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Significance The transmembrane electrical potential difference (Δψ), which is defined as membrane voltage, is used as the energy for many biological activities. For construction of the bacterial flagella on the cell surface, a specialized protein transporter utilizes Δψ to drive proton-coupled protein export, but it remains unknown how. Here, we report that an inactive flagellar protein transporter can be activated by an increase in Δψ above a threshold value through an interaction between FliJ and the transmembrane proton channel protein FlhA. Following activation, the protein transporter conducts protons through the FlhA channel to drive flagellar protein export. This report describes a Δψ-dependent activation mechanism used for a biological function other than voltage-gated ion channels., The proton motive force (PMF) consists of the electric potential difference (Δψ), which is measured as membrane voltage, and the proton concentration difference (ΔpH) across the cytoplasmic membrane. The flagellar protein export machinery is composed of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase ring complex consisting of FliH, FliI, and FliJ. ATP hydrolysis by the FliI ATPase activates the export gate complex to become an active protein transporter utilizing Δψ to drive proton-coupled protein export. An interaction between FliJ and a transmembrane ion channel protein, FlhA, is a critical step for Δψ-driven protein export. To clarify how Δψ is utilized for flagellar protein export, we analyzed the export properties of the export gate complex in the absence of FliH and FliI. The protein transport activity of the export gate complex was very low at external pH 7.0 but increased significantly with an increase in Δψ by an upward shift of external pH from 7.0 to 8.5. This observation suggests that the export gate complex is equipped with a voltage-gated mechanism. An increase in the cytoplasmic level of FliJ and a gain-of-function mutation in FlhA significantly reduced the Δψ dependency of flagellar protein export by the export gate complex. However, deletion of FliJ decreased Δψ-dependent protein export significantly. We propose that Δψ is required for efficient interaction between FliJ and FlhA to open the FlhA ion channel to conduct protons to drive flagellar protein export in a Δψ-dependent manner.

The chloride intracellular ion channel protein (CLIC) family are a unique set of ion channels that can exist as soluble and integral membrane proteins. New evidence has emerged that demonstrates CLICs' possess oxidoreductase enzymatic activity and may function as either membrane-spanning ion channels or as globular enzymes. To further characterize the enzymatic profile of members of the CLIC family and to expand our understanding of their functions, we expressed and purified recombinant CLIC1, CLIC3, and a non-functional CLIC1-Cys24A mutant using a Histidine tag, bacterial protein expression system. We demonstrate that the presence of the six-polyhistidine tag at the amino terminus of the proteins led to a decrease in their oxidoreductase enzymatic activity compared to their non-His-tagged counterparts, when assessed using 2-hydroxyethyl disulfide as a substrate. These results strongly suggest the six-polyhistidine tag alters CLIC's structure at the N-terminus, which also contains the enzyme active site. It also raises the need for caution in use of His-tagged proteins when assessing oxidoreductase protein enzymatic function., Highlights • CLIC proteins demonstrate oxidoreductase activity in the HEDS enzyme assay. • Presence of six histidine tag on recombinant CLIC proteins alters their enzymatic activity. • Presence of imidazole can also interfere with these enzymatic assays. • Recommend removal of His-tags when assessing a protein's oxidoreductase activity.

Although there are many reports on the regulation of ion channel expression in transcription and translation, few drugs have been studied to influence post-translational modification of ion channel proteins. The Kv1.5 channel is a potassium ion channel expressed in atrial muscle, belongs to the voltage-gated K(+) channel superfamily, and forms an ultrarapid delayed rectifier potassium ion current. It is important to understand the fate of these channel proteins, as cardiac Kv1.5 mutations can cause arrhythmias. Disruption of quantitative and qualitative control mechanisms of channels leads to stagnation and degradation of intracellular channel proteins. As a result, ion channel proteins are not transported to the cell membrane and are involved in the development of atrial fibrillation. This review takes the Kv1.5 channel as an example and focuses on the degradation mechanism of ion channel proteins, and discusses its application to the treatment of arrhythmia by drugs that control the mechanism of ion channel protein degradation.

A bioelectronic skin device based on nociceptive ion channels in nanovesicles is developed for the detection of chemical cold-pain stimuli and cold environments just like human somesthetic sensory systems. The human transient receptor potential ankyrin 1 (hTRPA1) is involved in transmission and modulation of cold-pain sensations. In the bioelectronic skin, the nanovesicles containing the hTRPA1 nociceptive ion channel protein reacts to cold-pain stimuli, and it is electrically monitored through carbon nanotube transistor devices based on floating electrodes. The bioelectronic skin devices sensitively detect chemical cold-pain stimuli like cinnamaldehyde at 10 fm, and selectively discriminate cinnamaldehyde among other chemical stimuli. Further, the bioelectronic skin is used to evaluate the effect of cold environments on the response of the hTRPA1, finding that the nociceptive ion channel responds more sensitively to cinnamaldehyde at lower temperatures than at higher temperatures. The bioelectronic skin device could be useful for a basic study on somesthetic systems such as cold-pain sensation, and should be used for versatile applications such as screening of foods and drugs.

The roles of surrounding membrane lipids in the functions of transmembrane and peripheral membrane proteins are largely unknown. Herein, we utilize the recently reported structures of the TRPV1 ion channel protein bound to its potent protein agonist, the double-knot toxin (DkTx), as a model system to investigate the roles of toxin-lipid interfaces in TRPV1 activation by characterizing a series of DkTx variants electrophysiologically. Together with membrane partitioning experiments, these studies reveal that toxin-lipid interfaces play an overwhelmingly dominant role in channel activation as compared to lipid-devoid toxin-channel interfaces. Additionally, we find that whereas the membrane interfaces formed by one of the knots of the toxin endow it with its low channel-dissociation rate, those formed by other knot contribute primarily to its potency. These studies establish that protein-lipid interfaces play nuanced yet profound roles in the function of protein-protein complexes within membranes.

Channelopathies are disorders caused by reduced expression or impaired function of ion channels. Most current therapies rely on symptomatic treatment without addressing the underlying cause. We have recently established proof of principle for delivery of functional ion channel protein into the membrane of target cells using fusogenic liposomes incorporating glycine receptor (GlyR)-containing cell membrane fragments (CMF) that were formulated by thin film hydration. Here, the effect of liposome size and the formulation technique on the performance of the delivery vehicle was assessed. Three types of liposomes were prepared using lecithin and cholesterol, (i) small (SL), and (ii) large (LL) liposomes made by thin film hydration, and (iii) small liposomes prepared by vortex agitation (V-SL). All liposomes were evaluated for their ability to (i) incorporate GlyR-rich CMF, (ii) fuse with the cell membrane of target cells and (iii) deliver functional GlyR, as assessed by patch-clamp electrophysiology. SL prepared by thin film hydration offered the most effective delivery of glycine receptors that gave clear glycine-mediated currents in target cells. LL showed higher incorporation of CMF, but did not effectively fuse with the target cell membrane, while V-SL did not incorporate sufficient amounts of CMF. Additionally, SL showed minimalin vivotoxicity upon intrathecal injection in mice. Thus, liposome-mediated delivery of membrane proteins may be a promising therapeutic approach for the treatment of channelopathies.

Atomic-level studies of protein activity represent a significant challenge as a result of the complexity of conformational changes occurring on wide-ranging timescales, often greatly exceeding that of even the longest simulations. A prime example is the elucidation of protein allosteric mechanisms, where localized perturbations transmit throughout a large macromolecule to generate a response signal. For example, the conversion of chemical to electrical signals during synaptic neurotransmission in the brain is achieved by specialized membrane proteins called pentameric ligand-gated ion channels. Here, the binding of a neurotransmitter results in a global conformational change to open an ion-conducting pore across the nerve cell membrane. X-ray crystallography has produced static structures of the open and closed states of the proton-gated GLIC pentameric ligand-gated ion channel protein, allowing for atomistic simulations that can uncover changes related to activation. We discuss a range of enhanced sampling approaches that could be used to explore activation mechanisms. In particular, we describe recent application of an atomistic string method, based on Roux's "swarms of trajectories" approach, to elucidate the sequence and interdependence of conformational changes during activation. We illustrate how this can be combined with transition analysis and Brownian dynamics to extract thermodynamic and kinetic information, leading to understanding of what controls ion channel function. © 2019 Wiley Periodicals, Inc.

Viroporins like influenza A virus M2, hepatitis C virus p7, HIV-1 Vpu and picornavirus 2B associate with host membranes, and create hydrophilic corridors, which are critical for viral entry, replication and egress. The 6K proteins from alphaviruses are conjectured to be viroporins, essential during egress of progeny viruses from host membranes, although the analogue in Chikungunya Virus (CHIKV) remains relatively uncharacterized. Using a combination of electrophysiology, confocal and electron microscopy, and molecular dynamics simulations we show for the first time that CHIKV 6K is an ion channel forming protein that primarily associates with endoplasmic reticulum (ER) membranes. The ion channel activity of 6K can be inhibited by amantadine, an antiviral developed against the M2 protein of Influenza A virus; and CHIKV infection of cultured cells can be effectively inhibited in presence of this drug. Our study provides crucial mechanistic insights into the functionality of 6K during CHIKV-host interaction and suggests that 6K is a potential therapeutic drug target, with amantadine and its derivatives being strong candidates for further development., Author summary Chikungunya fever is a severe crippling illness caused by the arthropod-borne virus CHIKV. Originally from the African subcontinent, the virus has now spread worldwide and is responsible for substantial morbidity and economic loss. The existing treatment against CHIKV is primarily symptomatic, and it is imperative that specific therapeutics be devised. The present study provides detailed insight into the functionality of 6K, an ion channel forming protein of CHIKV. Amantadine, a known antiviral against influenza virus, also inhibits CHIKV replication in cell culture and drastically alters the morphology of virus particles. This work highlights striking parallels among functionalities of virus-encoded membrane-interacting proteins, which may be exploited for developing broad-spectrum antivirals.

Ion channels are naturally occurring proteins that form hole in membrane. They play multiple roles in many important biological processes. Deletion or alteration of these channels often leads to serious problems in the physiological processes as it controls the flow of ions through it. The proper maintenance of the flow of ions, in turn, is required for normal health. The role of ions channels are now a proved factor behind nerve impulse. Here we have analyzed the nerve ion channel protein, with PDB entry 1BL8, which is basically an ion channel protein in Streptomyces Lividans. The equilibrium energy as well as molecular dynamics simulation is performed first. The possibility of ligand binding is investigated. The change of channel structure is found to be dependent on ligand binding. Implicit water model of the protein is subjected to molecular dynamics simulation to find their energy minimized value. Simulation of the protein in the environment of water and ions has given us important results too.

The α7 subtype of neuronal nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel protein that is vital to various neurological functions, including modulation of neurotransmitter release. A relatively high concentration of extracellular Ca2+ in the neuronal environment is likely to exert substantial structural and functional influence on nAChRs, which may affect their interactions with agonists and antagonists. In this work, we employed atomistic molecular dynamics (MD) simulations to examine the effects of elevated Ca2+ on the structure and dynamics of α7 nAChR embedded in a model phospholipid bilayer. Our results suggest that the presence of Ca2+ in the α7 nAChR environment results in closure of loop C-in the extracellular ligand-binding domain, a motion normally associated with agonist binding and receptor activation. Elevated Ca2+ also alters the conformation of key regions of the receptor, including the inter-helical loops, pore-lining helices and the "gate" residues, and causes partial channel opening in the absence of an agonist, leading to an attendant reduction in the free energy of Ca2+ permeation through the pore as elucidated by umbrella sampling simulations. Overall, the structural and permeability changes in α7 nAChR suggest that elevated Ca2+ induces a partially activated receptor state that is distinct from both the resting and the agonist-activated states. These results are consistent with the notion that divalent ions can serve as a potentiator of nAChRs, resulting in a higher rate of receptor activation (and subsequent desensitization) in the presence of agonists, with possible implications for diseases involving calcium dysregulation.

Genetic networks operate in the presence of local heterogeneities in single-cell transcription and translation rates. Bioelectrical networks and spatio-temporal maps of cell electric potentials can influence multicellular ensembles. Could cell-cell bioelectrical interactions mediated by intercellular gap junctions contribute to the stabilization of multicellular states against local genetic heterogeneities? We theoretically analyze this question on the basis of two well-established experimental facts: (i) the membrane potential is a reliable read-out of the single-cell electrical state and (ii) when the cells are coupled together, their individual cell potentials can be influenced by ensemble-averaged electrical potentials. We propose a minimal biophysical model for the coupling between genetic and bioelectrical networks that associates the local changes occurring in the transcription and translation rates of an ion channel protein with abnormally low (depolarized) cell potentials. We then analyze the conditions under which the depolarization of a small region (patch) in a multicellular ensemble can be reverted by its bioelectrical coupling with the (normally polarized) neighboring cells. We show also that the coupling between genetic and bioelectric networks of non-excitable cells, modulated by average electric potentials at the multicellular ensemble level, can produce oscillatory phenomena. The simulations show the importance of single-cell potentials characteristic of polarized and depolarized states, the relative sizes of the abnormally polarized patch and the rest of the normally polarized ensemble, and intercellular coupling.

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe the application of this novel label-free technique to study the interaction of human EAG1 (hEAG1) channel proteins with the small molecule PIP(2). hEAG1 channel has been recognized as potential therapeutic target because of its aberrant overexpression in cancers and a few gain-of-function mutations involved in some types of neurological diseases. We purified hEAG1 channel proteins from a mammalian stable expression system and measured the interaction with PIP(2) by BLI. The successful measurement of the kinetics of binding between hEAG1 protein and PIP(2 )demonstrates that the BLI assay is a potential high-throughput approach used for novel small-molecule ligand screening in ion channel pharmacology.

Ion channels are proteins forming pore inside the body of all living organisms. This potassium ion channel known as KcsA channel and it is found in the each cell and nervous system. Flow of various ions is regulated by the function of the ion channels. The nerve ion channel protein with protein data bank entry 1BL8, which is basically an ion channel protein in Streptomyces Lividans and which is taken up to form micelle-protein system and the system is analyzed by using molecular dynamics simulation. Firstly, ion channel pore is engineered by CHARMM potential and then Micelle-protein system is subjected to molecular dynamics simulation. For some specific micelle concentration, the protein unfolding is observed.

Bioelectricity is emerging as a crucial mechanism for signal transmission and processing from the single-cell level to multicellular domains. We explore theoretically the oscillatory dynamics that result from the coupling between the genetic and bioelectric descriptions of nonexcitable cells in multicellular ensembles, connecting the genetic prepatterns defined over the ensemble with the resulting spatio-temporal map of cell potentials. These prepatterns assume the existence of a small patch in the ensemble with locally low values of the genetic rate constants that produce a specific ion channel protein whose conductance promotes the cell-polarized state (inward-rectifying channel). In this way, the short-range interactions of the cells within the patch favor the depolarized membrane potential state, whereas the long-range interaction of the patch with the rest of the ensemble promotes the polarized state. The coupling between the local and long-range bioelectric signals allows a binary control of the patch membrane potentials, and alternating cell polarization and depolarization states can be maintained for optimal windows of the number of cells and the intercellular connectivity in the patch. The oscillatory phenomena emerge when the feedback between the single-cell bioelectric and genetic dynamics is coupled at the multicellular level. In this way, the intercellular connectivity acts as a regulatory mechanism for the bioelectrical oscillations. The simulation results are qualitatively discussed in the context of recent experimental studies.

Public exposure to electromagnetic radiation (EMR) emitted by radiofrequency (RF) and microwave (MW) technology devices such as mobile phones, Wi-Fi, smart meters and different medical equipment has increased significantly in the last decade. With more than 200 times increase in mobile phone use in the last 10 years, health concerns regarding significant increase in exposure to low power RF/MW radiation have been raised. In response, the World Health Organisation (WHO) designed a research agenda that is based on the views of the leading international experts in RF bioeffect research. Research agenda items include animal and human studies, dosimetry and risk communication research, and in vitro mechanistic studies. A number of studies were undertaken to investigate biological and health effects of low-level exposures in the range of 900–2600 MHz (3G and 4G mobile networks). It was shown that RF-EMF can target cellular plasma membrane enzymes and receptors, and hence can control the cell function. RF radiation can also alter the intracellular calcium homoeostasis and consequently target the cell proliferation and differentiation as well as modify bioactivity of different enzymes. TRPV4 is a non-selective cation channel that is expressed in various tissues, including epithelial and endothelial cells, which can be activated by different stimuli such as heat, hypotonic stress, GSK1016790A, derivatives of arachidonic acids and shear stress. This study is aimed to evaluate the effects of MW radiation at 1800 MHz and power of 17dBm (47 V/m) on TRPP4 ion channel protein expressed in Hek-293 cells to better understanding of thermal/non-thermal nature of RF-EMF interaction with normal epithelial cells. Here, we used Ca+2 imaging and confocal microscopy to investigate the effects of RF/MW radiation at 1800 MHz and power of 17dBm on TRPV4 channel gating in response to its selective agonist GSK1016790A. We found that 4hrs exposure to MWs at 1800 MHz and 17dBm modulates the response of TRPV4 when compared to the control group. These findings provide evidence that these particular non-thermal exposures induce changes in the biological activity of TRPV4.

Molecular dynamics (MD) simulations and excitation energy calculations of C1C2 chimera channelrhodopsin, a light-gated ion channel protein utilized as a biotechnological tool for optogenetics, based on a protein structure determined recently by X-ray crystallography were performed to investigate its structural and spectral properties. The MD simulations showed stability of hydrogen-bonds responsible for the channel gating observed in the crystallographic structural model. Analysis of electrostatic contribution of the surrounding protein groups to the absorption energy proposes several site-specific mutations that shift absorption maxima significantly, and provides a clear and controlled guide for engineering design of color variant proteins utilized in optogenetics.

The chloride intracellular channel (CLIC) family of proteins has the remarkable property of maintaining both a soluble form and an integral membrane form acting as an ion channel. The soluble form is structurally related to the glutathione-S-transferase family, and CLIC can covalently bind glutathione via an active site cysteine. We report approximately 0.6 s of molecular dynamics simulations, encompassing the three possible ligand-bound states of CLIC1, using the structure of GSH-bound human CLIC1. Noncovalently bound GSH was rapidly released from the protein, whereas the covalently ligand-bound protein remained close to the starting structure over 0.25 s of simulation. In the unliganded state, conformational changes in the vicinity of the glutathione-binding site resulted in reduced reactivity of the active site thiol. Elastic network analysis indicated that the changes in the unliganded state are intrinsic to the protein architecture and likely represent functional transitions. Overall, our results are consistent with a model of CLIC function in which covalent binding of glutathione does not occur spontaneously but requires interaction with another protein to stabilise the GSH binding site and/or transfer of the ligand. The results do not indicate how CLIC1 undergoes a radical conformational change to form a transmembrane chloride channel but further elucidate the mechanism by which CLICs are redox controlled.

Voltage-dependent potassium channels (Kv) go beyond the stabilization of the resting potential and regulate biochemical pathways, regulate intracellular signaling, and detect energy homeostasis. Because targeted deletion and pharmacological block of the Kv1.3 channel protein produce marked changes in metabolism, resistance to diet-induced obesity, and changes in olfactory structure and function, this investigation explored Nedd4-2-mediated ubiquitination and degradation to regulate Kv1.3 channel density. Heterologous coexpression of Nedd4-2 ligase and Kv1.3 in HEK 293 cells reduced Kv1.3 current density without modulation of kinetic properties as measured by patch-clamp electrophysiology. Modulation of current density was dependent on ligase activity and was lost through point mutation of cysteine 938 in the catalytic site of the ligase (Nedd4-2CS). Incorporation of adaptor protein Grb10 relieved Nedd4-2-induced current suppression as did application of the proteasome inhibitor Mg-132. SDS-PAGE and immunoprecipitation strategies demonstrated a channel/adaptor/ligase signalplex. Pixel immunodensity was reduced for Kv1.3 in the presence of Nedd4-2, which was eliminated upon additional incorporation of Grb10. We confirmed Nedd4-2/Grb10 coimmunoprecipitation and observed an increased immunodensity for Nedd4-2 in the presence of Kv1.3 plus Grb10, regardless of whether the catalytic site was active. Kv1.3/Nedd4-2 were reciprocally coimmunoprecipated, whereby mutation of the COOH-terminal, SH3-recognition (493–498), or ubiquitination sites on Kv1.3 (lysines 467, 476, 498) retained coimmunoprecipitation, while the latter prevented the reduction in channel density. A model is presented for which an atypical interaction outside the canonical PY motif may permit channel/ligase interaction to lead to protein degradation and reduced current density, which can involve Nedd4-2/Grb10 interactions to disrupt Kv1.3 loss of current density.

© 2016 by the authors; licensee MDPI, Basel, Switzerland. CLIC1 is a Chloride Intracellular Ion Channel protein that exists either in a soluble state in the cytoplasm or as a membrane bound protein. Members of the CLIC family are largely soluble proteins that possess the intriguing property of spontaneous insertion into phospholipid bilayers to form integral membrane ion channels. The regulatory role of cholesterol in the ion‐channel activity of CLIC1 in tethered lipid bilayers was previously assessed using impedance spectroscopy. Here we extend this investigation by evaluating the influence of cholesterol on the spontaneous membrane insertion of CLIC1 into Langmuir film monolayers prepared using 1‐palmitoyl‐2‐oleoylphosphatidylcholine, 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phospho‐ethanolamine and 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phospho‐L‐serine alone or in combination with cholesterol. The spontaneous membrane insertion of CLIC1 was shown to be dependent on the presence of cholesterol in the membrane. Furthermore, pre‐incubation of CLIC1 with cholesterol prior to its addition to the Langmuir film, showed no membrane insertion even in monolayers containing cholesterol, suggesting the formation of a CLIC1‐cholesterol pre‐complex. Our results therefore suggest that CLIC1 membrane interaction involves CLIC1 binding to cholesterol located in the membrane for its initial docking followed by insertion. Subsequent structural rearrangements of the protein would likely also be required along with oligomerisation to form functional ion channels.

The purpose of this study was to obtain functional hERG ion channel protein for use in a non-cell-based ion channel assay. hERG was expressed in Sf9 insect cells. Attempts to solubilise the hERG protein from Sf9 insect cell membranes using non-ionic detergents (NP40 and DDM) were not successful. We therefore generated liposomes from the unpurified membrane fraction and incorporated these into porous Teflon-supported bilayer lipid membranes. Macroscopic potassium currents (1 nA) were recorded that approximated those in whole-cell patch-clamping, but the channels were bidirectional in the bilayer lipid membrane (BLM). Currents were partially inhibited by the hERG blockers E4031, verapamil, and clofilium, indicating that the protein of interest is present at high levels in the BLM relative to endogenous channels. Cell liposomes produced from Sf9 insect cell membranes expressing voltage-gated sodium channels also gave current responses that were activated by veratridine and inhibited by saxitoxin. These results demonstrate that purification of the ion channel of interest is not always necessary for liposomes used in macro-current BLM systems.

The stabilization of suspended planar lipid membranes, or black lipid membranes (BLMs), through polymerization of mono- and bis-functionalized dienoyl lipids was investigated. Electrical properties, including capacitance, conductance, and dielectric breakdown voltage, were determined for BLMs composed of mono-DenPC, bis-DenPC, mono-SorbPC, and bis-SorbPC both prior to and following photopolymerization, with diphytanoyl phosphocholine (DPhPC) serving as a control. Poly(lipid) BLMs exhibited significantly longer lifetimes and increased the stability to air-water transfers. BLM stability followed the order: bis-DenPC > mono-DenPC ≈ mono-SorbPC > bis-SorbPC. The conductance of bis-SorbPC BLMs was significantly higher than that of the other lipids, which is attributed to a high density of hydrophilic pores, resulting in relatively unstable membranes. The use of poly(lipid) BLMs as matrices for supporting the activity of an ion channel protein (IC) was explored using α – hemolysin (α-HL), a model IC. Characteristic i-V plots of α-HL were maintained following photopolymerization of bis-DenPC, mono-DenPC, and mono-SorbPC, demonstrating the utility of these materials for preparing more durable BLMs for single channel recordings of reconstituted ICs.

A striking feature of the CLIC (chloride intracellular channel) protein family is the ability of its members to convert between a soluble state and an integral membrane channel form. Direct evidence of the structural transition required for the CLIC protein to autonomously insert into the membrane is lacking, largely because of the challenge of probing the conformation of the membrane-bound protein. However, insights into the CLIC transmembrane form can be gained by biophysical methods such as fluorescence resonance energy transfer (FRET) spectroscopy. This approach was used to measure distances from tryptophan 35, located within the CLIC1 putative N-domain transmembrane region, to three native cysteine residues within the C-terminal domain. These distances were computed both in aqueous solution and upon the addition of membrane vesicles. The FRET distances were used as constraints for modeling of a structure for the CLIC1 integral membrane form. The data are suggestive of a large conformational unfolding occurring between the N- and C-domains of CLIC1 upon interaction with the membrane. Consistent with previous findings, the N-terminal domain of CLIC1 is likely to insert into the lipid bilayer, while the C-domain remains in solution on the extravesicular side of the membrane.

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel protein whose transmembrane domain (TM-domain) is believed to be responsible for channel gating via a hydrophobic effect. In this work, we perform molecular dynamics and Brownian dynamics simulations to investigate the effect of transmembrane potential on the conformation and water occupancy of TM-domain, and the resulting ion permeation events. The results show that the behavior of the hydrophobic gate is voltage-dependent. Large hyperpolarized membrane potential can change the conformation of TM-domain and water occupancy in this region, which may enable ion conduction. An electrostatic gating mechanism is also proposed from our simulations, which seems to play a role in addition to the well-known hydrophobic effect.

We demonstrate that cyano-phenylalanine (Phe(CN)) can be utilized to probe the binding of the inhalational anesthetic halothane to an anesthetic-binding, model ion channel protein hbAP-Phe(CN). The Trp to Phe(CN) mutation alters neither the alpha-helical conformation nor the 4-helix bundle structure. The halothane binding properties of this Phe(CN) mutant hbAP-Phe(CN), based on fluorescence quenching, are consistent with those of the prototype, hbAP1. The dependence of fluorescence lifetime as a function of halothane concentration implies that the diffusion of halothane in the nonpolar core of the protein bundle is one-dimensional. As a consequence, at low halothane concentrations, the quenching of the fluorescence is dynamic, whereas at high concentrations the quenching becomes static. The 4-helix bundle structure present in aqueous detergent solution and at the air-water interface, is preserved in multilayer films of hbAP-Phe(CN), enabling vibrational spectroscopy of both the protein and its nitrile label (-CN). The nitrile groups' stretching vibration band shifts to higher frequency in the presence of halothane, and this blue-shift is largely reversible. Due to the complexity of this amphiphilic 4-helix bundle model membrane protein, where four Phe(CN) probes are present adjacent to the designed cavity forming the binding site within each bundle, all contributing to the infrared absorption, molecular dynamics (MD) simulation is required to interpret the infrared results. The MD simulations indicate that the blue-shift of -CN stretching vibration induced by halothane arises from an indirect effect, namely an induced change in the electrostatic protein environment averaged over the four probe oscillators, rather than a direct interaction with the oscillators. hbAP-Phe(CN) therefore provides a successful template for extending these investigations of the interactions of halothane with the model membrane protein via vibrational spectroscopy, using cyano-alanine residues to form the anesthetic binding cavity.

Synopsis and pictogram: Gated pores in the ferritin family of protein nanocages, illustrated in the pictogram, control transfer of ferrous iron into and out of the cages by regulating contact between hydrated ferric oxide mineral inside the protein cage, and reductants such as FMNH(2) on the outside. The structural and functional homology between the gated ion channel proteins in inaccessible membranes and gated ferritin pores in the stable, water soluble nanoprotein, make studies of ferritin pores models for gated pores in many ion channel proteins.Properties of ferritin gated pores, which control rates of FMNH(2) reduction of ferric iron in hydrated oxide minerals inside the protein nanocage, are discussed in terms of the conserved pore gate residues (arginine 72-apspartate 122 and leucine 110-leucine 134), of pore sensitivity to heat at temperatures 30 °C below that of the nanocage itself, and of pore sensitivity to physiological changes in urea (1-10 mM). Conditions which alter ferritin pore structure/function in solution, coupled with the high evolutionary conservation of the pore gates, suggest the presence of molecular regulators in vivo that recognize the pore gates and hold them either closed or open, depending on biological iron need. The apparent homology between ferrous ion transport through gated pores in the ferritin nanocage and ion transport through gated pores in ion channel proteins embedded in cell membranes, make studies of water soluble ferritin and the pore gating folding/unfolding a useful model for other gated pores.

Influenza A virus and influenza B virus particles both contain small integral membrane proteins (A/M2 and BM2, respectively) that function as a pH-sensitive proton channel and are essential for virus replication. The mechanism of action of the M2 channels is a subject of scientific interest particularly as A/M2 channel was shown to be a target for the action of the antiviral drug amantadine. Unfortunately, an inhibitor of the BM2 channel activity is not known. Thus, knowledge of the structural and functional properties of the BM2 channel is essential for the development of potent antiviral drugs. The characterization of the oligomeric state of the BM2 channel is an essential first step in the understanding of channel function. Here we describe determination of the stoichiometry of the BM2 proton channel by utilizing three different approaches. 1) We demonstrated that BM2 monomers can be chemically cross-linked to yield species consistent with dimers, trimers, and tetramers. 2) We studied electrophysiological and biochemical properties of mixed oligomers consisting of wild-type and mutated BM2 subunits and related these data to predicted binomial distribution models. 3) We used fluorescence resonance energy transfer (FRET) in combination with biochemical measurements to estimate the relationships between BM2 channel subunits expressed in the plasma membrane. Our experimental data are consistent with a tetrameric structure of the BM2 channel. Finally, we demonstrated that BM2 transmembrane domain is responsible for the channel oligomerization.

The structure of CLIC4, a member of the CLIC family of putative intracellular chloride ion channel proteins, has been determined at 1.8 Angstroms resolution by X-ray crystallography. The protein is monomeric and it is structurally similar to CLIC1, belonging to the GST fold class. Differences between the structures of CLIC1 and CLIC4 are localized to helix 2 in the glutaredoxin-like N-terminal domain, which has previously been shown to undergo a dramatic structural change in CLIC1 upon oxidation. The structural differences in this region correlate with the sequence differences, where the CLIC1 sequence appears to be atypical of the family. Purified, recombinant, wild-type CLIC4 is shown to bind to artificial lipid bilayers, induce a chloride efflux current when associated with artificial liposomes and produce an ion channel in artificial bilayers with a conductance of 30 pS. Membrane binding is enhanced by oxidation of CLIC4 while no channels were observed via tip-dip electrophysiology in the presence of a reducing agent. Thus, recombinant CLIC4 appears to be able to form a redox-regulated ion channel in the absence of any partner proteins.

We report on a silicon wafer-based device that can be used for recording macroscopic ion channel protein activities across a diverse group of cell-types. Gigaohm seals were achieved for CHO-K1 and RIN m5F cells, and both cell-attached and whole-cell mode configurations were also demonstrated. Two distinct intrinsic potassium ion channels were recorded in whole-cell mode for HIT-T15 and RAW 264.7 cells. Polydimethylsiloxane (PDMS) microfluidics were also coupled with the micromachined silicon chips in order to demonstrate that a single cell could be selectively directed to a micropore, and membrane protein currents could subsequently be recorded. These silicon chip-based devices have significant advantages over traditional micropipette approaches, and may serve as combinatorial tools for investigating membrane biophysics, pharmaceutical screening, and other bio-sensing tasks.

The use of solid supported membranes (SSM) was investigated for reconstitution of ion channels and for potential application to screen pharmacological reagents affecting ion channel function. The voltage-gated Kv1.5 K+ channel was reconstituted on an SSM and a current was measured. This current was dependent on the presence of K+, but not Na+, indicating that the Kv1.5 K+ channel maintained cation specificity when reconstituted on SSM. Two pharmacological reagents applied to Kv1.5 K+ channels reconstituted on SSM had similar inhibitory effects as those measured using Kv1.5 in biological membranes. SSM-mounted ion channels were stable enough to be washed with buffer solution and reused many times, allowing solution exchange essential for pharmacological drug screening.

Voltage-gated sodium channels are members of a large family with similar pore structures. The mechanism of opening and closing is unknown, but structural studies suggest gating via bending of the inner pore helix at a glycine hinge. Here we provide functional evidence for this gating model for the bacterial sodium channel NaChBac. Mutation of glycine 219 to proline, which would strongly favor bending of the alpha helix, greatly enhances activation by shifting its voltage dependence -51 mV and slowing deactivation by 2000-fold. The mutation also slows voltage-dependent inactivation by 1200-fold. The effects are specific because substitutions of proline at neighboring positions and substitutions of other amino acids at position 219 have much smaller functional effects. Our results fit a model in which concerted bending at glycine 219 in the S6 segments of NaChBac serves as a gating hinge. This gating motion may be conserved in most members of this large ion channel protein family.

The first objective is to better understand how the size and charge of an analyte affect the flow of ions through a transmembrane nanopore to support the development of a bio-nanosensor that can measure the mass and identify of complex macromolecules such as DNA. To this end, molecular dynamics (MD) simulations were conducted on a transmembrane nanopore system composed of the β-barrel region of the transmembrane protein α-hemolysin (α-HL), a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer membrane, and an aqueous solution of potassium chloride (KCl), in which the β-barrel forms a channel through the membrane. After the system was allowed to equilibrate, a single molecule of polyethylene glycol (PEG) was added into the channel and a transmembrane potential was applied to one side of the membrane. The ionic current through the channel was measured, and compared to the measured current without a PEG molecule present. This work concluded that a system composed of only the β-barrel region of α-HL (not including the head region) can accurately depict the flow of ions through the nanopore in response to an applied transmembrane potential with greater computational ease than with the entire protein.The second objective is to better understand the response of the 5HT3 receptor to the ligand 5HT (serotonin) in an environment that mimics the surface of a proposed microelectromechanical system (MEMS) device designed to measure the presence of ligands such as neurotransmitters, neuropeptides and drug molecules. To this end, MD simulations were conducted on a system composed of the Cys-loop ligand-gated ion channel protein receptor 5HT3, a 7:7:6 ratio POPC/1-stearoyl-2-docosahexaenoyl-sn-glyerco-3-phosphocholine (SDPC)/cholesterol membrane, and 5HT to determine if binding of the ligand causes protein reassembly and channel opening.

Successful uncoating of the influenza B virus in endosomes is predicted to require acidification of the interior of the virus particle. We report that a virion component, the BM2 integral membrane protein, when expressed in Xenopus oocytes or in mammalian cells, causes acidification of the cells and possesses ion channel activity consistent with proton conduction. Furthermore, coexpression of BM2 with hemagglutinin (HA) glycoprotein prevents HA from adopting its low-pH-induced conformation during transport to the cell surface, and overexpression of BM2 causes a delay in intracellular transport in the exocytic pathway and causes morphological changes in the Golgi. These data are consistent with BM2 equilibrating the pH gradient between the Golgi and the cytoplasm. The transmembrane domain of BM2 protein and the influenza A virus A/M2 ion channel protein both contain the motif HXXXW, and, for both proteins, the His and Trp residues are important for channel function.

Significance: Voltage-gated K+ channels are a large family of K+-selective ion channel protein complexes that open on membrane depolarization. These K+ channels are expressed in diverse tissues and their function is vital for numerous physiological processes, in particular of neurons and muscle cells. Potentially reversible oxidative regulation of voltage-gated K+ channels by reactive species such as reactive oxygen species (ROS) represents a contributing mechanism of normal cellular plasticity and may play important roles in diverse pathologies including neurodegenerative diseases. Recent Advances: Studies using various protocols of oxidative modification, site-directed mutagenesis, and structural and kinetic modeling provide a broader phenomenology and emerging mechanistic insights. Critical Issues: Physicochemical mechanisms of the functional consequences of oxidative modifications of voltage-gated K+ channels are only beginning to be revealed. In vivo documentation of oxidative modifications of specific amino-acid residues of various voltage-gated K+ channel proteins, including the target specificity issue, is largely absent. Future Directions: High-resolution chemical and proteomic analysis of ion channel proteins with respect to oxidative modification combined with ongoing studies on channel structure and function will provide a better understanding of how the function of voltage-gated K+ channels is tuned by ROS and the corresponding reducing enzymes to meet cellular needs. Antioxid. Redox Signal. 21, 933–952.

This paper describes the formation of giant proteoliposomes containing P-glycoprotein (P-gp) from a solution of small proteoliposomes that had been deposited and partially dried on a film of agarose. This preparation method generated a significant fraction of giant proteoliposomes that were free of internalized vesicles, making it possible to determine the accessible liposome volume. Measuring the intensity of the fluorescent substrate rhodamine 123 (Rho123) inside and outside these giant proteoliposomes determined the concentration of transported substrates of P-gp. Fitting a kinetic model to the fluorescence data revealed the rate of passive diffusion as well as active transport by reconstituted P-gp in the membrane. This approach determined estimates for the membrane permeability coefficient (Ps) of passive diffusion and rate constants of active transport (kT) by P-gp as a result of different experimental conditions. The Ps value for Rho123 was larger in membranes containing P-gp under all assay conditions than in membranes without P-gp indicating increased leakiness in the presence of reconstituted transmembrane proteins. For P-gp liposomes, the kT value was significantly higher in the presence of ATP than in its absence or in the presence of ATP and the competitive inhibitor verapamil. This difference in kT values verified that P-gp was functionally active after reconstitution and quantified the rate of active transport. Lastly, patch clamp experiments on giant proteoliposomes showed ion channel activity consistent with a chloride ion channel protein that co-purified with P-gp. Together, these results demonstrate several advantages of using giant rather than small proteoliposomes to characterize transport properties of transport proteins and ion channels.

The viral ion channel protein M2 supports the transit of influenza virus and its glycoproteins through acidic compartments of the cell. M2 conducts endosomal protons into the virion to initiate uncoating and, by equilibrating the pH at trans -Golgi membranes, preserves the native conformation of acid-sensitive viral hemagglutinin. The exceptionally low conductance of the M2 channel thwarted resolution of single channels by electrophysiological techniques. Assays of liposome-reconstituted M2 yielded the average unitary channel current of the M2 tetramer—1.2 aA (1.2 × 10 −18 A) at neutral pH and 2.7 to 4.1 aA at pH 5.7—which activates the channel. Extrapolation to physiological temperature predicts 4.8 and 40 aA, respectively, and a unitary conductance of 0.03 versus 0.4 fS. This minute activity, below previous estimates, appears sufficient for virus reproduction, but low enough to avert abortive cytotoxicity. The unitary permeability of M2 was within the range reported for other proton channels. To address the ion selectivity of M2, we exploited the coupling of ionic influx and efflux in sealed liposomes. Metal ion fluxes were monitored by proton counterflow, employing a pH probe 1,000 times more sensitive than available Na + or K + probes. Even low-pH-activated M2 did not conduct Na + and K + . The proton selectivity of M2 was estimated to be at least 3 × 10 6 (over sodium or potassium ions), in agreement with electrophysiological studies. The stringent proton selectivity of M2 suggests that the cytopathology of influenza virus does not involve direct perturbation of cellular sodium or potassium gradients.

The M2 ion channel protein of influenza A virus is essential for mediating protein-protein dissociation during the virus uncoating process that occurs when the virus is in the acidic environment of the lumen of the secondary endosome. The difficulty of determining the ion selectivity of this minimalistic ion channel is due in part to the fact that the channel activity is so great that it causes local acidification in the expressing cells and a consequent alteration of reversal voltage, Vrev. We have confirmed the high proton selectivity of the channel (1.5‐2.0 3 10 6 ) in both oocytes and mammalian cells by using four methods as follows: 1) comparison of Vrev with proton equilibrium potential; 2) measurement of pHin and Vrev while Na 1 out was replaced; 3) measurements with limiting external buffer concentration to limit proton currents specifically; and 4) comparison of measurements of M2-expressing cells with cells exposed to a protonophore. Increased currents at low pHout are due to true activation and not merely increased [H 1 ]out because increased pHout stops the outward current of acidified cells. Although the proton conductance is the biologically relevant conductance in an influenza virusinfected cell, experiments employing methods 1‐3 show that the channel is also capable of conducting NH4 , probably by a different mechanism from H 1 . The M2 protein of influenza A virus is thought to function as an ion channel that permits protons to enter virus particles during virion uncoating in endosomes. In addition, in influenza virus-infected cells, the M2 protein causes the equilibration of pH between the acidic lumen of the trans-Golgi network and the cytoplasm (reviewed in Refs. 1 and 2). The activity of the M2 ion channel is inhibited by the antiviral drug amantadine (3‐ 5). The mature M2 protein consists of a 23-residue N-terminal extracellular domain, a single internal hydrophobic domain of 19 residues that acts as a transmembrane domain and forms the pore of the channel, and a 54-residue cytoplasmic tail (6). Chemical cross-linking studies showed the M2 protein to exist minimally as a homotetramer (7‐9). Statistical analysis of the ion channel activity of mixed oligomers also indicated that a homotetramer is the minimal active oligomeric form of the protein (10). Despite the small size of the active M2 oligomer, several lines of evidence indicate that ion channel activity is intrinsic to the M2 protein. First, ion channel activity has been observed in three different expression systems, Xenopus oocytes (3, 11, 12), mammalian cells (5, 13), and yeast (14). Second, M2 channel activity has also been recorded in artificial lipid bilayers from a reconstituted peptide corresponding to the transmembrane domain of the M2 protein (15) and from purified M2 protein (16). Thus, due to its structural simplicity, the M2 ion channel is a potentially useful model for the study of ion channels in general. Although a great deal of evidence indicates H 1 is the biologically relevant ion for the role of M2 protein in the life cycle of the influenza virus (1, 3, 17‐22), other ions have been shown to

NCC27 belongs to a family of small, highly conserved, organellar ion channel proteins. It is constitutively expressed by native CHO-K1 and dominantly localized to the nucleus and nuclear membrane. When CHO-K1 cells are transfected with NCC27-expressing constructs, synthesized proteins spill over into the cytoplasm and ion channel activity can then be detected on the plasma as well as nuclear membrane. This provided a unique opportunity to directly compare electrophysiological characteristics of the one cloned channel, both on the nuclear and cytoplasmic membranes. At the same time, as NCC27 is unusually small for an ion channel protein, we wished to directly determine whether it is a membrane-resident channel in its own right. In CHO-K1 cells transfected with epitope-tagged NCC27 constructs, we have demonstrated that the NCC27 conductance is chloride dependent and that the electrophysiological characteristics of the channels are essentially identical whether expressed on plasma or nuclear membranes. In addition, we show that a monoclonal antibody directed at an epitope tag added to NCC27 rapidly inhibits the ability of the expressed protein to conduct chloride, but only when the antibody has access to the tag epitope. By selectively tagging either the amino or carboxyl terminus of NCC27 and varying the side of the membrane from which we record channel activity, we have demonstrated conclusively that NCC27 is a transmembrane protein that directly forms part of the ion channel and, further, that the amino terminus projects outward and the carboxyl terminus inward. We conclude that despite its relatively small size, NCC27 must form an integral part of an ion channel complex.

The deadly toxins produced by many creatures, including spiders, snakes, and scorpions, work by blocking the ion channels that are essential for the normal operation of many different types of cells. Ion channels are proteins and, as their name suggests, they allow ions—usually sodium, potassium, or calcium ions—to move in and out of cells. They are especially important for cells that generate or respond to electrical signals, such as neurons and the cells in heart muscle. Ion channels are located in the lipid membranes that surround all cells, and the ions enter or leave the cell via a pore that runs through the channel protein. They can be opened and closed (or ‘gated’) in different ways: some ion channels open and close in response to voltages, whereas others are gated by biomolecules, such as neurotransmitters, that bind to them. Now, Banerjee et al. have used x-ray crystallography to study the structure of the complex that is formed when charybdotoxin (CTX), a toxin that is found in scorpion venom, blocks a voltage-gated potassium channel. Previous studies have shown that CTX binds to the channel on the extracellular side of the pore. Banerjee et al. show that the toxin fits into the entrance to the channel like a key into a lock, which means the toxin is preformed to fit the shape of the channel. The potassium ion channel is made up of four subunits, and the pore contains four ion-binding sites that form a ‘selectivity filter’: it is this filter that ensures that only potassium ions can pass through the channel when it is open. When CTX binds to the channel, a lysine residue poised at a critical position on the toxin is so close to the outermost ion-binding site that it prevents potassium ions binding to the site. The structure determined by Banerjee et al. explains many previous findings, including the fact that ions entering the pore from inside the cell can disrupt the binding between the toxin and the ion channel protein. It remains to be seen if the toxins that target the pore of other types of ion channels work in the same way.

Chloride intracellular Channel 1 (CLIC1) is a metamorphic protein that changes from a soluble cytoplasmic protein into a transmembrane protein. Once inserted into membranes, CLIC1 multimerises and is able to form chloride selective ion channels. Whilst CLIC1 behaves as an ion channel both in cells and in artificial lipid bilayers, its structure in the soluble form has led to some uncertainty as to whether it really is an ion channel protein. CLIC1 has a single putative transmembrane region that contains only two charged residues: arginine 29 (Arg29) and lysine 37 (Lys37). As charged residues are likely to have a key role in ion channel function, we hypothesized that mutating them to neutral alanine to generate K37A and R29A CLIC1 would alter the electrophysiological characteristics of CLIC1. By using three different electrophysiological approaches: i) single channel Tip-Dip in artificial bilayers using soluble recombinant CLIC1, ii) cell-attached and iii) whole-cell patch clamp recordings in transiently transfected HEK cells, we determined that the K37A mutation altered the single-channel conductance while the R29A mutation affected the single-channel open probability in response to variation in membrane potential. Our results show that mutation of the two charged amino acids (K37 and R29) in the putative transmembrane region of CLIC1 alters the biophysical properties of the ion channel in both artificial bilayers and cells. Hence these charged residues are directly involved in regulating its ion channel activity. This strongly suggests that, despite its unusual structure, CLIC1 itself is able to form a chloride ion channel.

The Chloride Intracellular ion channel protein CLIC1 has the ability to spontaneously insert into lipid membranes from a soluble, globular state. The precise mechanism of how this occurs and what regulates this insertion is still largely unknown, although factors such as pH and redox environment are known contributors. In the current study, we demonstrate that the presence and concentration of cholesterol in the membrane regulates the spontaneous insertion of CLIC1 into the membrane as well as its ion channel activity. The study employed pressure versus area change measurements of Langmuir lipid monolayer films; and impedance spectroscopy measurements using tethered bilayer membranes to monitor membrane conductance during and following the addition of CLIC1 protein. The observed cholesterol dependent behaviour of CLIC1 is highly reminiscent of the cholesterol-dependent-cytolysin family of bacterial pore-forming proteins, suggesting common regulatory mechanisms for spontaneous protein insertion into the membrane bilayer. © 2013 Valenzuela et al.

Planar lipid bilayer is an electrophysiological technique that enables study of functional activities of ion channels, porins, and other pore-forming molecular complexes. The main purpose of this method is to monitor ion channels’ behavior at the single molecule level in the artificial membranes. Here, I describe the details of this technique that will underline formation of the lipid bilayers and incorporation and activation of the ion channel protein.

Conductivity measurements and theoretical electronic structure calculations have been made in proteins, the calculations giving allowed energies for electrons that would enable them to participate in charge transport processes, probably by hopping and with close similarity to amorphous semiconductors. These theoretical computations have confirmed that the experimental results are probably due to electronic conduction. The foregoing considerations allow us to describe the protein-electrolyte interface as a semiconductor-electrolyte interface. Electron-transfer (ET) at such interfaces has been studied and experimentally established to occur from semiconductor electronic energy states to redox energy states in solution, or to redox surface states. ET has also been measured between organic and biochemical conducting compounds (particularly proteins and conducting membranes) and electrodes or redox solutions. Therefore, it is assumed in this paper that ET occurs at the ion channel protein interfaces with the cytoplasm and extracellular electrolytes, and that electron transport processes occur through the channel α-helices. A possible ET mechanism is proposed involving surface state oxygen-derived free radicals like Superoxide. The mathematical expressions for the biophysical phenomena discussed give two time-varying interface potentials and charge distributions that could provide the energy for the conformational changes that open and close the channel; these potentials could also change the ion permeation energy barriers by electrostatic influence. An expression is thus obtained for channel conductance as a function of membrane and interface potentials. Using this expression for sodium and potassium complemented by the usual passive components, gives a mathematical model that produces action potentials, intrinsic repetitive firing, and bursting, all of which can be modulated by changes in the biophysical parameters. Implications for ionic channel voltage gating are discussed and compared with existing experimental results and models. The possible relationships with redox and oxygen sensitivities, superoxide oxidative stress and neurotoxicity, drug and neurotransmitter effects, and ionic channel modulation are also discussed.

High level expression of the M2 ion channel protein of influenza virus inhibits the rate of intracellular transport of the influenza virus hemagglutinin (HA) and that of other integral membrane glycoproteins. HA coexpressed with M2 is properly folded, is not associated with GRP78-BiP, and trimerizes with the same kinetics as when HA is expressed alone. Analysis of the rate of transport of HA from the ER to the cis and medial golgi compartments and the TGN indicated that transport through the Golgi apparatus is delayed. Uncleaved HA0 was not expressed at the cell surface, and accumulation HA at the plasma membrane was reduced to 75-80% of control cells. The delay in intracellular transport of HA on coexpression of M2 was not observed in the presence of the M2-specific ion channel blocker, amantadine, indicating that the Golgi transport delay is due to the M2 protein ion channel activity equilibrating pH between the Golgi lumen and the cytoplasm, and not due to saturation of the intracellular transport machinery. The Na+/H+ ionophore, monensin, which also equilibrates pH between the Golgi lumen and the cytoplasm, caused a similar inhibition of intracellular transport as M2 protein expression did for HA and other integral membrane glycoproteins. EM data showed a dilation of Golgi cisternae in cells expressing the M2 ion channel protein. Taken together, the data suggest a similarity of effects of M2 ion channel activity and monensin on intracellular transport through the Golgi apparatus.

Three examples are given of how concepts from fractals and nonlinear dynamics have been used to analyze the voltages and currents recorded through ion channels in an attempt to determine the physical properties of ion channel proteins. (1) Early models had assumed that the switching of the ion channel protein from one conformational state to another can be represented by a Markov process that has no long-term correlations. However, one support for the existence of long-term correlations in channel function is that the currents recorded through individual ion channels have self-similar properties. These fractal properties can be characterized by a scaling function determined from the distribution of open and closed time intervals, which provides information on the distribution of activation energy barriers between the open and closed conformational substates of the ion channel protein and/or on how those energy barriers change in time. (2) Another support for such long-term correlations is that the whole-cell membrane voltage recorded across many channels at once may also have a fractal form. The Hurst rescaled range analysis of these fluctuations provides information on the type and degree of correlation in time of the functioning of ion channels. (3) The early models had also assumed that the switching from one state to another is an inherently random process driven by the energy from thermal fluctuations. More recently developed models have shown that deterministic dynamics may also produce the same distributions of open and closed times as those previously attributed to random events. This raises the possibility that the deterministic atomic and electrostatic forces play a role in switching the channel protein from one conformational shape to another. Debate exists about whether random, fractal, or deterministic models best represent the functioning of ion channels. However, fractal and deterministic dynamics provide a new approach to the study of ion channels that should be seriously considered by neuroscientists.

Most cystic fibrosis is caused by a deletion of a single residue (F508) in CFTR (cystic fibrosis transmembrane conductance regulator) that disrupts the folding and biosynthetic maturation of the ion channel protein. Progress towards understanding the underlying mechanisms and overcoming the defect remains incomplete. Here, we show that the thermal instability of human ΔF508 CFTR channel activity evident in both cell-attached membrane patches and planar phospholipid bilayers is not observed in corresponding mutant CFTRs of several non-mammalian species. These more stable orthologs are distinguished from their mammalian counterparts by the substitution of proline residues at several key dynamic locations in first N-terminal nucleotide-binding domain (NBD1), including the structurally diverse region, the γ-phosphate switch loop, and the regulatory insertion. Molecular dynamics analyses revealed that addition of the prolines could reduce flexibility at these locations and increase the temperatures of unfolding transitions of ΔF508 NBD1 to that of the wild type. Introduction of these prolines experimentally into full-length human ΔF508 CFTR together with the already recognized I539T suppressor mutation, also in the structurally diverse region, restored channel function and thermodynamic stability as well as its trafficking to and lifetime at the cell surface. Thus, while cellular manipulations that circumvent its culling by quality control systems leave ΔF508 CFTR dysfunctional at physiological temperature, restoration of the delicate balance between the dynamic protein's inherent stability and channel activity returns a near-normal state.

BACKGROUND Ion permeation through the nicotinic acetylcholine receptor channel is inhibited by general anesthetics. This inhibition could be mediated either by binding of anesthetic molecules to the channel protein itself or by the effects of anesthetics on the lipid environment of the protein. METHODS Patch clamp recording techniques were used to investigate the effects of ether and propofol on acetylcholine receptor channels in outside-out patches from BC3H-1 cells. The kinetic and conductance properties of single channels were measured. A rapid perfusion system was used to make rapid changes in anesthetic concentration during patch clamp recording to determine the kinetics of inhibition by anesthetics. RESULTS Ether, isoflurane (results from previous studies), and propofol produce distinct kinetic patterns of single acetylcholine receptor channel activity. Ether reduces the apparent current amplitude of channels, isoflurane induces flickering channel activity and propofol merely decreases the open time of the channel. The kinetics of inhibition are also different for these anesthetics. Ether ( or = 2 ms). CONCLUSIONS These diverse patterns can be interpreted in terms of a unitary mechanism in which the anesthetics interact directly with the channel protein. Each anesthetic is considered to bind to a site on the protein (perhaps, but not necessarily within the pore of the channel) and interrupt the flow of ions through the pore. Anesthetics have access to this inhibitory binding site even when the gate of the channel is closed. The pattern of channel activity induced by an anesthetic is determined by the frequency and duration of binding events.

Highlights ► Computational modeling of PEDV OFR3 protein. ► Wild-type PEDV ORF3 protein functions as an ion channel and regulates virus production. ► The truncated protein encoded by an attenuated-type PEDV ORF3 shows less channel activity., Several studies suggest that the open reading frame 3 (ORF3) gene of porcine epidemic diarrhea virus (PEDV) is related to viral infectivity and pathogenicity, but its function remains unknown. Here, we propose a structure model of the ORF3 protein consisting of four TM domains and forming a tetrameric assembly. ORF3 protein can be detected in PEDV-infected cells and it functions as an ion channel in both Xenopus laevis oocytes and yeast. Mutation analysis showed that Tyr170 in TM4 is important for potassium channel activity. Furthermore, viral production is reduced in infected Vero cells when ORF3 gene is silenced by siRNA. Interestingly, the ORF3 gene from an attenuated PEDV encodes a truncated protein with 49 nucleotide deletions, which lacks the ion channel activity.

Coarse-grained molecular dynamics provides a means for simulating the assembly and the interactions of membrane protein/lipid complexes at a reduced level of representation, allowing longer and larger simulations. We describe a fragment-based protocol for converting membrane simulation systems, comprising a membrane protein embedded in a phospholipid bilayer, from coarse-grained to atomistic resolution, for further refinement and analysis via atomistic simulations. Overall, this provides a method for generating an accurate and well equilibrated membrane protein/lipid complex. We exemplify the protocol using the acid-sensing/amiloride-sensitive ion channel protein (ASIC) channel protein, a trimeric integral membrane protein. The method is further evaluated using a test set of 10 different membrane proteins of differing size and complexity. Simulations are assessed in terms of protein conformational drift, lipid/protein interactions, and lipid dynamics. © 2011 American Chemical Society.

The structural basis of general anesthesia has remained hypothetical. Ion channels are plausible targets, as general anesthetics interact directly with neuronal ion channels and modulate their function. Investigating these interactions at the molecular level will further the understanding of general anesthesia and aid in the design of safer and more effective general anesthetics. The voltage-gated K+ channel Shaw2 is inhibited by relevant doses of n-alcohols and inhaled anesthetics at a discrete site. To map the residues that constitute this site in the activation gate of the channel, we conducted alanine-scanning mutagenesis in the S4-S5 linker and the S6 segment (post PVPV) of the Shaw2 protein and expressed the resulting mutants in Xenopus oocytes. Characterization of the 1-butanol and halothane dose-inhibition relations under voltage-clamp conditions and double mutant cycle analysis revealed significant energetic effects of single and double mutations on the inferred drug binding. Double mutants, such as Q320A/Y420A and A326V/Y419A (between the S4-S5 and S6), and Q320A/A326V (within the S4-S5 linker), exhibited the following coupling coefficients: 1.6, 4.3 and 2.8, respectively. Additional mutations suggested that these effects are not the result of altered gating. Exploiting these results, molecular dynamics simulations and docking calculations demonstrate the structural contributions of the S4-S5 linker and the distal S6 segment to a putative amphiphilic cavity that binds general anesthetics in the Shaw2 channel. Photolabeling of the Shaw2 protein is currently being explored as a method to test the hypothesis that this cavity is an anesthetic binding site. In conclusion, this work provides compelling evidence for a molecular mechanism of general anesthesia in which inhaled anesthetics modulate function through direct binding to a discrete cavity in the ion channel protein. Supported by NIH T04324 (AF), CNPg 141009/2009-8 (CA), NIH GM55876 (RGE), NIH AA010615 (MC).