Viral ion channels: structure and function

Viral ion channels are short auxiliary membrane proteins with a length of ca. 100 amino acids. They are found in enveloped viruses from influenza A, influenza B and influenza C (Orthomyxoviridae), and the human immunodeficiency virus type 1 (HIV-1, Retroviridae). The channels are called M2 (influenza A), NB (influenza B), CM2 (influenza C) and Vpu (HIV-1). Recently, in Paramecium bursaria chlorella virus (PBCV-1, Phycodnaviridae), a K+ selective ion channel has been discovered. The viral channels form homo oligomers to allow an ion flux and represent miniaturised systems. Proton conductivity of M2 is established; NB, Vpu and the potassium channel from PBC-1 conduct ions; for CM2 ion conductivity is still under proof. This review summarises the current knowledge of these short viral membrane proteins. Their discovery is outlined and experimental evidence for their structure and function is discussed. Studies using computational methods are presented as well as investigations of drug-protein interactions.     

The structure and function of glutamate receptor ion channels

As in the case of many ligand-gated ion channels, the biochemical and electrophysiological properties of the ionotropic glutamate receptors have been studied extensively. Nevertheless, we still do not understand the molecular mechanisms that harness the free energy of agonist binding, first to drive channel opening, and then to allow the channel to close (desensitize) even though agonist remains bound. Recent crystallographic analyses of the ligand-binding domains of these receptors have identified conformational changes associated with agonist binding, yielding a working hypothesis of channel function. This opens the way to determining how the domains and subunits are assembled into an oligomeric channel, how the domains are connected, how the channel is formed, and where it is located relative to the ligand-binding domains, all of which govern the processes of channel activation and desensitization.     

L-type voltage-gated calcium channels: understanding function through structure

L-type voltage-gated calcium channels (VGCCs) are multisubunit membrane proteins that regulate calcium influx into excitable cells. Within the last two years there have been four separate reports describing the structure of the skeletal muscle VGCC determined by electron microscopy and single particle analysis methods. There are some discrepancies between the structures, as well as reports for both monomeric and dimeric forms of the channel. This article considers each of the VGCC structures in terms of similarities and differences with an emphasis upon translation of data into a biological context.     

Phylogeny of ion channels: clues to structure and function

Voltage-gated ion channels are responsible for the electrical activity in a variety of cell types in modern-day animals. However, they represent the result of many millions of years of evolution of a family of ion channel proteins that are also found in prokaryotes and diverse eukaryotes, and probably exist in all life forms. This review traces the evolution of ion channels, with particular emphasis on the factors and evolutionary pathways that may have given rise to voltage-gated potassium (K+), calcium (Ca2+), and sodium (Na+) channels. The review also highlights the utility of comparing phylogenetically distinct versions of the same protein as a means to better understand the structure and function of proteins.     

Toward an understanding of the physiological function of Mammalian stem cells

Stem cell biology has the potential to yield new therapies, new insights into disease, and a clearer understanding of tissue formation and maintenance. However, much of what we know about many stem cells is based upon experiments performed in culture. Stem cells sometimes exhibit critical differences in their properties or regulation between the culture and in vivo environments. Though cell lines with stem cell properties can be derived from the long-term culture of diverse tissues, it is not clear whether cells with similar properties exist in vivo. If the goal is to use differentiated cells for therapy or drug screening, it may not matter whether these stem cells exist in vivo. However, to understand tissue development/maintenance or the role of stem cells in disease, it is important to characterize progenitor function in vivo to evaluate physiological significance.     

Toward understanding the structure and interactions of microtubules and motor proteins

To obtain an overall three-dimensional picture of the interaction between microtubules and the motor proteins of the kinesin family it will be necessary to take account of both atomic resolution structures obtained by X-ray crystallography and medium resolution reconstructions obtained by electron cryomicroscopy. We examine the problems associated with obtaining the required structural information from electron micrographs of vitreous ice-embedded microtubules decorated with motor domains. We find that the minus-end directed motor, ncd, decorates microtubules with an 80 Å periodicity as for kinesin. Our theoretical analysis and experiments with ncd illustrate the difficulty in determining unambiguously the surface lattice organization by diffraction analysis of micrographs. 3D reconstructions of decorated microtubules are required to accurately locate the motor domains. Helical diffraction theory is not usually applicable because microtubules are cylindrical structures that rarely have complete helical symmetry. We propose using a back-projection method based on the long pitch helices formed by individual protofilaments. Model reconstructions show that this approach is feasible. © 1995 Wiley-Liss, Inc.     

The structure and regulation of magnesium selective ion channels

The magnesium ion (Mg^2 +) is the most abundant divalent cation within cells. In man, Mg^2 +-deficiency is associated with diseases affecting the heart, muscle, bone, immune, and nervous systems. Despite its impact on human health, little is known about the molecular mechanisms that regulate magnesium transport and storage. Complete structural information on eukaryotic Mg^2 +-transport proteins is currently lacking due to associated technical challenges. The prokaryotic MgtE and CorA magnesium transport systems have recently succumbed to structure determination by X-ray crystallography, providing first views of these ubiquitous and essential Mg^2 +-channels. MgtE and CorA are unique among known membrane protein structures, each revealing a novel protein fold containing distinct arrangements of ten transmembrane-spanning α-helices. Structural and functional analyses have established that Mg^2 +-selectivity in MgtE and CorA occurs through distinct mechanisms. Conserved acidic side-chains appear to form the selectivity filter in MgtE, whereas conserved asparagines coordinate hydrated Mg^2 +-ions within the selectivity filter of CorA. Common structural themes have also emerged whereby MgtE and CorA sense and respond to physiologically relevant, intracellular Mg^2 +-levels through dedicated regulatory domains. Within these domains, multiple primary and secondary Mg^2 +-binding sites serve to staple these ion channels into their respective closed conformations, implying that Mg^2 +-transport is well guarded and very tightly regulated. The MgtE and CorA proteins represent valuable structural templates to better understand the related eukaryotic SLC41 and Mrs2–Alr1 magnesium channels. Herein, we review the structure, function and regulation of MgtE and CorA and consider these unique proteins within the expanding universe of ion channel and transporter structural biology.     

The construction of an amino acid network for understanding protein structure and function

Amino acid networks (AANs) are undirected networks consisting of amino acid residues and their interactions in three-dimensional protein structures. The analysis of AANs provides novel insight into protein science, and several common amino acid network properties have revealed diverse classes of proteins. In this review, we first summarize methods for the construction and characterization of AANs. We then compare software tools for the construction and analysis of AANs. Finally, we review the application of AANs for understanding protein structure and function, including the identification of functional residues, the prediction of protein folding, analyzing protein stability and protein–protein interactions, and for understanding communication within and between proteins.     

Differential regulation of ion channels function by proteolysis

Ion channels are pore-forming protein complexes in membranes that play essential roles in a diverse array of biological activities. Ion channel activity is strictly regulated at multiple levels and by numerous cellular events to selectively activate downstream effectors involved in specific biological activities. For example, ions, binding proteins, nucleotides, phosphorylation, the redox state, channel subunit composition have all been shown to regulate channel activity and subsequently allow channels to participate in distinct cellular events. While these forms of modulation are well documented and have been extensively reviewed, in this article, we will first review and summarize channel proteolysis as a novel and quite widespread mechanism for altering channel activity. We will then highlight the recent findings demonstrating that proteolysis profoundly alters Inositol 1,4,5 trisphosphate receptor activity, and then discuss its potential functional ramifications in various developmental and pathological conditions.     

Toward an understanding of the structural basis of translation

The recently solved X-ray crystal structures of the ribosome have provided opportunities for studying the molecular basis of translation with a variety of methods including cryo-electron microscopy - where maps give the first glimpses of ribosomal evolution - and fluorescence spectroscopy techniques.     

Toward an understanding of biochemical equilibria within living cells

Four types of environmental effects that can affect macromolecular reactions in a living cell are defined: nonspecific intermolecular interactions, side reactions, partitioning between microenvironments, and surface interactions. Methods for investigating these interactions and their influence on target reactions in vitro are reviewed. Methods employed to characterize conformational and association equilibria in vivo are reviewed and difficulties in their interpretation cataloged. It is concluded that, in order to be amenable to unambiguous interpretation, in vivo studies must be complemented by in vitro studies carried out in well-characterized and controllable media designed to contain key elements of selected intracellular microenvironments.     

Toward better understanding of protein secondary structure: extracting prediction rules

Although numerous computational techniques have been applied to predict protein secondary structure (PSS), only limited studies have dealt with discovery of logic rules underlying the prediction itself. Such rules offer interesting links between the prediction model and the underlying biology. In addition, they enhance interpretability of PSS prediction by providing a degree of transparency to the predicting model usually regarded as a black box. In this paper, we explore the generation and use of C4.5 decision trees to extract relevant rules from PSS predictions modeled with two-stage support vector machines (TS-SVM). The proposed rules were derived on the RS126 data set of 126 nonhomologous globular proteins and on the PSIPRED data set of 1,923 protein sequences. Our approach has produced sets of comprehensible, and often interpretable, rules underlying the PSS predictions. Moreover, many of the rules seem to be strongly supported by biological evidence. Further, our approach resulted in good prediction accuracy, few and usually compact rules, and rules that are generally of higher confidence levels than those generated by other rule extraction techniques.     

Predicting the transmembrane secondary structure of ligand-gated ion channels

Recent mutational analyses of ligand-gated ion channels (LGICs) have demonstrated a plausible site of anesthetic action within their transmembrane domains. Although there is a consensus that the transmembrane domain is formed from four membrane-spanning segments, the secondary structure of these segments is not known. We utilized 10 state-of-the-art bioinformatics techniques to predict the transmembrane topology of the tetrameric regions within six members of the LGIC family that are relevant to anesthetic action. They are the human forms of the GABA alpha 1 receptor, the glycine alpha 1 receptor, the 5HT3 serotonin receptor, the nicotinic AChR alpha 4 and alpha 7 receptors and the Torpedo nAChR alpha 1 receptor. The algorithms utilized were HMMTOP, TMHMM, TMPred, PHDhtm, DAS, TMFinder, SOSUI, TMAP, MEMSAT and TOPPred2. The resulting predictions were superimposed on to a multiple sequence alignment of the six amino acid sequences created using the CLUSTAL W algorithm. There was a clear statistical consensus for the presence of four alpha helices in those regions experimentally thought to span the membrane. The consensus of 10 topology prediction techniques supports the hypothesis that the transmembrane subunits of the LGICs are tetrameric bundles of alpha helices.     

Toward a molecular understanding of the structure-function of ryanodine receptor Ca2+ release channels

Identification of the genetic basis of human diseases linked to dysfunctional free calcium (Ca2+) signaling has triggered an explosion of interest in the functional characterization of the molecular components regulating intracellular Ca2+ homeostasis. There is a growing appreciation of the central role of intracellular ryanodine-sensitive Ca2+ release channel (RyR) regulation in skeletal and cardiac muscle pathologies, including malignant hyperthermia, heart failure, and sudden cardiac death. The use of cloned RyR isoforms and recombinant expression techniques has greatly facilitated the elucidation of the molecular basis of RyR Ca2+ release functionality. This review will focus on the recombinant techniques used in the functional characterization of recombinant RyR isoforms and the insights that these approaches have yielded in unraveling the mechanistic basis of RyR channel functionality.     

The structure and function of Na+ channels.

The past year has seen major advances in our understanding of voltage-gated ion channels through a powerful combination of patch-clamp and molecular biological techniques. These approaches have identified regions (in some cases single amino acid residues) that are essential for voltage-dependent activation and inactivation, lining of the pore, and regulation of channel function.