Potassium ion channels and human disease: phenotypes to drug targets?

A significant difficulty faced by the pharmaceutical industry is the initial identification and selection of macromolecular targets upon which de novo drug discovery programs can be initiated. A drug target should have several characteristics: known biological function; robust assay systems for in vitro characterization and high-throughput screening; and be specifically modified by and accessible to small molecular weight compounds in vivo. Ion channels have many of these attributes and can be viewed as suitable targets for small molecule drugs. Potassium (K⁺) ion channels form a large and diverse gene family responsible for critical functions in numerous cell types, tissues and organs. Recent discoveries, facilitated by genomics technologies combined with advanced biophysical characterization methods, have identified novel K⁺ channels that are involved in important physiologic processes, or mutated in human inherited disease. These findings, coupled with a rapidly growing body of information regarding modulatory channel subunits and high resolution channel structures, are providing the critical information necessary for validation of K⁺ channels as drug targets.     

α2-Interferon- and oligoadenylate-induced morphological differentiation and modulation of the ion channels in neuroblastoma cells

The experiments were carried out on the IMR-32 human neuroblastoma and NIE-115 murine neuroblastoma cultured cells. Peculiarities of the ion channel expression and their correlation with the main morphological parameters characterizing neuronal differentiation were studied under conditions of incubation of the cells with 2-interferon and 2,5-oligoadenylate, 2–5A. Twenty-four hours after addition of 1000 U act./ml interferon to the culture medium, a 56% increase in the mean projective surface of the IMR-32 cells was observed, and after a nine-day-long exposition this increase was 132%, as compared with the control. Mean total length of the cellular protrusions nearly doubled after nine-day-long incubation. Morphological and electrophysiological properties of the N1E-115 murine neuroblastoma cells showed practically no changes after incubation with human 2-interferon. Cultivation of the IMR-32 human neuroblastoma cells in the presence of 2–5A evoked insignificant changes in their morphological parameters. By contrast, the mean total length of neurites of the N1E-115 neuroblastoma protrusion-supplied cells increased more than a factor of five after eight-day-long incubation with 2–5A in a 1.0 µM concentration, and at a 0.01 µM 2–5A concentration this increase was about fourfold. At the same time, the projective surface exhibited no significant changes either in the neurite-supplied or the neurite-free cellular subpopulations. Twenty-four hours after the incubation with human 2-interferon had been begun, the density of sodium current in the IMR-32 human neuroblastoma cells increased by 250% compared with the control. A similar effect was observed after the addition of 2–5A to the medium: the density of sodium current approximately doubled. Cultivation of the neurite-supplied N1E-115 murine neuroblastoma cells was followed by a gradual increase in the density of fast sodium current both in control and 2–5A-influenced samples, but in the latter case this increase was significantly faster. In the neurite-free cells, the density of sodium current was 27% higher after their 24-h-long incubation with 2–5A, as compared with the control values (11.05±0.9 and 8.7±0.9 pA/pF, respectively). Longer incubation resulted in a sharp decrease in the density of potassium current. The results of our study are in agreement with the data about species-related individuality of 2-interferon and different intensity of its effects on the cells passing different stages of cellular differentiation.Neirofiziologiya/Neurophysiology, Vol. 27, No. 3, pp. 199–207, May–June, 1995.     

β-Barrel topology of Alzheimer's β-amyloid ion channels

Emerging evidence supports the ion channel mechanism for Alzheimer's disease pathophysiology wherein small β-amyloid (Aβ) oligomers insert into the cell membrane, forming toxic ion channels and destabilizing the cellular ionic homeostasis. Solid-state NMR-based data of amyloid oligomers in solution indicate that they consist of a double-layered β-sheets where each monomer folds into β-strand-turn-β-strand and the monomers are stacked atop each other. In the membrane, Aβ peptides are proposed to be β-type structures. Experimental structural data available from atomic force microscopy (AFM) imaging of Aβ oligomers in membranes reveal heterogeneous channel morphologies. Previously, we modeled the channels in a non-tilted organization, parallel with the cross-membrane normal. Here, we modeled a β-barrel-like organization. β-Barrels are common in transmembrane toxin pores, typically consisting of a monomeric chain forming a pore, organized in a single-layered β-sheet with antiparallel β-strands and a right-handed twist. Our explicit solvent molecular dynamics simulations of a range of channel sizes and polymorphic turns and comparisons of these with AFM image dimensions support a β-barrel channel organization. Different from the transmembrane β-barrels where the monomers are folded into a circular β-sheet with antiparallel β-strands stabilized by the connecting loops, these Aβ barrels consist of multimeric chains forming double β-sheets with parallel β-strands, where the strands of each monomer are connected by a turn. Although the Aβ barrels adopt the right-handed β-sheet twist, the barrels still break into heterogeneous, loosely attached subunits, in good agreement with AFM images and previous modeling. The subunits appear mobile, allowing unregulated, hence toxic, ion flux.     

PACAP stimulates insulin secretion by PAC1 receptor and ion channels in β-cells

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) plays a crucial role in the endocrine system. The present study aimed to investigate the effect of PACAP38 on insulin secretion and the underlying mechanism in rat pancreatic β-cells. The insulin secretion results showed that PACAP38 stimulated insulin secretion in a glucose- and dose-dependent manner. The insulinotropic effect was mediated by PAC₁ receptor, but not by VPAC₁ and VPAC₂ receptors. Inhibition of adenylyl cyclase and protein kinase A suppressed PACAP38-augmented insulin secretion. Glucose-regulated insulin secretion is dependent on a series of electrophysiological activities. Current-clamp technology suggested that PACAP38 prolonged action potential duration. Voltage-clamp recordings revealed that PACAP38 blocked voltage-dependent potassium currents, and this effect was reversed by inhibition of PAC₁ receptor, adenylyl cyclase, or protein kinase A. Activation of Ca²⁺ channels by PACAP38 was also observed, which could be antagonized by the PAC₁ receptor antagonist. In addition, calcium-imaging analysis indicated that PACAP38 increased intracellular Ca²⁺ concentration, which was decreased by PAC₁ receptor antagonist. These findings demonstrate that PACAP38 stimulates glucose-induced insulin secretion mainly by acting on PAC₁ receptor, inhibiting voltage-dependent potassium channels, activating Ca²⁺ channels and increasing intracellular Ca²⁺ concentration. Further, PACAP blocks voltage-dependent potassium currents via the adenylyl cyclase/protein kinase A signaling pathway.     

Engineering aspects of biological ion channels—from biosensors to computational models for permeation

Molecular sequencing has helped resolve the phylogenetic relationships amongst the diverse groups of algal, fungal-like and protist organisms that constitute the Chromalveolate “superkingdom” clade. It is thought that the whole clade evolved from a photosynthetic ancestor and that there have been at least three independent plastid losses during their evolutionary history. The fungal-like oomycetes and hyphochytrids, together with the marine flagellates Pirsonia and Developayella, form part of the clade defined by Cavalier-Smith and Chao (2006) as the phylum “Pseudofungi”, which is a sister to the photosynthetic chromistan algae (phylum Ochrophyta). Within the oomycetes, a number of predominantly marine holocarpic genera appear to diverge before the main “saprolegnian” and “peronosporalean” lines, into which all oomycetes had been traditionally placed. It is now clear that oomycetes have their evolutionary roots in the sea. The earliest diverging oomycete genera so far documented, Eurychasma and Haptoglossa, are both obligate parasites that show a high degree of complexity and sophistication in their host parasite interactions and infection structures. Key morphological and cytological features of the oomycetes will be reviewed in the context of our revised understanding of their likely phylogeny. Recent genomic studies have revealed a number of intriguing similarities in host–pathogen interactions between the oomycetes with their distant apicocomplexan cousins. Therefore, the earlier view that oomycetes evolved from the largely saprotrophic “saprolegnian line” is not supported and current evidence shows these organisms evolved from simple holocarpic marine parasites. Both the hyphal-like pattern of growth and the acquisition of oogamous sexual reproduction probably developed largely after the migration of these organisms from the sea to land.     

Effect of nifedipine on the ion channels formed by α1 subunits of the cardiac and vascular muscle L-type Ca channels

We investigated the voltage dependence of nifedipine sensitivity of the ion channels formed by α₁ subunits of the cardiac and smooth muscles (CM and SM, respectively) L-type Ca²⁺ channels stably expressed in Chinese hamster ovary (CHO) cells. Equilibrium inhibition of the α₁ subunits, directing Ba²⁺ current (I _α1), by different concentrations of nifedipine was measured at the holding potentials (V _h ) of −100 mV and −50 mV. AtV _h =−100 mV, the SM α₁ subunit was found to be 6-fold more sensitive for nifedipine than the subunit (K ₋₁₀₀=8.3 and 50.4 nM, respectively). Depolarization to −50 mV resulted in about sevenfold increase in the nifedipine potency for both subunits (K ₋₅₀=1.25 and 6.95 nM, respectively). The voltage dependence of steady-state inactivation could be fitted by a sum of two Boltzmann’s equations with slope factors of about 12 and 5 mV. The midpoints of both components in the CM α₁ subunit (−75.6 and −42.8 mV) were more negative than those in the SM subunit (−63.7 and −37.7 mV). The relative contribution of the less sloped component in the control was rather low, being less pronounced in the CM (0.15) than in the SM (0.34) subunits. Nifedipine shifted the midpoints of inactivation curves to more negative potentials. The shift was more pronounced for the SM α₁ subunit (−24.8 mV compared with −11.8 mV for the CM subunit in the presence of 10 nM nifedipine). Nifedipine differentially affected the two Boltzmann components of inactivation curves, more effectively inhibiting the steeper component. In the presence of 10 nM nifedipine, this component completely disappeared in the SM subunit, while its relative contribution in the CM subunit decreased from 0.85 to 0. 57, resulting in an apparent decrease in the steepness. These results are inconsistent with the receptor modulated hypothesis and suggest the existence of two mechanisms of inactivation characterized by different voltage dependence.     

BioMOCA—a Boltzmann transport Monte Carlo model for ion channel simulation

With the recent availability of high-resolution structural information for several key ion channel proteins and large-scale computational resources, Molecular Dynamics has become an increasingly popular tool for ion channel simulation. However, the CPU requirements for simulating ion transport on time scales relevant to conduction still exceed the resources presently available. To address this problem, we have developed Biology Monte Carlo (BioMOCA), a three-dimensional (3D) coarse-grained particle ion channel simulator based on the Boltzmann Transport Monte Carlo (BTMC) methodology. Although this approach is widely employed in the engineering community to study charge transport in electron devices, its application to molecular biology and electrolytes in general is new and hence must be validated. The pair correlation function, which is a measure of the microscopic structure of matter, provides a suitable benchmark to compare the BTMC method against the well-established Equilibrium Monte Carlo (EMC) approach. For validation purposes BioMOCA is used to simulate several simple homogeneous equilibrium electrolytes at concentrations of physiological interest. The ion–ion pair correlation functions computed from these simulations compare very well with those obtained from EMC simulations. We also demonstrate several performance-improving techniques that result in a several-fold speed-up without compromising the pair correlation function. BioMOCA is then used to perform full 3D simulations of ion transport in the gramicidin A channel in situ in a membrane environment, as well as to study the link between the electrostatic and dielectric properties of the protein and the channel's selectivity.     

Presynaptic AMPA receptors: more than just ion channels?

AMPA receptor ion channels are of paramount importance for postsynaptic excitation. Several reports demonstrate that AMPA receptors are present in the presynaptic compartment and point to a role of these receptors in the modulation of presynaptic function. We discuss here the possibility that not only ion influx through the receptor, but also biochemical cascades, activated by ligand binding and independent from ion flux, might contribute to AMPA mediated presynaptic modulation.     

Amyloid-β-induced reactive oxygen species production and priming are differentially regulated by ion channels in microglia

Production of reactive oxygen species (ROS) by microglial cells and subsequent oxidative stress are strongly implicated in the pathogenesis of Alzheimer's disease. Although it is recognized that amyloid‐β (Aβ) plays a major role in inducing and regulating microglial ROS production in Alzheimer's disease, to date little is known about cellular mechanisms underlying Aβ‐stimulated ROS production. Here, we identified ion channels involved in Aβ‐induced microglial ROS production and in Aβ‐induced microglial priming. Acute stimulation of microglial cells with either fibrillar Aβ_1–42 (fAβ_1–42) or soluble Aβ_1–42 (sAβ_1–42) caused significant increases in microglial ROS production, which were abolished by inhibition of TRPV1 cation channels with 5‐iodo‐resiniferatoxin (I‐RTX), but were unaffected by inhibition of K⁺ channels with charybdotoxin (CTX). Furthermore, pretreatment with either fAβ_1–42 or sAβ_1–42 induced microglial priming, that is, increased ROS production upon secondary stimulation with the phorbol ester PMA. Microglial priming induced by fAβ_1–42 or sAβ_1–42 remained unaffected by TRPV1 channel inhibition with I‐RTX. However, sAβ_1–42‐induced priming was inhibited by CTX and margatoxin, but not by TRAM‐34 or paxilline, indicating a role of Kv1.3 voltage‐gated K⁺ channels, but not of Ca²⁺‐activated K⁺ channels, in the priming process. In summary, our data suggest that in microglia Aβ‐induced ROS production and priming are differentially regulated by ion channels, and that TRPV1 cation channels and Kv1.3 K⁺ channels may provide potential therapeutic targets to reduce microglia‐induced oxidative stress in Alzheimer's disease. J. Cell. Physiol. 226: 3295–3302, 2011. © 2011 Wiley Periodicals, Inc.     

Novel actions of ryanodine and analogues—Perturbers of potassium channels

The effects of ryanodine, 9,21-didehydroryanodine and 9,21-didehydroryanodol on two types of K⁺ channel (a maxi, Ca²⁺-activated, 170 pS channel (BK channel) and an inward rectifier, stretch-sensitive channel of 35 pS conductance (IK channel) found in the plasma membrane of locust skeletal muscle have been investigated. 10^–9M-10^–5M ryanodine irreversibly induced a dose-dependent reduction of the reversal potential (V_rev) of the currents of both channels, i.e. from 60 mV in the absence of the alkaloid to 15 mV for 10^–5M ryanodine, measured under physiologically normal K⁺ and Na⁺ gradients. In both cases the change in the ionic selectivity was Ca²⁺-independent. 9,21-didehydroryanodine and 9,21-didehyroryanodol also reduced V_rev, but only to 35 mV during application of 10^–5M of these compounds. Additionally, 9,21-didehydroryanodine reversibly diminished the conductances of the two K⁺ channels. To test the hypothesis that ryanoids increase Na⁺ permeability by enlarging the K⁺ channels, the channels were probed with quaternary ammonium ions during ryanoid application. When applied to the cytoplasmic face of inside-out patches exised from locust muscle membrane, TEA blocked the K⁺ channels in a voltage-dependent fashion. The dissociation constant (K_d(0)) for TEA block of the IK channel was reduced from 44 mM to 1 mM by 10^–7 M ryanodine, but the voltage-dependence of the block was unaffected. Qualitatively similar data were obtained for the BK channel. Ryanodine had no effect on the K_d for cytoplasmically-applied TMA. However, the voltage-dependence for TMA block was increased for both K⁺ channels, from 0.47 to 0.8 with 10^–6M ryanodine. The effects of ryanodine on TEA and TMA block support the hypothesis that ryanodine enlarges the K⁺ channels so as to facilitate permeation of partially hydrated Na⁺ ions.     

Phylogenetic conservation of protein–lipid motifs in pentameric ligand-gated ion channels

Using the crosstalk between the nicotinic acetylcholine receptor (nAChR) and its lipid microenvironment as a paradigm, this short overview analyzes the occurrence of structural motifs which appear not only to be conserved within the nAChR family and contemporary eukaryotic members of the pentameric ligand-gated ion channel (pLGIC) superfamily, but also extend to prokaryotic homologues found in bacteria. The evolutionarily conserved design is manifested in: 1) the concentric three-ring architecture of the transmembrane region, 2) the occurrence in this region of distinct lipid consensus motifs in prokaryotic and eukaryotic pLGIC and 3) the key participation of the outer TM4 ring in conveying the influence of the lipid membrane environment to the middle TM1–TM3 ring and this, in turn, to the inner TM2 channel-lining ring, which determines the ion selectivity of the channel. The preservation of these constant structural–functional features throughout such a long phylogenetic span likely points to the successful gain-of-function conferred by their early acquisition. This article is part of a Special Issue entitled: Lipid–protein interactions.     

Sexual dimorphism and the differential mortality model: is behaviour related to survival?

There are numerous hypotheses to explain the evolution of sexual dimorphism in spiders. One of the most controversial is the differential mortality model (DMM) which proposes that differing rates of (adult) male and female mortality can result in a skewed operational sex ratio and lead to the evolution of small males. This hypothesis has been examined using a comparative approach which assumes that the behaviour of males and females could be used as a surrogate measure of mortality. We tested this assumption using two model species, Hogna helluo and Pardosa milvina (Araneae: Lycosidae) that differ in the degree of sexual dimorphism both in terms of body size and level of activity. Our data demonstrate that differences in male and female behaviour are not predictive of differences in mortality. Rather, as in other organisms, mortality is a complex phenomenon dependent on activity as well as size. These data call into question the methods previously used to test the DMM and suggest that understanding sexual size dimorphism (SSD) in spiders will require evaluation of historical constraints as well as how size currently influences fitness in each sex.  © 2003 The Linnean Society of London . Biological Journal of the Linnean Society , 2003, 78 , 97−103.     

“Divide and conquer” approach to the structural studies of multidomain ion channels by the example of isolated voltage sensing domains of human Kv2.1 and Nav1.4 channels

Voltage-gated K⁺ and Na⁺ channels are involved in diverse physiological processes including excitability of heart, muscular and neuronal cells, as well as release of hormones and neurotransmitters. These channels have modular structure and contain five membrane domains: four voltage-sensing domains (VSDs) and one pore domain. VSDs of different channels contain unique ligand-binding sites and are considered as potential pharmacological targets. Modular organization of ion channels points to the possibility of NMR structural studies of isolated VSDs apart from the pore. Here, the feasibility of such studies is considered by the example of VSD of human Kv2.1 channel and VSD-I of human Nav1.4 channel. Cell-free protein expression systems based on the S30 bacterial extract from E. coli, which allow us to produce milligram quantities of VSD samples, including their analogues labeled with stable isotopes, were developed. The choice of membrane- mimicking media that provide long-term stability of the native structure of the membrane protein and high-quality of NMR spectra is a crucial step in NMR studies. Screening of various environments showed that the domains of the Kv2.1 and Nav1.4 channels are unstable in media containing phospholipids: micelles of short-chain lipid DC7PC and lipid-detergent bicelles based on zwitterionic or anionic saturated lipids (DMPC and DMPG). It was demonstrated that the optimal media for NMR studies are the mixtures of zwitterionic and weakly cationic detergents (FOS-12/LDAO). The VSD sample of the Nav1.4 channel in FOS- 12/LDAO environment aggregated irreversibly within a few days despite the high-quality spectra. It is likely that VSDs of human K⁺ and Na⁺ channels are not completely autonomous membrane domains and the contacts with other domains of the channel are required for their stabilization.     

Subtilisin DY—Kinetic characterization and comparison with related proteinases

Subtilisin DY, a serine proteinase fromBacillus subtilis, variant DY, is compared with subtilisin Carlsberg, proteinase K, thermitase, as well as with -chymotrypsin by use of kinetic parameters of the reactions with the synthetic peptidyl substrates succinyl-Ala₃-4-nitroanilide, succinyl-Ala₂-Phe-4-nitroanilide, MeO-succinyl-Ala₂-Phe-4-nitroanilide, and with the irreversible inhibitors of the general structure benzyloxycarbonyl-Ala_n-PheCH₂Cl (n=0, 1, 2, 3, 4) and benzyloxycarbonyl-Ala-AaCH₂Cl (Aa=Gly, Ala, Val, Leu, Phe), respectively. The data demonstrated that subtilisin DY is very closely related to the Carlsberg enzyme. Thermitase and -chymotrypsin deviate in their behavior from that of subtilisin DY more than proteinase K. The best inhibitor (highest k_inact/K_i) for the two subtilisins and proteinase K is benzyloxycarbonyl-Ala₂-PheCH₂Cl; for thermitase and -chymotrypsin, benzyloxycarbonyl-Ala₃-PheCH₂Cl. In the series of the benzyloxycarbonyl-protected dipeptidyl chloromethyl ketones, the following order of reactivity was obtained for subtilisin DY as well as for subtilisin Carlsberg and proteinase K: Aa=LeuPhe>AlaVal>Gly. Thermitase and -chymotrypsin prefer the aromatic phenylalanyl rather than the also hydrophobic leucyl residue at S₁. Succinyl-Ala₂-Phe-4-nitroanilide was the most suitable substrate to indicate the inhibition reactions.     

In vitro analysis of ion channels in periaxolemmal-myelin and white matter clathrin coated vesicles: Modulation by calcium and GTPγS

This study reports the analysis of K⁺ channel activity in bovine periaxolemmal-myelin and white matter-derived clathrin-coated vesicles. Channel activity was evaluated by the fusion of membrane vesicles with phospholipid bilayers formed across a patch-clamp pipette. In periaxolemmal myelin spontaneous K⁺ channels were observed with amplitudes of 25–30, 45–55, and 80–100 pS, all of which exhibited mean open-times of 1–2 msec. The open state probability of the 50 pS channel in periaxolemmal-myelin was increased by 6-methyldihydro-pyran-2-one. Periaxolemmal-myelin K⁺ channel activity was regulated by Ca²⁺. Little or no change in activity was observed when Ca²⁺ was added to thecis side of the bilayer. Addition of 10 M total Ca²⁺ also resulted in little change in K⁺ channel activity. However, at 80 M total Ca²⁺ all K⁺ channel activity was suppressed along with the activation of a 100 pS Cl^– channel. The K⁺ channel activity in periaxolemmal myelin was also regulated through a G-protein. Addition of GTPS to thetrans side of the bilayer resulted in a restriction of activity to the 45–50 pS channel which was present at all holding potentials. Endocytic coated vesicles, form in part through G-protein mediated events; white matter coated vesicles were analyzed for G proteins and for K⁺ channel activity. These vesicles, which previous studies had shown are derived from periaxolemmal domains, were found to be enriched in the subunits of G₀, G_s, and G_i and the low molecular weight G protein,ras. As with periaxolemmal-myelin treated with GTPS, the vesicle membrane exhibited only the 50 pS channel. The channel was active at all holding potentials and had open times of 1–6 msec. Addition of GTPS to the bilayer fused with vesicle membrane appeared to suppress this channel activity at low voltages yet induced a hyperactive state at holding potentials of 45 mV or greater. The vesicle 50 pS K⁺ channel was also activated by the 6-methyl-dihydropyron-2-one (20 M).Abbreviations CNPase 2–3 cyclic nucleotide phosphohydrolase - EDTA ethylenediamine N,N,N,N-tetraacetic acid - G-protein GTP(guanosine triphosphate) binding protein - GTPS guanosine 5-O-(3-thiotriphosphate) - MAG myelin associated glycoprotein - Na⁺ K⁺ ATPase, Na⁺ and K⁺ stimulated adenosine triphosphatase - PLP myelin proteolipid protein Special issue dedicated to Dr. Majorie B. Lees.