Functional engineered channels and pores (Review)

Significant progress has been made in membrane protein engineering over the last 5 years, based largely on the re-design of existing scaffolds. Engineering techniques that have been employed include direct genetic engineering, both covalent and non-covalent modification, unnatural amino acid mutagenesis and total synthesis aided by chemical ligation of unprotected fragments. Combinatorial mutagenesis and directed evolution remain, by contrast, underemployed. Techniques for assembling and purifying heteromeric multisubunit pores have been improved. Progress in the de novo design of channels and pores has been slower. But, we are at the beginning of a new era in membrane protein engineering based on the accelerating acquisition of structural information, a better understanding of molecular motion in membrane proteins, technical improvements in membrane protein refolding and the application of computational approaches developed for soluble proteins. In addition, the next 5 years should see further advances in the applications of engineered channels and pores, notably in therapeutics and sensor technology.     

Mechanosensitive channels in bacteria: signs of closure?

Bacterial mechanosensitive channels are activated by increases in tension in the lipid bilayer of the cytoplasmic membrane, where they transiently create large pores in a controlled manner. Mechanosensitive channel research has benefited from advances in electrophysiology, genomics and molecular genetics as well as from the application of biophysical techniques. Most recently, new analytical methods have been used to complement existing knowledge and generate insights into the molecular interactions that take place between mechanosensitive channel proteins and the surrounding membrane lipids. This article reviews the latest developments.     

Classification of projection images of crystalline arrays of the mitochondrial, voltage-dependent anion-selective channel embedded in aurothioglucose.

Low-dose electron microscopic images have been recorded from membrane crystals of the mitochondrial, voltage-dependent anion-selective channel, embedded in aurothioglucose. There is considerable variation in the high-resolution detail present in correlation averages computed from these images. Correspondence analysis reveals three classes of "control" averages, with main components of variation involving projected size of the pores and density modulations around the pores and in the corners of the unit cells away from the pores. Pretreatments that affect the functional state of the channel also affect the array averages. In particular, there appears to be a general correlation between the expected effector-induced state (i.e., open and closed) and the projected diameter of the channel lumens in the crystalline arrays.     

Simplified bacterial "pore" channel provides insight into the assembly, stability, and structure of sodium channels

Eukaryotic sodium channels are important membrane proteins involved in ion permeation, homeostasis, and electrical signaling. They are long, multidomain proteins that do not express well in heterologous systems, and hence, structure/function and biochemical studies on purified sodium channel proteins have been limited. Bacteria produce smaller, homologous tetrameric single domain channels specific for the conductance of sodium ions. They consist of N-terminal voltage sensor and C-terminal pore subdomains. We designed a functional pore-only channel consisting of the final two transmembrane helices, the intervening P-region, and the C-terminal extramembranous region of the sodium channel from the marine bacterium Silicibacter pomeroyi. This sodium "pore" channel forms a tetrameric, folded structure that is capable of supporting sodium flux in phospholipid vesicles. The pore-only channel is more thermally stable than its full-length counterpart, suggesting that the voltage sensor subdomain may destabilize the full-length channel. The pore subdomains can assemble, fold, and function independently from the voltage sensor and exhibit similar ligand-blocking characteristics as the intact channel. The availability of this simple pore-only construct should enable high-level expression for the testing of potential new ligands and enhance our understanding of the structural features that govern sodium selectivity and permeability.     

Some assembly required: constructing the elementary units of store-operated Ca2+ entry

The means by which Ca(2+) store depletion evokes the opening of store-operated Ca(2+) channels (SOCs) in the plasma membrane of excitable and non-excitable cells has been a longstanding mystery. Indirect evidence has supported local interactions between the ER and SOCs as well as long-range interactions mediated through a diffusible activator. The recent molecular identification of the ER Ca(2+) sensor (STIM1) and a subunit of the CRAC channel (Orai1), a prototypic SOC, has now made it possible to visualize directly the sequence of events that links store depletion to CRAC channel opening. Following store depletion, STIM1 moves from locations throughout the ER to accumulate in ER subregions positioned within 10-25nm of the plasma membrane. Simultaneously, Orai1 gathers at discrete sites in the plasma membrane directly opposite STIM1, resulting in local CRAC channel activation. These new studies define the elementary units of store-operated Ca(2+) entry, and reveal an unprecedented mechanism for channel activation in which the stimulus brings a channel and its activator/sensor together for interaction across apposed membrane compartments. We discuss the implications of this choreographic mechanism with regard to Ca(2+) dynamics, specificity of Ca(2+) signaling, and the existence of a specialized ER subset dedicated to the control of the CRAC channel.     

Effect of sensor domain mutations on the properties of voltage-gated ion channels: molecular dynamics studies of the potassium channel Kv1.2

The effects on the structural and functional properties of the Kv1.2 voltage-gated ion channel, caused by selective mutation of voltage sensor domain residues, have been investigated using classical molecular dynamics simulations. Following experiments that have identified mutations of voltage-gated ion channels involved in state-dependent omega currents, we observe for both the open and closed conformations of the Kv1.2 that specific mutations of S4 gating-charge residues destabilize the electrostatic network between helices of the voltage sensor domain, resulting in the formation of hydrophilic pathways linking the intra- and extracellular media. When such mutant channels are subject to transmembrane potentials, they conduct cations via these so-called “omega pores.” This study provides therefore further insight into the molecular mechanisms that lead to omega currents, which have been linked to certain channelopathies.     

A tetrameric porin limits the cell wall permeability of Mycobacterium smegmatis

Mycobacteria protect themselves with an outer lipid bilayer, which is the thickest biological membrane hitherto known and has an exceptionally low permeability rendering mycobacteria intrinsically resistant against many antibiotics. Pore proteins mediate the diffusion of hydrophilic nutrients across this membrane. Electron microscopy revealed that the outer membrane of Mycobacterium smegmatis contained about 1000 protein pores per microm(2), which are about 50-fold fewer pores per microm(2) than in Gram-negative bacteria. The projection structure of the major porin MspA of M. smegmatis was determined at 17 A resolution. MspA forms a cone-like tetrameric complex of 10 nm in length with a single central pore. Thus, MspA is drastically different from the trimeric porins of Gram-negative bacteria and represents a new class of channel proteins. The formation of MspA micelles indicated that the ends of MspA have different hydrophobicities. Oriented insertion of MspA into membranes was demonstrated in lipid bilayer experiments, which revealed a strongly asymmetrical voltage gating of MspA channels at -30 mV. The length of MspA is sufficient to span the outer membrane and contributes in combination with the tapering end of the pore and the low number of pores to the low permeability of the cell wall of M. smegmatis for hydrophilic compounds.     

How far will you go to sense voltage?

Despite tremendous progress in the study of voltage-gated channels, the molecular mechanism underlying voltage sensing has remained a matter of debate. We review five new studies that make major progress in the field. The studies employ a battery of distinct approaches that have the common aim of measuring the motion of the voltage sensor. We interpret the results in light of the recent crystal structure of the mammalian potassium channel Kv1.2. We focus on the transmembrane movement of the voltage sensor as a key element to the detection of membrane potential and to the control of channel gating.     

瞬时受体电位M8离子通道功能及其翻译后修饰调节机制

瞬时受体电位M8(transient receptor potential melastatin 8, TRPM8)又称冷及薄荷醇感受器,位于细胞膜或细胞器膜上,是瞬时受体电位(transient receptor potential, TRP)通道超家族中的一员。TRPM8通道分布广泛,是一个非选择性阳离子通道,可作为冷热传感器和冷痛传感器进行信号传导,参与众多生物过程的调节,在维持细胞内外稳态、控制离子进出细胞方面具有重要作用。研究发现,蛋白质翻译后修饰(post-translational modification, PTM)通过调控TRPM8通道的功能,进而影响多种疾病的发生和发展。因此,探究TRPM8的翻译后修饰的过程,对深入了解TRPM8的功能及调控机制是十分必要的。目前,已报道的TRPM8翻译后修饰包括磷酸化、泛素化和糖基化等,它们能够调控蛋白质的相互作用和改变TRPM8离子通道的活性,从而调控细胞增殖、迁移和凋亡。值得注意的是,TRPM8的表达与前列腺癌、膀胱癌和乳腺癌等多种癌症密切相关。本文将从TRPM8离子通道的结构出发,系统地阐述TRPM8蛋白翻译后修饰和激动剂、...     

Ion transport across transmembrane pores

To study the pore-mediated transport of ionic species across a lipid membrane, a series of molecular dynamics simulations have been performed of a dipalmitoyl-phosphatidyl-choline bilayer containing a preformed water pore in the presence of sodium and chloride ions. It is found that the stability of the transient water pores is greatly reduced in the presence of the ions. Specifically, the binding of sodium cations at the lipid/water interface increases the pore line tension, resulting in a destabilization of the pore. However, the application of mechanical stress opposes this effect. The flux of ions through these mechanically stabilized pores has been analyzed. Simulations indicate that the transport of the ions through the pores depends strongly on the size of the water channel. In the presence of small pores (radius <1.5 nm) permeation is slow, with both sodium and chloride permeating at similar rates. In the case in which the pores are larger (radius >1.5 nm), a crossover is observed to a regime where the anion flux is greatly enhanced. Based on these observations, a mechanism for the basal membrane permeability of ions is discussed.     

Permeability Increase Induced by Escherichia coli Hemolysin A in Human Macrophages is Due to the Formation of Ionic Pores: A Patch Clamp Characterization

Escherichia coli hemolysin is known to cause hemolysis of red blood cells by forming hydrophilic pores in their cell membrane. Hemolysin-induced pores have been directly visualized in model systems such as planar lipid membranes and unilamellar vesicles. However this hemolysin, like all the members of a related family of toxins called Repeat Toxins, is a potent leukotoxin. To investigate whether the formation of channels is involved also in its leukotoxic activity, we used patch-clamped human macrophages as targets. Indeed, when exposed to the hemolysin, these cells developed additional pores into their membrane. Such exogenous pores had properties very different from the endogenous channels already present in the cell membrane (primarily K⁺ channels), but very similar to the pores formed by the toxin in purely lipidic model membranes. Observed properties were: large single channel conductance, cation over anion selectivity but weak discrimination among different cations, quasilinear current-voltage characteristic and the existence of a flickering pre-open state of small conductance. The selectivity properties of the toxin channels appearing in phospholipid vesicles were also investigated, using a specially adapted polarization/depolarization assay, and were found to be completely consistent with that of the current fluctuations observed in excised macrophage patches. Received: 14 August 1995/Revised: 2 October 1995     

Structural insights into eukaryotic aquaporin regulation

Aquaporin-mediated water transport across cellular membranes is an ancient, ubiquitous mechanism within cell biology. This family of integral membrane proteins includes both water selective pores (aquaporins) and transport facilitators of other small molecules such as glycerol and urea (aquaglyceroporins). Eukaryotic aquaporins are frequently regulated post-translationally by gating, whereby the rate of flux through the channel is controlled, or by trafficking, whereby aquaporins are shuttled from intracellular storage sites to the plasma membrane. A number of high-resolution X-ray structures of eukaryotic aquaporins have recently been reported and the new structural insights into gating and trafficking that emerged from these studies are described. Basic structural themes reoccur, illustrating how the problem of regulation in diverse biological contexts builds upon a limited set of possible solutions.     

Porin is present in the plasma membrane where it is concentrated in caveolae and caveolae-related domains.

Mitochondrial porin, or voltage-dependent anion channel, is a pore-forming protein first discovered in the outer mitochondrial membrane. Later investigations have provided indications for its presence also in other cellular membranes, including the plasma membrane, and in caveolae. This extra-mitochondrial localization is debated and no clear-cut conclusion has been reached up to now. In this work, we used biochemical and electrophysiological techniques to detect and characterize porin within isolated caveolae and caveolae-like domains (low density Triton-insoluble fractions). A new procedure was used to isolate porin from plasma membrane. The outer surface of cultured CEM cells was biotinylated by an impermeable reagent. Low density Triton-insoluble fractions were prepared from the labeled cells and used as starting material to purify a biotinylated protein with the same electrophoretic mobility and immunoreactivity of mitochondrial porin. In planar bilayers, the porin from these sources formed slightly anion-selective pores with properties indistinguishable from those of mitochondrial porin. This work thus provides a strong indication of the presence of porin in the plasma membrane, and specifically in caveolae and caveolae-like domains.     

Probing amphotericin B single channel activity by membrane dipole modifiers

The effects of dipole modifiers and their structural analogs on the single channel activity of amphotericin B in sterol-containing planar phosphocholine membranes are studied. It is shown that the addition of phloretin in solutions bathing membranes containing cholesterol or ergosterol decreases the conductance of single amphotericin B channels. Quercetin decreases the channel conductance in cholesterol-containing bilayers while it does not affect the channel conductance in ergosterol-containing membranes. It is demonstrated that the insertion of styryl dyes, such as RH 421, RH 237 or RH 160, in bilayers with either cholesterol or ergosterol leads to the increase of the current amplitude of amphotericin B pores. Introduction of 5α-androstan-3β-ol into a membrane-forming solution increases the amphotericin B channel conductance in a concentration-dependent manner. All the effects are likely to be attributed to the influence of the membrane dipole potential on the conductance of single amphotericin B channels. However, specific interactions of some dipole modifiers with polyene-sterol complexes might also contribute to the activity of single amphotericin B pores. It has been shown that the channel dwell time increases with increasing sterol concentration, and it is higher for cholesterol-containing membranes than for bilayers including ergosterol, 6-ketocholestanol, 7-ketocholestanol or 5α-androstan-3β-ol. These findings suggest that the processes of association/dissociation of channel forming molecules depend on the membrane fluidity.     

Inward and outward potassium currents through the same chimeric human Kv channel

Voltage-gated ion channels are among the most intensely studied membrane proteins today and a variety of techniques has led to a basic mapping of functional roles onto specific regions of their structure. The architecture of the proteins appears to be modular and segments associated with voltage sensing and the pore lining have been identified. However, the means by which movement of the sensor is transduced into channel opening is still unclear. In this communication, we report on a chimeric potassium channel construct which can function in two distinct operating voltage ranges, spanning both inward and outward currents with a non-conducting intervening regime. The observed changes in operating range could be brought about by perturbing either the direction of sensor movement or the process of transducing movements of the sensor into channel opening and closing. The construct could thus provide a means to identify the machinery underlying these processes.     

CHAP: A Versatile Tool for the Structural and Functional Annotation of Ion Channel Pores

The control of ion channel permeation requires the modulation of energetic barriers or “gates” within their pores. However, such barriers are often simply identified from the physical dimensions of the pore. Such approaches have worked well in the past, but there is now evidence that the unusual behavior of water within narrow hydrophobic pores can produce an energetic barrier to permeation without requiring steric occlusion of the pathway. Many different ion channels have now been shown to exploit “hydrophobic gating” to regulate ion flow, and it is clear that new tools are required for more accurate functional annotation of the increasing number of ion channel structures becoming available. We have previously shown how molecular dynamics simulations of water can be used as a proxy to predict hydrophobic gates, and we now present a new and highly versatile computational tool, the Channel Annotation Package (CHAP) that implements this methodology.     

Oriented channel insertion reveals the motion of a transmembrane beta strand during voltage gating of VDAC

Yeast VDAC channels (isolated from the mitochondrial outer membrane) form large aqueous pores whose walls are believed to consist of 1 a helix and 12 strands. Each channel has two voltage-gating processes: one closes the channels at positive potentials, the other at negative. When VDAC is reconstituted into phospholipid (soybean) membranes, the two gating processes have virtually the same steepness of voltage dependence and the same midpoint voltage. Substituting lysine for glutamate at either end of one putative strand (E145K or E152K) made the channels behave asymmetrically, increasing the voltage dependence of one gating process but not the other. The asymmetry was the same whether 1 or 100 channels were in the membrane, indicating oriented channel insertion. However, the direction of insertion varied from membrane to membrane, indicating that the insertion of the first channel was random and subsequent insertions were directed by the previously inserted channel (s). This raises the prospect of an auto-directed insertion with possible implications to protein targeting in cells. Each of the mutations affected a different gating process because the double mutant increased voltage dependence of both processes. Thus this strand may slide through the membrane in one direction or the other depending on the gating process. We propose that the model of folding for VDAC be altered to move this strand into the sensor region of the protein where it may act as a tether and guide/restrict the motion of the sensor.This work was supported by grants from the Office of Naval Research (N00014-90-J-1024) and the National Institutes of Health (GM 35759). Present address: Department of Physiology, 6811 Med. Sciences Bldg 2, University of Michigan, Ann Arbor, MI 48109 Present address: Department of Physiology, K.U. Leuven Medical School, Gasthuijsberg, 3000 Leuven, Belgium     

Functional properties and toxin pharmacology of a dorsal root ganglion sodium channel viewed through its voltage sensors

The voltage-activated sodium (Nav) channel Nav1.9 is expressed in dorsal root ganglion (DRG) neurons where it is believed to play an important role in nociception. Progress in revealing the functional properties and pharmacological sensitivities of this non-canonical Nav channel has been slow because attempts to express this channel in a heterologous expression system have been unsuccessful. Here, we use a protein engineering approach to dissect the contributions of the four Nav1.9 voltage sensors to channel function and pharmacology. We define individual S3b-S4 paddle motifs within each voltage sensor, and show that they can sense changes in membrane voltage and drive voltage sensor activation when transplanted into voltage-activated potassium channels. We also find that the paddle motifs in Nav1.9 are targeted by animal toxins, and that these toxins alter Nav1.9-mediated currents in DRG neurons. Our results demonstrate that slowly activating and inactivating Nav1.9 channels have functional and pharmacological properties in common with canonical Nav channels, but also show distinctive pharmacological sensitivities that can potentially be exploited for developing novel treatments for pain.     

Studies on the structural properties of porous membranes: measurement of linear dimensions of solutes

This work is concerned with a novel way of studying the basic structural and transport properties of porous membranes. Formulae have been derived for: the total number (N) of pores in the membrane; and mean square radius (r(k)) of pores and for selective membranes: the mean square radius (r(ka)) of pores impermeable to solutes and mean square radius (r(kb)) of permeable pores. The result of the investigation is the proposal of a new research procedure (osmotic) for the determination of the linear dimensions of solutes.