvent the host immune response and to survive in the intracellular environment. Pore-forming proteins are among the innumerable tools used by intracellular microorganisms to achieve these goals. Remarkably, this seems to be a multipurpose group of proteins that can act in several ways. Making channels may signify entering into host cells, inhibiting phagocytosis, escaping phagosomes or promoting pathogen dissemination. In certain cases, pore-forming proteins are double-edged tools and may benefit the host by eliminating infected cells and/or inducing inflammation.
Vandenberg, J. I., B. D. Walker, et al. (2001). "HERG K+ channels: friend and foe." Trends Pharmacol Sci 22(5): 240-6.
The K+ channel encoded by the human ether-a-go-go related gene (HERG) is one of many ion channels that are crucial for normal action potential repolarization in cardiac myocytes. HERG encodes the pore-forming subunit of the rapid component of the delayed rectifier K+ channel, I(K(Vr)). HERG K+ channels are of considerable pharmaceutical interest as possible therapeutic targets for anti-arrhythmic agents and as the molecular target responsible for the cardiac toxicity of a wide range of pharmaceutical agents. Recent studies of the molecular basis of the promiscuity of HERG K+ channel drug binding has not only started to shed light on this tricky pharmaceutical problem but has also provided further insights into the structure and function of HERG K+ channels.
Tseng, G. N. (2001). "I(Kr): the hERG channel." J Mol Cell Cardiol 33(5): 835-49.
G.-N. Tseng. I(Kr): The hERG Channel. Journal of Molecular and Cellular Cardiology (2001) 33, 835-849. The rapid delayed rectifier (I(Kr)) channel is important for cardiac action potential repolarization. Suppressing I(Kr)function, due to either genetic defects in its pore-forming subunit (hERG) or adverse drug effects, can lead to long-QT (LQT) syndrome that carries increased risk of life-threatening arrhythmias. The implication of I(Kr)in cardiac arrhythmias and in anti-arrhythmic/pro-arrhythmic actions of drugs has driven intensive research interests in its structure-function relationship, the linkage between LQT-associated mutations and changes in channel function, and the mechanism of drug actions. This review will cover the following topics: (1) heterogeneous contribution of I(Kr)to action potential repolarization in the heart, (2) structure-function relationship of I(Kr)/hERG channels, (3) role of regulatory & bgr; subunits in I(Kr)/hERG channel function, (4) structural basis for the unique pharmacological properties of I(Kr)/hERG channels, and (5) I(Kr)/hERG channel modulation by changes in cellular milieu under physiological and pathological conditions of the heart. It is anticipated that further advances in our understanding of I(Kr)/hERG, particularly in the areas of roles of different (& agr; and & bgr;) subunits in native I(Kr)function, alterations in I(Kr)function in diseased hearts, and the 3-dimensional structure of the I(Kr)/hERG pore based on homology modeling using the KcsA model, will help us better define the role of I(Kr)in arrhythmias and design therapeutic agents that can increase I(Kr)and are useful for LQT syndrome.
Smyth, M. J., J. M. Kelly, et al. (2001). "Unlocking the secrets of cytotoxic granule proteins." J Leukoc Biol 70(1): 18-29.
Cytotoxic lymphocytes largely comprise CD8(+) cytotoxic T cells and natural killer cells and form the major defense of higher organisms against virus-infected and transformed cells. A key function of cytotoxic lymphocytes is to detect and eliminate potentially harmful cells by inducing them to undergo apoptosis. This is achieved through two principal pathways, both of which require direct but transient contact between the killer cell and its target. The first, involving ligation of TNF receptor-like molecules such as Fas/CD95 by their cognate ligands, results in mobilization of conventional, programmed cell-death pathways centered on activation of pro-apoptotic caspases. This review concentrates on the second pathway, in which the toxic contents of secretory vesicles of the cytotoxic lymphocyte are secreted toward the target cell, and some toxins penetrate into the target cell cytoplasm and nucleus. In addition to invoking a powerful stimulus to caspase activation, this "granule-exocytosis mechanism" provides a variety of additional strategies for overcoming inhibitors of the caspase cascade that may be elaborated by viruses. The key molecular players in this process are the pore-forming protein perforin and a family of granule-bound serine proteases or granzymes. The molecular functions of perforin and granzymes are under intense investigation in many laboratories including our own, and recent advances will be discussed. In addition, this review discusses the evidence pointing to the importance of perforin and granzyme function in pathophysiological situations as diverse as infection with intracellular pathogens, graft versus host disease, susceptibility to transplantable and spontaneous malignancies, lymphoid homeostasis, and the tendency to auto-immune diseases.
Sela, M. N. (2001). "Role of Treponema denticola in periodontal diseases." Crit Rev Oral Biol Med 12(5): 399-413.
Among periodontal anaerobic pathogens, the oral spirochetes, and especially Treponema denticola, have been associated with periodontal diseases such as early-onset periodontitis, necrotizing ulcerative gingivitis, and acute pericoronitis. Basic research as well as clinical evidence suggest that the prevalence of T denticola, together with other proteolytic gram-negative bacteria in high numbers in periodontal pockets, may play an important role in the progression of periodontal disease. The accumulation of these bacteria and their products in the pocket may render the surface lining periodontal cells highly susceptible to lysis and damage. T. denticola has been shown to adhere to fibroblasts and epithelial cells, as well as to extracellular matrix components present in periodontal tissues, and to produce several deleterious factors that may contribute to the virulence of the bacteria. These bacterial components include outer-sheath-associated peptidases, chymotrypsin-like and trypsin-like proteinases, hemolytic and hemagglutinating activities, adhesins that bind to matrix proteins and cells, and an outer-sheath protein with pore-forming properties. The effects of T. denticola whole cells and their products on a variety of host mucosal and immunological cells has been studied extensively (Fig. 1). The clinical data regarding the presence of T. denticola in periodontal health and disease, together with the basic research results involving the role of T. denticola factors and products in relation to periodontal diseases, are reviewed and discussed in this article.
Prevost, G., L. Mourey, et al. (2001). "Staphylococcal pore-forming toxins." Curr Top Microbiol Immunol 257: 53-83.
Pond, A. L. and J. M. Nerbonne (2001). "ERG proteins and functional cardiac I(Kr) channels in rat, mouse, and human heart." Trends Cardiovasc Med 11(7): 286-94.
The voltage-gated K(+) channel (Kv) pore forming alpha subunit, ERG1 (KCNH2), has been identified as the locus of mutations in one type of inherited long QT syndrome, LQT2. Heterologous expression of ERG1 reveals rapidly activating and inactivating K(+) currents, characterized by marked inward rectification at potentials positive to 0 mV, which are similar to the rapid component of cardiac delayed rectification I(Kr). There are, however, marked differences in the properties of expressed ERG1 and endogenous cardiac I(Kr), suggesting that functional I(Kr) channels reflect the coassembly of full-length ERG1 with splice variants and /or accessory subunits. Consistent with these hypotheses, N- and C-terminal variants of ERG1 have been identified, and it has been demonstrated that heterologously expressed ERG1 and minK (or MiRP1) coimmunoprecipitate. Recent biochemical studies, however, suggest that only full-length ERG1 is expressed in adult mouse, rat, or human heart. Clearly, further studies, focused on identifying the subunits that coassemble with ERG1 in vivo, as well as on post-translational processing of the full-length ERG1 protein will be necessary to define the molecular composition of functional cardiac I(Kr) channels.
Oudit, G. Y., Z. Kassiri, et al. (2001). "The molecular physiology of the cardiac transient outward potassium current (I(to)) in normal and diseased myocardium." J Mol Cell Cardiol 33(5): 851-72.
G. Y. Oudit, Z. Kassiri, R. Sah, R. J. Ramirez, C. Zobel and P. H. Backx. The Molecular Physiology of the Cardiac Transient Outward Potassium Current (I(to)) in Normal and Diseased Myocardium. Journal of Molecular and Cellular Cardiology (2001) 33, 851-872. The Ca(2+)-independent transient outward potassium current (I(to)) plays an important role in early repolarization of the cardiac action potential. I(to)has been clearly demonstrated in myocytes from different cardiac regions and species. Two kinetic variants of cardiac I(to)have been identified: fast I(to), called I(to,f), and slow I(to), called I(to,s). Recent findings suggest that I(to,f)is formed by assembly of K(v4.2)and/or K(v4.3)alpha pore-forming voltage-gated subunits while I(to,s)is comprised of K(v1.4)and possibly K(v1.7)subunits. In addition, several regulatory subunits and pathways modulating the level and biophysical properties of cardiac I(to)have been identified. Experimental findings and data from computer modeling of cardiac action potentials have conclusively established an important physiological role of I(to)in rodents, with its role in large mammals being less well defined due to complex interplay between a multitude of cardiac ionic currents. A central and consistent electrophysiological change in cardiac disease is the reduction in I(to)density with a loss of heterogeneity of I(to)expression and associated action potential prolongation. Alterations of I(to)in rodent cardiac disease have been linked to repolarization abnormalities and alterations in intracellular Ca(2+)homeostasis, while in larger mammals the link with functional changes is far less certain. We review the current literature on the molecular basis for cardiac I(to)and the functional consequences of changes in I(to)that occur in cardiovascular disease.
Nerbonne, J. M., C. G. Nichols, et al. (2001). "Genetic manipulation of cardiac K(+) channel function in mice: what have we learned, and where do we go from here?" Circ Res 89(11): 944-56.
In the mammalian myocardium, potassium (K(+)) channels control resting potentials, action potential waveforms, automaticity, and refractory periods and, in most cardiac cells, multiple types of K(+) channels that subserve these functions are expressed. Molecular cloning has revealed the presence of a large number of K(+) channel pore forming (alpha) and accessory (beta) subunits in the heart, and considerable progress has been made recently in defining the relationships between expressed K(+) channel subunits and functional cardiac K(+) channels. To date, more than 20 mouse models with altered K(+) channel expression/functioning have been generated using dominant-negative transgenic and targeted gene deletion approaches. In several instances, the genetic manipulation of K(+) channel subunit expression has revealed the role of specific K(+) channel subunit subfamilies or individual K(+) channel subunit genes in the generation of myocardial K(+) channels. In other cases, however, the phenotypic consequences have been unexpected. This review summarizes what has been learned from the in situ genetic manipulation of cardiac K(+) channel functioning in the mouse, discusses the limitations of the models developed to date, and explores the likely directions of future research.
Menestrina, G., M. D. Serra, et al. (2001). "Mode of action of beta-barrel pore-forming toxins of the staphylococcal alpha-hemolysin family." Toxicon 39(11): 1661-72.
Staphylococcal alpha-hemolysin is the prototype of a family of bacterial exotoxins with membrane-damaging function, which share sequence and structure homology. These toxins are secreted in a soluble form which finally converts into a transmembrane pore by assembling an oligomeric beta-barrel, with hydrophobic residues facing the lipids and hydrophilic residues facing the lumen of the channel. Besides alpha-hemolysin the family includes other single chain toxins forming homo-oligomers, e.g. beta-toxin of Clostridium perfringens, hemolysin II and cytotoxin K of Bacillus cereus, but also the staphylococcal bi-component toxins, like gamma-hemolysins and leucocidins, which are only active as the combination of two similar proteins which form hetero-oligomers. The molecular basis of membrane insertion has become clearer after the determination of the crystal structure of both the oligomeric pore and the soluble monomer. Studies on this family of beta-barrel pore-forming toxins are important for many aspects: (i) they are involved in serious pathologies of humans and farmed animals, (ii) they are a good model system to investigate protein-membrane interaction and (iii) they are the basic elements for the construction of nanopores with biotechnological applications in various fields.
Liss, B. and J. Roeper (2001). "Molecular physiology of neuronal K-ATP channels (review)." Mol Membr Biol 18(2): 117-27.
ATP sensitive potassium (K-ATP) channels are widely expressed in many cell types including neurons. K-ATP channels are heteromeric membrane proteins that consist of two very different subunits: the pore-forming, two-transmembrane spanning potassium channel subunit (Kir6) and the regulatory, 17 transmembrane spanning sulphonylurea receptor (SUR). This ensemble--joined together in a 4:4 stoichiometry--endows this channel with a unique combination of functional properties. The open probability of K-ATP channels directly depends on the intracellular ATP/ADP levels allowing the channels to directly couple the metabolic state of a cell to its electrical activity. Here, recent progress on the molecular composition and functional diversity of neuronal K-ATP channels is reviewed. One is particular concerned with single-cell mRNA expression studies that give insight to the coexpression patterns of Kir6 and SUR isoforms in identified neurons. In addition, the physiological roles of neuronal K-ATP channels in glucose sensing and adapting neuronal activity to metabolic demands are discussed, as well as their emerging pathophysiological functions in acute brain ischemia and chronic neurodegenerative diseases.
Levin, B. E. (2001). "Glucosensing neurons do more than just sense glucose." Int J Obes Relat Metab Disord 25 Suppl 5: S68-72.
The brain regulates energy homeostasis by balancing energy intake, expenditure and storage. To accomplish this, it has evolved specialized neurons that receive and integrate afferent neural and metabolic signals conveying information about the energy status of the body. These sensor-integrator-effector neurons are located in brain areas involved in homeostatic functions such as the hypothalamus, locus coeruleus, basal ganglia, limbic system and nucleus tractus solitarius. The ability to sense and regulate glucose metabolism is critical because of glucose's primacy as a metabolic substrate for neural function. Most neurons use glucose as an energy substrate, but glucosensing neurons also use glucose as a signaling molecule to regulate neuronal firing and transmitter release. There are two types of glucosensing neurons that either increase (glucose responsive, GR) or decrease (glucose sensitive, GS) their firing rate as brain glucose levels rise. Little is known about the mechanism by which GS neurons sense glucose. However, GR neurons appear to function much like the pancreatic beta-cell where glycolysis regulates the activity of an ATP-sensitive K(+) (K(ATP)) channel. The K(ATP) channel is composed of four pore-forming units (Kir6.2) and four sulfonylurea binding sites (SUR). Glucokinase (GK) appears to modulate K(ATP) channel activity via its gatekeeper role in the glycolytic production of ATP. Thus, GK may serve as a marker for GR neurons. Neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) neurons in the hypothalamic arcuate nucleus are critical components of the energy homeostasis pathways in the brain. Both express Kir6.2 and GK, as well as leptin receptors. They also receive visceral neural and intrinsic neuropeptide and transmitter inputs. Such metabolism-related signals can summate upon K(ATP) channel activity which then alters membrane potential, neuronal firing rate and peptide/transmitter release. The outputs of these neurons are integral components of effector systems which regulate energy homeostasis. Thus, arcuate NPY and POMC neurons are probably prototypes of this important class of sensor-integrator-effector neurons.
Lakey, J. H. and S. L. Slatin (2001). "Pore-forming colicins and their relatives." Curr Top Microbiol Immunol 257: 131-61.
The pore-forming colicins, the first proteins that were capable of forming voltage-dependent ion channels to be sequenced, have turned out to be both less tractable and more mysterious than imagined; yet they have proved interesting at every step of their short journey from producing cell to vanquished target cell. Starting out as a remarkably extended water-soluble protein, the colicin molecule is designed to interact simultaneously with several components of the complex membrane of the target cell, transform itself into a membrane protein, and become an ion channel with inscrutable properties. Unraveling how it does all this appears to be leading us into the dark recesses of protein/protein and protein/membrane interaction, where lurk fundamental processes reluctantly waiting to be revealed.
Isom, L. L. (2001). "Sodium channel beta subunits: anything but auxiliary." Neuroscientist 7(1): 42-54.
Voltage-gated sodium channels are glycoprotein complexes responsible for initiation and propagation of action potentials in excitable cells such as central and peripheral neurons, cardiac and skeletal muscle myocytes, and neuroendocrine cells. Mammalian sodium channels are heterotrimers, composed of a central, pore-forming alpha subunit and two auxiliary beta subunits. The alpha subunits form a gene family with at least 10 members. Mutations in alpha subunit genes have been linked to paroxysmal disorders such as epilepsy, long QT syndrome, and hyperkalemic periodic paralysis in humans, and motor endplate disease and cerebellar ataxia in mice. Three genes encode sodium channel beta subunits with at least one alternative splice product. A mutation in the beta 1 subunit gene has been linked to generalized epilepsy with febrile seizures plus type 1 (GEFS + 1) in a human family with this disease. Sodium channel beta subunits are multifunctional. They modulate channel gating and regulate the level of channel expression at the plasma membrane. More recently, they have been shown to function as cell adhesion molecules in terms of interaction with extracellular matrix, regulation of cell migration, cellular aggregation, and interaction with the cytoskeleton. Structure-function studies have resulted in the preliminary assignment of functional domains in the beta 1 subunit. A sodium channel signaling complex is proposed that involves beta subunits as channel modulators as well as cell adhesion molecules, other cell adhesion molecules such as neurofascin and contactin, RPTP beta, and extracellular matrix molecules such as tenascin.
Heuck, A. P., R. K. Tweten, et al. (2001). "Beta-barrel pore-forming toxins: intriguing dimorphic proteins." Biochemistry 40(31): 9065-73.
Dietrich, P., D. Sanders, et al. (2001). "The role of ion channels in light-dependent stomatal opening." J Exp Bot 52(363): 1959-67.
Stomatal opening represents a major determinant of plant productivity and stress management. Because plants lose water essentially through open stomata, volume control of the pore-forming guard cells represents a key step in the regulation of plant water status. These sensory cells are able to integrate various signals such as light, auxin, abscisic acid, and CO(2). Following signal perception, changes in membrane potential and activity of ion transporters finally lead to the accumulation of potassium salts and turgor pressure formation. This review analyses recent progress in molecular aspects of ion channel regulation and suggests how these developments impact on our understanding of light- and auxin-dependent stomatal action.
Blachly-Dyson, E. and M. Forte (2001). "VDAC channels." IUBMB Life 52(3-5): 113-8.
Trafficking of metabolites across the outer mitochondrial membrane is believed to be mediated primarily by the pore-forming voltage-dependent anion channel, VDAC (also known as mitochondrial porin). An expanding body of in vitro studies have strongly suggest that the pore formed by VDAC can be regulated in a number of ways that implicate it as a site for the regulation of mitochondrial function, yet technical limitations have prevented the extension these studies to a relevant cellular context. The goal of this brief review is to summarize recent data that examine the role of VDAC and its regulation in the context not of the isolated protein or organelles but in cells, focusing on the application of genetic strategies in a number of experimental systems.
Aguilar-Bryan, L., J. Bryan, et al. (2001). "Of mice and men: K(ATP) channels and insulin secretion." Recent Prog Horm Res 56: 47-68.
K(ATP) channels are a unique, small family of potassium (K+)-selective ion channels assembled from four inward rectifier pore-forming subunits, K(IR)6.x, paired with four sulfonylurea receptors (SURs), members of the adenosine triphosphate (ATP)-binding cassette superfamily. The activity of these channels can be regulated by metabolically driven changes in the ratio of adenosine diphosphate (ADP) to ATP, providing a means to couple membrane electrical activity with metabolism. In pancreatic beta cells in the islets of Langerhans, K(ATP) channels are part of an ionic mechanism that couples glucose metabolism to insulin secretion. This chapter 1) briefly describes the properties of K(ATP) channels; 2) discusses data on a genetically recessive form of persistent hyperinsulinemic hypoglycemia of infancy (PHHI), caused by loss of beta-cell K(ATP) channel activity; and 3) compares the severe impairment of glucose homeostasis that characterizes the human phenotype with the near-normal phenotype observed in K(ATP) channel null mice.
van Os, C. H., E. J. Kamsteeg, et al. (2000). "Phsyiological relevance of aquaporins: luxury or necessity?" Pflugers Arch 440(4): 513-20.
Aquaporins are members of a large family of pore-forming intrinsic membrane proteins, the MIP family. Based on their permeability properties they are now further subdivided into aquaporins, with real water-selective pores, and aquaglyceroporins with slightly less selective pores. Aquaporins are expressed in a large variety of tissues throughout the body but in most situations it is not clear whether their presence is necessary for the proper physiological function of these tissues. This review focuses on recent insight into the physiological relevance of aquaporins gained from studying aquaporin knockout mouse models and from diseases, on new surprising findings related to gating and selectivity, and on the consequences of tetramerization for routing and the genetics of nephrogenic diabetes insipidus. The active fluid transport in proximal tubules and in salivary glands is seriously compromised by aquaporin deletion. This is in contrast to lung, airways and stomach, where active fluid transport proceeds unhindered in the face of greatly reduced water permeabilities due to aquaporin deletion. Therefore, aquaporins seem to be a necessity at extreme high rates of active fluid transport but appear to be more of a luxury at medium or low fluid transport rates.
Toth, V. and L. Emody (2000). "Proteus virulence: involvement of the pore forming alpha-hemolysin (a short review)." Acta Microbiol Immunol Hung 47(4): 457-70.
The genus Proteus belongs to the tribe of Proteae in the family of Enterobacteriaceae, and consists of five species: P. mirabilis, P. vulgaris, P. morganii, P. penneri and P. myxofaciens. They are distinguished from the rest of Enterobacteriaceae by their ability to deaminate phenylalanine and tryptophane. They hydrolyze urea and gelatin and fail to ferment lactose, mannose, dulcitol and malonate; and do not form lysine and arginine decarboxylase or beta-galactosidase [1]. Colonies produce distinct "burned chocolate" odor and frequently show the characteristics of swarming motility on solid media. P. mirabilis, P. vulgaris and P. morganii are widely recognized human pathogens. They have been isolated from urinary tract infections, wounds, ear, and nosocomial bacteremic infections, often in immuncompromised patients [2-6]. P. myxofaciens has no clinical interest to this time. P. penneri as species nova was nominated by the recommendation of Hickman and co-workers [7]. Formerly it was recognized as P. vulgaris biogroup 1 or indole negative P. vulgaris [8, 9]. Although it has been less commonly isolated from clinical samples than the other three human pathogenic Proteus species, it has nevertheless been connected with infections of the urinary tract, wounds and has been isolated from the feces of both healthy and diarrheic individuals [10-12]. Potential virulence factors responsible for virulence of Proteae are: IgA protease, urease, type3 fimbriae associated with MR/K haemagglutinins of at least two antigenic types, endotoxin, swarming motility and HlyA and/or HpmA type hemolysins [for review see ref. 13]. In the followings we give a survey of accumulated concepts about the position and characteristics of HlyA type alpha-hemolysins both in general and with emphasis on virulence functions in the tribe of Proteae.
Reusch, R. N. (2000). "Transmembrane ion transport by polyphosphate/poly-(R)-3-hydroxybutyrate complexes." Biochemistry (Mosc) 65(3): 280-95.
Transmembrane ion transport, a critical process in providing energy for cell functions, is carried out by pore-forming macromolecules capable of discriminating among very similar ions and responding to changes in membrane potential. It is widely regarded that ion channels are exclusively proteins, relatively late arrivals in cell evolution. Here we discuss the formation of ion-selective, voltage-activated channels by complexes of two simple homopolymers, namely, inorganic polyphosphates (polyPs) and poly-(R)-3-hydroxybutyrates (PHBs), derived from phosphate and acetate, respectively. Each has unique molecular characteristics that facilitate ion selection, solvation, and transport. Complexes of the two polymers, isolated from bacterial plasma membranes or prepared from the synthetic polymers, form voltage-dependent, Ca2+-selective channels in planar lipid bilayers that are selective for divalent over monovalent cations, permeant to Ca2+, Sr2+, and Ba2+, and blocked by transition metal cations in a concentration-dependent manner. Recently, both polyP and PHB have been found to be components of ion-conducting proteins: namely, the human erythrocyte Ca2+-ATPase pump and the Streptomyces lividans potassium channel. The contribution of polyP and PHB to ion selection and/or transport in these proteins is yet unknown, but their presence gives rise to the hypothesis that these and other ion transporters are supramolecular structures in which proteins, polyP, and PHB cooperate in forming well-regulated and specific cation transfer systems.
Provoda, C. J. and K. D. Lee (2000). "Bacterial pore-forming hemolysins and their use in the cytosolic delivery of macromolecules." Adv Drug Deliv Rev 41(2): 209-21.
Advances in our understanding of fundamental cell biological processes have facilitated an expansion of therapeutic approaches to altering cellular physiology and phenotype. As many of these methods involve macromolecular agents that act on targets within the nucleus or cytoplasm, achieving their full potential ultimately requires the efficient delivery of these agents across the cell membrane barrier into the cytosol. Various strategies have been employed to enhance cytosolic delivery. These include either directly penetrating the plasma membrane, or avoiding degradation within the hydrolytic environment of the endosomal/lysosomal pathway after endocytic uptake. Some of the more promising methods in this regard have exploited the mechanisms utilized by certain viruses and bacteria for escaping into their host cell's cytosol. In this review, we will discuss some of these methods with an emphasis on the use of pore-forming proteins from bacteria. Particular attention will be drawn to the pH-sensitive endosomolytic bacterial hemolysins, such as listeriolysin O, and the potentiol for their use in cytosolic drug delivery systems.
Nerbonne, J. M. (2000). "Molecular basis of functional voltage-gated K+ channel diversity in the mammalian myocardium." J Physiol 525 Pt 2: 285-98.
In the mammalian heart, Ca2+-independent, depolarization-activated potassium (K+) currents contribute importantly to shaping the waveforms of action potentials, and several distinct types of voltage-gated K+ currents that subserve this role have been characterized. In most cardiac cells, transient outward currents, Ito,f and/or Ito,s, and several components of delayed reactivation, including IKr, IKs, IKur and IK,slow, are expressed. Nevertheless, there are species, as well as cell-type and regional, differences in the expression patterns of these currents, and these differences are manifested as variations in action potential waveforms. A large number of voltage-gated K+ channel pore-forming (alpha) and accessory (beta, minK, MiRP) subunits have been cloned from or shown to be expressed in heart, and a variety of experimental approaches are being exploited in vitro and in vivo to define the relationship(s) between these subunits and functional voltage-gated cardiac K+ channels. Considerable progress has been made in defining these relationships recently, and it is now clear that distinct molecular entities underlie the various electrophysiologically distinct repolarizing K+ currents (i.e. Ito,f, Ito,s, IKr, IKs, IKur, IK,slow, etc.) in myocyardial cells.
Lory, P., A. Monteil, et al. (2000). "[Molecular diversity of calcium channel activities by depolarization]." Therapie 55(2): 249-54.
Voltage-gated calcium channels are involved in a large variety of cellular functions such as excitation-contraction coupling, hormone secretion, firing and pacemaker activity, gene activation and proliferation. Cloning of complementary DNAs encoding for calcium channel subunits has challenged the study of the functional properties of calcium channels and has allowed analysis of the molecular basis of calcium channel diversity. Recently, pore-forming subunits of T-type calcium channels have been cloned. Recent data describing the genes encoding calcium channels, their molecular and pharmacological studies, as well as their linkage to human genetic diseases are reviewed in this article.
Lopez, L. B., M. B. Braga, et al. (2000). "Strategies by which some pathogenic trichomonads integrate diverse signals in the decision-making process." An Acad Bras Cienc 72(2): 173-86.
The interaction between each one of Trichomonas vaginalis and Tritrichomonas foetus with their hosts is a complex process in which components associated to the cell surfaces of both parasites and host epithelial cells, and also to soluble components found in vaginal/urethral secretions, are involved. Either cytoadhesion or the cytotoxicity exerted by parasites to host cells can be dictated by virulence factors such as adhesins, cysteine proteinases, laminin-binding proteins, integrins, integrin-like molecules, a cell detachment factor, a pore-forming protein, and glycosidases among others. How trichomonads manipulate informations from the extracellular medium, transduce such informations, and respond to them by stimulating the activities of some surface molecules and/or releasing enzymes are the aspects concerning trichomonal virulence which are here briefly reviewed and discussed.
Korsmeyer, S. J., M. C. Wei, et al. (2000). "Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c." Cell Death Differ 7(12): 1166-73.
We review data supporting a model in which activated tBID results in an allosteric activation of BAK, inducing its intramembranous oligomerization into a proposed pore for cytochrome c efflux. The BH3 domain of tBID is not required for targeting but remains on the mitochondrial surface where it is required to trigger BAK to release cytochrome c. tBID functions not as a pore-forming protein but as a membrane targeted and concentrated death ligand. tBID induces oligomerization of BAK, and both Bid and Bak knockout mice indicate the importance of this event in the release of cytochrome c. In parallel, the full pro-apoptotic member BAX, which is highly homologous to BAK, rapidly forms pores in liposomes that release intravesicular FITC-cytochrome c approximately 20A. A definable pore progressed from approximately 11A consisting of two BAX molecules to a approximately 22A pore comprised of four BAX molecules, which transported cytochrome c. Thus, an activation cascade of pro-apoptotic proteins from BID to BAK or BAX integrates the pathway from surface death receptors to the irreversible efflux of cytochrome c. Cell Death and Differentiation (2000) 7, 1166 - 1173
Kam, C. M., D. Hudig, et al. (2000). "Granzymes (lymphocyte serine proteases): characterization with natural and synthetic substrates and inhibitors." Biochim Biophys Acta 1477(1-2): 307-23.
Natural killer (NK) and cytotoxic T-lymphocytes (CTLs) kill cells within an organism to defend it against viral infections and the growth of tumors. One mechanism of killing involves exocytosis of lymphocyte granules which causes pores to form in the membranes of the attacked cells, fragments nuclear DNA and leads to cell death. The cytotoxic granules contain perforin, a pore-forming protein, and a family of at least 11 serine proteases termed granzymes. Both perforin and granzymes are involved in the lytic activity. Although the biological functions of most granzymes remain to be resolved, granzyme B clearly promotes DNA fragmentation and is directly involved in cell death. Potential natural substrates for Gr B include procaspases and other proteins involved in cell death. Activated caspases are involved in apoptosis. The search continues for natural substrates for the other granzymes. The first granzyme crystal structure remains to be resolved, but in the interim, molecular models of granzymes have provided valuable structural information about their substrate binding sites. The information has been useful to predict the amino acid sequences that immediately flank each side of the scissile peptide bond of peptide and protein substrates. Synthetic substrates, such as peptide thioesters, nitroanilides and aminomethylcoumarins, have also been used to study the substrate specificity of granzymes. The different granzymes have one of four primary substrate specificities: tryptase (cleaving after Arg or Lys), Asp-ase (cleaving after Asp), Met-ase (cleaving after Met or Leu), and chymase (cleaving after Phe, Tyr, or Trp). Natural serpins and synthetic inhibitors (including isocoumarins, peptide chloromethyl ketones, and peptide phosphonates) inhibit granzymes. Studies of substrate and inhibitor kinetics are providing valuable information to identify the most likely natural granzyme substrates and provide tools for the study of key reactions in the cytolytic mechanism.
January, C. T., Q. Gong, et al. (2000). "Long QT syndrome: cellular basis and arrhythmia mechanism in LQT2." J Cardiovasc Electrophysiol 11(12): 1413-8.
LQT2 is one form of the congenital long QT syndrome. It results from mutations in the human ether-a-go-go-related gene (HERG), and more than 80 mutations, usually causing single amino acid substitutions in the HERG protein, are known. HERG encodes the ion channel pore-forming subunit protein for the rapidly activating delayed rectifier K+ channel (I(Kr)) in the heart. This review summarizes current findings about mutations causing LQT2, the mechanisms by which mutations may cause the clinical phenotype of a reduction in I(Kr) and a prolonged QT interval, and how this may be involved in the generation of ventricular arrhythmias.
Hertle, R. (2000). "Serratia type pore forming toxins." Curr Protein Pept Sci 1(1): 75-89.
The Serratia marcescens hemolysin represents a new type of hemolysin and has been studied in great molecular detail with regard to structure, activation and secretion. It has nothing in common with the pore forming toxins of E. coli type (RTX toxins), the Staphylococcus aureus alpha-toxin or the thiol activated toxin of group A beta-hemolytic streptococci (Streptolysin O). Studies on erythrocytes, eukaryotic cells and artificial black lipid membranes, have shown that the mechanism of pore formation of ShlA is different form other pore forming toxins. The S. marcescens hemolysin proteins ShlB and ShlA exhibit protein sequence homologues in Proteus mirabilis, Haemophilus ducreyi, Edwardsiella tarda and Erwinia chrysantemi. Furthermore, sequence motifs present in ShlA and Shlb have been shown to be important for activity and secretion of the S. marcescens hemolysin. Thus, the S. marcescens hemolysin forms the prototype of a new class of hemolysins and of a new secretory mechanism. The uniqueness of this new mechanism is underlined by the fact that activation of ShlA by ShlB strictly requires phosphatidylethanolamine as a cofactor. New data implicate a conformational change in ShlA during activation. In addition, ShlA not only forms pores in erythrocytes but also in fibroblasts and epithelial cells. The cytotoxic action of ShlA is mainly determined by lysis of infected cells in vitro. In sublytic doses, as will normally be the situation in vivo, ShlA exerts additionally effects which are currently under investigation. The knowledge of the structure, activation, secretion and mode of action of S. marcescens hemolysin has implications for proteins, related in sequence or in mode of secretion and activation.
Hering, S., S. Berjukow, et al. (2000). "Molecular determinants of inactivation in voltage-gated Ca2+ channels." J Physiol 528 Pt 2: 237-49.
Evolution has created a large family of different classes of voltage-gated Ca2+ channels and a variety of additional splice variants with different inactivation properties. Inactivation controls the amount of Ca2+ entry during an action potential and is, therefore, believed to play an important role in tissue-specific Ca2+ signalling. Furthermore, mutations in a neuronal Ca2+ channel (Ca(v)2.1) that are associated with the aetiology of neurological disorders such as familial hemiplegic migraine and ataxia cause significant changes in the process of channel inactivation. Ca2+ channels of a given subtype may inactivate by three different conformational changes: a fast and a slow voltage-dependent inactivation process and in some channel types by an additional Ca2+-dependent inactivation mechanism. Inactivation kinetics of Ca2+ channels are determined by the intrinsic properties of their pore-forming alpha1-subunits and by interactions with other channel subunits. This review focuses on structural determinants of Ca2+ channel inactivation in different parts of Ca2+ channel alpha1-subunits, including pore-forming transmembrane segments and loops, intracellular domain linkers and the carboxyl terminus. Inactivation is also affected by the interaction of the alpha1-subunits with auxiliary beta-subunits and intracellular regulator proteins. The evidence shows that pore-forming S6 segments and conformational changes in extra- (pore loop) and intracellular linkers connected to pore-forming segments may play a principal role in the modulation of Ca2+ channel inactivation. Structural concepts of Ca2+ channel inactivation are discussed.
Gulbins, E., A. Jekle, et al. (2000). "Physiology of apoptosis." Am J Physiol Renal Physiol 279(4): F605-15.
Ion fluxes and volume changes of the whole cell as well as of organelles belong to the hallmarks of apoptosis; however, the molecular mechanism regulating these changes is only poorly characterized. Several ion channels in the plasma membrane, in particular the N-type K(+) channel, the chloride channel cystic fibrosis conductance regulator, and an outward rectifying chloride channel, as well as the mitochondrial permeability transition pore, have been implicated to be involved in signal transduction cascades regulating apoptosis. Furthermore, Bcl-2-like proteins have been suggested to function, at least in part, as ion channels, because they display some homology to bacterial pore-forming toxins. In contrast to the demonstration of the involvement of these different ion channels in apoptosis, the molecular consequences regulated by these ion channels, and finally triggering apoptosis, are almost completely unknown.
Catterall, W. A. (2000). "Structure and regulation of voltage-gated Ca2+ channels." Annu Rev Cell Dev Biol 16: 521-55.
Voltage-gated Ca(2+) channels mediate Ca(2+) entry into cells in response to membrane depolarization. Electrophysiological studies reveal different Ca(2+) currents designated L-, N-, P-, Q-, R-, and T-type. The high-voltage-activated Ca(2+) channels that have been characterized biochemically are complexes of a pore-forming alpha1 subunit of approximately 190-250 kDa; a transmembrane, disulfide-linked complex of alpha2 and delta subunits; an intracellular beta subunit; and in some cases a transmembrane gamma subunit. Ten alpha1 subunits, four alpha2delta complexes, four beta subunits, and two gamma subunits are known. The Cav1 family of alpha1 subunits conduct L-type Ca(2+) currents, which initiate muscle contraction, endocrine secretion, and gene transcription, and are regulated primarily by second messenger-activated protein phosphorylation pathways. The Cav2 family of alpha1 subunits conduct N-type, P/Q-type, and R-type Ca(2+) currents, which initiate rapid synaptic transmission and are regulated primarily by direct interaction with G proteins and SNARE proteins and secondarily by protein phosphorylation. The Cav3 family of alpha1 subunits conduct T-type Ca(2+) currents, which are activated and inactivated more rapidly and at more negative membrane potentials than other Ca(2+) current types. The distinct structures and patterns of regulation of these three families of Ca(2+) channels provide a flexible array of Ca(2+) entry pathways in response to changes in membrane potential and a range of possibilities for regulation of Ca(2+) entry by second messenger pathways and interacting proteins.
Bathori, G., I. Parolini, et al. (2000). "Extramitochondrial porin: facts and hypotheses." J Bioenerg Biomembr 32(1): 79-89.
Mitochondrial porin, or VDAC, is a pore-forming protein abundant in the outer mitochondrial membrane. Several publications have reported extramitochondrial localizations as well, but the evidence was considered insufficient by many, and the presence of porin in nonmitochondrial cellular compartments has remained in doubt for a long time. We have now obtained new data indicating that the plasma membrane of hematopoietic cells contains porin, probably located mostly in caveolae or caveolae-like domains. Porin was purified from the plasma membrane of intact cells by a procedure utilizing the membrane-impermeable labeling reagent NH-SS-biotin and streptavidin affinity chromatography, and shown to have the same properties as mitochondrial porin. A channel with properties similar to that of isolated VDAC was observed by patch-clamping intact cells. This review discusses the evidence supporting extramitochondrial localization, the putative identification of the plasma membrane porin with the "maxi" chloride channel, the hypothetical mechanisms of sorting porin to various cellular membrane structures, and its possible functions.
Abrami, L., M. Fivaz, et al. (2000). "Adventures of a pore-forming toxin at the target cell surface." Trends Microbiol 8(4): 168-72.
The past three years have shed light on how the pore-forming toxin aerolysin binds to its target cell and then hijacks cellular devices to promote its own polymerization and pore formation. This selective permeabilization of the plasma membrane has unexpected intracellular consequences that might explain the importance of aerolysin in Aeromonas pathogenicity.
Sandford, R., S. Mulroy, et al. (1999). "The polycystins: a novel class of membrane-associated proteins involved in renal cystic disease." Cell Mol Life Sci 56(7-8): 567-79.
Polycystin-1, polycystin-2 and polycystin-L are the predicted protein products of the PKD1, PKD2 and PKDL genes, respectively. Mutations in PKD1 and PKD2 are responsible for almost all cases of autosomal dominant polycystic kidney disease (ADPKD). This condition is one of the commonest mendelian disorders of man with a prevalence of 1:800 and is responsible for nearly 10% of cases of end-stage renal failure in adults. The cloning of PKD1 and PKD2 in recent years has provided the initial steps in defining the mechanisms underlying renal cyst formation in this condition, with the aim of defining pharmacological and genetic interventions that may ameliorate the diverse and often serious clinical manifestations of this disease. The PKD genes share regions of sequence similarity, and all predictintegral membrane proteins. Whilst the predicted protein domain structure of polycystin-1 suggests it is involved in cell-cell or cell-matrix interactions, the similarity of polycystin-2 and polycystin-L to the pore-forming domains of some cation channels suggests that they all form subunits of a large plasma membrane ion channel. In the few years since the cloning of the PKD genes, a consensus that defines the range of mutations, expression pattern, interactions and functional domains of these genes and their protein products is emerging. This review will therefore attempt to summarise these data and provide an insight in to the key areas in which polycystin research is unravelling the mechanisms involved in renal cyst formation.
Meisinger, C., J. Brix, et al. (1999). "The preprotein translocase of the outer mitochondrial membrane: receptors and a general import pore." Cell Mol Life Sci 56(9-10): 817-24.
Cytosol-synthesized preproteins destined for the mitochondria are transported across the outer membrane by the translocase of the mitochondrial outer membrane (TOM complex). This dynamic transport machinery can be divided into receptors that recognize preprotein targeting signals and components of the general import pore complex that mediate preprotein transport across the outer membrane. This review focuses on recent studies dealing with the central questions regarding the pore-forming subunits, and architecture and gating of the translocation channel of the outer membrane.
Baumeister, S., A. Burgwedel, et al. (1999). "Reconstitution of protein transport across the vacuolar membrane in Plasmodium falciparum-infected permeabilized erythrocytes." Novartis Found Symp 226: 145-54; discussion 154-6.
The parasite Plasmodium falciparum induces morphological and biochemical alterations of its host erythrocyte. Some of these changes are mediated by parasite proteins that are transported to specific destinations within the erythrocyte or to the erythrocyte plasma membrane. The pathways underlying this transport are still unknown. We anticipate that at least some aspects of these pathways may be biologically unique and therefore potential targets for chemotherapeutic intervention. We have utilized bacterial pore-forming proteins to establish an experimental system that allows selective permeabilization of the erythrocyte plasma membrane, without affecting the integrity of the vacuolar membrane and the parasite plasma membrane, in order to study protein transport from the parasite into the host erythrocyte. Physiological properties of the parasite within permeabilized erythrocytes, such as the ability to synthesize proteins, will be described. The permeabilization of infected erythrocytes has allowed the dissection of individual steps in protein transport from the parasite surface across the vacuolar membrane. Possible pathways involved in the trafficking of parasite proteins within the erythrocyte cytosol, i.e. in a cell that normally has no need to transport proteins, will be discussed.