The International Symposium on Receptors and Ion Channels--Including Ion Pumps and Second Messenger Systems. March 6-9, 1989, West Berlin.
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Animals↗
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Ligand-gated ion channels: mechanisms underlying ion selectivity.
Anion/cation selectivity is a critical property of ion channels and underpins their physiological function. Recently, there have been numerous mutagenesis studies, which have mapped sites within the ion channel-forming segments of ligand-gated ion channels that are determinants of the ion selectivity. Site-directed mutations to specific amino acids within or flanking the M2 transmembrane segments of the anion-selective glycine, GABA(A) and GABA(C) receptors and the cation-selective nicotinic acetylcholine and serotonin (type 3) receptors have revealed discrete, equivalent regions within the ion channel that form the principal selectivity filter, leading to plausible molecular mechanisms and mathematical models to describe how ions preferentially permeate these channels. In particular, the dominant factor determining anion/cation selectivity seems to be the sign and exposure of charged amino acids lining the selectivity filter region of the open channel. In addition, the minimum pore diameter, which can be influenced by the presence of a local proline residue, also makes a contribution to such ion selectivity in LGICs with smaller diameters increasing anion/cation selectivity and larger ones decreasing it.
Animals↗
Correlation studies of open and closed state fluctuations in an ion channel: Analysis of ion current through a large-conductance locust potassium channel.
Ion current fluctuations occurring within open and closed states of a large-conductance locust potassium channel (BK channel) were investigated for the existence of correlation. Both the time series, extracted from the ion current signal, were studied by the autocorrelation function and the detrended fluctuation analysis (DFA) methods. The persistent character of the short- and middle-range correlations of time series is shown by the slow decay of the autocorrelation function. The DFA exponent alpha is significantly larger than 0.5. The existence of strongly persistent long-range correlations was detected only for closed state fluctuations, with alpha=0.98+/-0.02. The long-range correlation of the BK channel action is therefore determined by the character of closed states. The main outcome of this study reveals that the memory effect is present not only between successive conducting states of the channel but also independently within the open and closed states themselves. As the ion current fluctuations give information about the dynamics of the channel protein, our results point to the correlated character of the protein movement regardless of whether the channel is in its open or closed state.
Animals↗
The sensory mechanotransduction ion channel ASIC2 (acid sensitive ion channel 2) is regulated by neurotrophin availability.
Almost all sensory neurons of the dorsal root ganglia have a mechanosensitive receptive field in the periphery. We have shown that the sensitivity to mechanical stimuli of a subset of sensory neurons that are slowly adapting mechanoreceptors (SAM) is strongly dependent on the availability of brain-derived neurotrophic factor (BDNF). Here we have investigated whether the ASIC2 sodium channel, recently shown by us to be necessary for normal SAM sensitivity, might be regulated by BDNF and thus partially account for the down-regulation of SAM sensitivity seen in BDNF deficient mice. We show that the mRNA for ASIC2 channels is reduced in the DRG of BDNF deficient mice indicating that BDNF might maintain its expression in vivo. We also made short-term cultures of sensory neurons from adult BDNF deficient mice and used a specific antibody to detect the presence of ASIC2 channels in different classes of sensory neurons. We observed that the channel protein was dramatically down-regulated selectively in medium and large diameter neurons and this expression could be rescued in a dose and time dependent manner by addition of BDNF to the culture (10-100 ng/ml). Drugs that block new transcription or protein synthesis also prevented the rescue effects of BDNF. We observed that ASIC2 channels were down-regulated in sensory neurons taken from neurotrophin-4 and neurotrophin-3 deficient mice; these effects might be due to a selective loss of neurons that normally express large amounts of ASIC2 channels. In summary, our data identify the ASIC2 channel as a target of BDNF signaling in vivo and suggest that the functional down-regulation of sensory mechanotransduction in BDNF deficient mice is in part due to loss of ASIC2 expression.
Acid Sensing Ion Channels↗
Phenotype variation and newcomers in ion channel disorders.
Ion channels are part of a large family of macromolecules whose functions include the control and maintenance of electrical potential across cell membranes, secretion and signal transduction. Close inspection of the physiological processes involved in channel function and the secondary structure of various ion channels has served as a basis for subdividing ion channels into a number of superfamilies. The voltage-gated ion channels are one of these superfamilies. Recent work has shown that mutations in various ion channel genes are responsible for a number of neuromuscular and neurological disorders. Correlation of the various mutations with the clinical phenotype is providing us with insight into the pathophysiology of these channel proteins. Interestingly, different mutations within the same gene may cause quite distinct clinical disorders, while mutations in different channel genes may result in very similar phenotypes (genetic heterogeneity). Examples of phenotypic variation and genetic heterogeneity are presented in the context of the periodic paralytic disorders of skeletal muscle, episodic ataxia, migraine, long QT syndrome and paroxysmal dyskinesia. Some of these disorders are known to be caused by mutations in ion channel genes, while in the episodic movement disorders, ion channel genes are considered excellent candidate genes.
Animals↗
ATP depletion and inactivation of an ATP-sensitive taurine channel by classic ion channel blockers.
Cell volume regulation in different cell types is mediated in part by plasma membrane channel(s) that allow taurine and other important intracellular organic osmolytes to efflux from the cell. Previous studies have demonstrated that intracellular ATP is required for activation of a volume-sensitive taurine-permeable channel. The present study examined the relation between cellular ATP and ADP concentrations and swelling-induced [14C]taurine efflux and anion current (whole-cell patch-clamp) after exposure of isolated skate (Raja erinacea) hepatocytes to metabolic poisons and a series of ion channel blockers. When intracellular ATP content was lowered with gradually increasing concentrations of 2,4-dinitrophenol, a sigmoidal relation between ATP content and volume-activated [14C]taurine efflux was observed. Taurine efflux was progressively inhibited over a relatively narrow range of intracellular ATP levels, indicating that physiologic alterations in cellular nucleotides may modulate the opening of the channel. Surprisingly, the inhibition of [14C]taurine efflux by a number of ion channel blockers [glibenclamide, 5-nitro-2-(3-phenylpropylamino)benzoate, diphenylamine-2-carboxylate, ketoconazole, gossypol, niflumic acid, and quinine] was related to a decrease in cellular ATP concentrations and ATP/ADP ratios, rather than to a direct interaction with the channel. In contrast, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and pyridoxal-5-phosphate inhibited volume-activated anion channels but had no effect on cellular ATP levels. These findings suggest multiple sites for regulation of volume-sensitive osmolyte channels and indicate that some putative ion channel blockers may actually alter the activity of ATP-regulated transporters by depleting cellular ATP.
2,4-Dinitrophenol↗
Comprehensive analysis of the ascidian genome reveals novel insights into the molecular evolution of ion channel genes.
Ion fluxes through membrane ion channels play crucial roles both in neuronal signaling and the homeostatic control of body electrolytes. Despite our knowledge about the respective ion channels, just how diversification of ion channel genes underlies adaptation of animals to the physical environment remains unknown. Here we systematically survey up to 160 putative ion channel genes in the genome of Ciona intestinalis and compare them with corresponding gene sets from the genomes of the nematode Chaenorhabditis elegans, the fruit fly Drosophila melanogaster, and the more closely related genomes of vertebrates. Ciona has a set of so-called "prototype" genes for ion channels regulating neuronal excitability, or for neurotransmitter receptors, suggesting that genes responsible for neuronal signaling in mammals appear to have diversified mainly via gene duplications of the more restricted members of ancestral genomes before the ascidian/vertebrate divergence. Most genes responsible for modulation of neuronal excitability and pain sensation are absent from the ascidian genome, suggesting that these genes arose after the divergence of urochordates. In contrast, the divergent genes encoding connexins, transient receptor potential-related channels and chloride channels, channels involved rather in homeostatic control, indicate gene duplication events unique to the ascidian lineage. Because several invertebrate-unique channel genes exist in Ciona genome, the crown group of extant vertebrates not only acquired novel channel genes via gene/genome duplications but also discarded some ancient genes that have persisted in invertebrates. Such genome-wide information of ion channel genes in basal chordates enables us to begin correlating the innovation and remodeling of genes with the adaptation of more recent chordates to their physical environment.
Animals↗
Diffusion model in ion channel gating. Extension to agonist-activated ion channels.
Previously, we described a model which treats ion channel gating as a discrete diffusion problem. In the case of agonist-activated channels at high agonist concentration, the model predicts that the closed lifetime probability density function from single channel recording approximates a power law with an exponent of -3/2 (Millhauser, G. L., E. E. Salpeter, and R. E. Oswald. 1988a. Proc. Natl. Acad. Sci. USA. 85: 1503-1507). This prediction is consistent with distributions derived from a number of ligand-gated channels at high agonist concentration (Millhauser, G. L., E. E. Salpeter, and R. E. Oswald. 1988b. Biophys. J. 54: 1165-1168.) but does not describe the behavior of ion channels at low activator concentrations. We examine here an extension of this model to include an agonist binding step. This extended model is consistent with the closed time distributions generated from the BC3H-1 nicotinic acetylcholine receptor for agonist concentrations varying over three orders of magnitude.
Animals↗
Stretch-inactivated ion channels coexist with stretch-activated ion channels.
Stretch-activated ion channels of animal, plant, bacterial, and fungal cells are implicated in mechanotransduction and osmoregulation. A new class of channel has now been described that is stretch-inactivated. These channels occur in neurons, where they coexist with stretch-activated channels. Both channels are potassium selective. The differing stretch sensitivities of the two channels minimize potassium conductance over an intermediate range of tension, with the consequence that, over this same range, voltage-gated calcium channels are most readily opened. Thus, by setting the relation between membrane tension and transmembrane calcium fluxes, stretch-sensitive potassium channels may participate in the control of calcium-dependent motility in differentiating, regenerating, or migrating neurons.
Animals↗
Conductivity noise in transmembrane ion channels due to ion concentration fluctuations via diffusion.
A Green's function approach is developed from first principles to evaluate the power spectral density of conductance fluctuations caused by ion concentration fluctuations via diffusion in an electrolyte system. This is applied to simple geometric models of transmembrane ion channels to obtain an estimate of the magnitude of ion concentration fluctuation noise in the channel current. Pure polypeptide alamethicin forms stable ion channels with multiple conductance states in artificial phospholipid bilayers isolated onto tips of micropipettes with gigaohm seals. In the single-channel current recorded by voltage-clamp techniques, excess noise was found after the background instrumental noise and the intrinsic Johnson and shot noises were removed. The noise que to ion concentration fluctuations via diffusion was isolated by the dependence of the excess current noise on buffer ion concentration. The magnitude of the concentration fluctuation noise derived from experimental data lies within limits estimated using our simple geometric channel models. Variation of the noise magnitude for alamethicin channels in various conductance states agrees with theoretical prediction.
Alamethicin↗
Influenza A virus can undergo multiple cycles of replication without M2 ion channel activity.
Ion channel proteins are common constituents of cells and have even been identified in some viruses. For example, the M2 protein of influenza A virus has proton ion channel activity that is thought to play an important role in viral replication. Because direct support for this function is lacking, we attempted to generate viruses with defective M2 ion channel activity. Unexpectedly, mutants with apparent loss of M2 ion channel activity by an in vitro assay replicated as efficiently as the wild-type virus in cell culture. We also generated a chimeric mutant containing an M2 protein whose transmembrane domain was replaced with that from the hemagglutinin glycoprotein. This virus replicated reasonably well in cell culture but showed no growth in mice. Finally, a mutant lacking both the transmembrane and cytoplasmic domains of M2 protein grew poorly in cell culture and showed no growth in mice. Thus, influenza A virus can undergo multiple cycles of replication without the M2 transmembrane domain responsible for ion channel activity, although this activity promotes efficient viral replication.
Animals↗
Cell-based assays and instrumentation for screening ion-channel targets.
Ion channels are an important class of drug targets. They comprise the molecular basis for essential physiological functions including fluid secretion, electrolyte balance, bioenergetics and membrane excitability. High-throughput screening for ion-channel function requires sensitive, simple assays and instrumentation that will report ion channel activity in living cells. This article will review relevant assay technologies for ion channels and describe voltage-sensitive probes and instruments based on fluorescence resonance energy transfer (FRET) that enable ion-channel drug discovery.
Journal Article↗
High-throughput screening for ion channel modulators.
Ion channels present a group of targets for major clinical indications, which have been difficult to address due to the lack of suitable rapid but biologically significant methodologies. To address the need for increased throughput in primary screening, the authors have set up a Beckman/Sagian core system to fully automate functional fluorescence-based assays that measure ion channel function. They apply voltage-sensitive fluorescent probes, and the activity of channels is monitored using Aurora's Voltage/Ion Probe Reader (VIPR). The system provides a platform for fully automated high-throughput screening as well as pharmacological characterization of ion channel modulators. The application of voltage-sensitive fluorescence dyes coupled with fluorescence resonance energy transfer is the basis of robust assays, which can be adapted to the study of a variety of ion channels to screen for both inhibitors and activators of voltage-gated and other ion channels.
Automation↗
The rhythmic consequences of ion channel stochasticity.
Ion channels are membrane-spanning proteins with central pores through which ions cross neuronal membranes. The pores through each ion channel flicker between open and closed states, starting and stopping the flow of ions and the electrical current they carry. Hence the current flickers on and off, varying widely on very short time scales. Recent evidence suggests that this noisy current is a source of rhythmic behaviors in neurons. In this review, we begin by providing an illustrative model that links the stochastic flicker of ion channels to neuronal rhythms. The author explores recent experimental work that shows channel flicker is necessary for at least one rhythm that characterizes a class of cortical neurons in vitro. Finally, the author highlights a number of novel studies that link ion channel stochasticity to neuronal rhythmic behaviors in other interesting ways.
Animals↗
Mechanisms of valence selectivity in biological ion channels.
Transmembrane ion channels play a crucial role in the existence of all living organisms. They partition the exterior from the interior of the cell, maintain the proper ionic gradient across the cell membrane and facilitate signaling between cells. To perform these functions, ion channels must be highly selective, allowing some types of ions to pass while blocking the passage of others. Here we review a number of studies that have helped to elucidate the mechanisms by which ion channels discriminate between ions of differing charge, focusing on four channel families as examples: gramicidin, ClC chloride, voltage-gated calcium and potassium channels. The recent availability of high-resolution structural data has meant that the specific inter-atomic interactions responsible for valence selectivity can be pinpointed. Not surprisingly, electrostatic considerations have been shown to play an important role in ion specificity, although many details of the origins of this discrimination remain to be determined.
Calcium Channels↗
Functional genomics of cardiac ion channel genes.
Ion channels are an ensemble of specialized membrane proteins that act in concert to create and modulate the electrical activity of many excitable cells, including cardiac myocytes. Following completion of the sequencing of various genomes, including that of the human, the complete repertoire of ion channel genes has been elucidated for different species. How transcripts issued from this gene collection are expressed and modulated in relation to variable physiological and pathological situations is the subject of functional or physiological genomics. Specialized microarrays (IonChips) comprising probes for the ensemble of ion channel and regulatory genes were developed as an alternative to whole-genome DNA chips. Physiological genomics of cardiac ion channel genes is a growing field that, in combination with genetics, should markedly increase our comprehension of the molecular mechanisms leading to arrhythmias.
Amiodarone↗
Phasic activation and state-dependent inhibition: an explicit solution for a three-state ion channel system.
Ion channels can exist in three broad classes of states: closed (C), open (O), and desensitized or inactivated (I). Many ion channel modulators interact preferentially with one of these states giving rise to use or state dependent effects and often complex interactions with phasic stimulation. Although mathematical descriptions of three-state systems at steady-state or following a single perturbation are well known, a solution to the boundary problem of how such a system interacts with regular phasic perturbations or stimuli has not previously been reported. In physiological systems, ion channels typically experience phasic stimulation and an explicit mathematical description of the interaction between phasic activation and use-dependent modulation within the framework of a three-state system should be useful. Here we present derivations of generalized, recurrent and explicit formulae describing this interaction that allow prediction of the degree of use dependent modulation at any point during a train of repeated stimuli. Each state is defined by two functions of time (y or z) that define the fraction of channels in that state during the alternating stimulation and resting phases, respectively. For a train of repeated stimuli we defined vector Z2n that has coordinates Z2nO and Z(2n)I representing the values for O and I states at the end of the n-th resting phase. We then defined a recurrent relationship, [symbol: see formula]. Therefore, for the steady state: [symbol: see formula], where [symbol: see formula] E is the identity matrix. Matrix and vector elements, Cif, are defined in terms of duration of the repeated stimulation and resting phases and the two sets of six rate constants that describe the three-state model during those two phases. Several conclusions can be deduced from the formulation: (I) in order to determine an occupancy of any state under the cyclic stimulus-rest protocol it is necessary to know at least two occupancy levels-either of the same state but related to different phases of the stimulus protocol or of different states at the same point in the stimulus protocol, for instance: [symbol: see text] (2) the solution Z2n can be approximated by a matrix-exponential function, with the precision of the approximation depending on the interval between stimuli; (3) for all steady-state solutions, the matrix F is such that [symbol: see text] is a zero-matrix. Application of this approach is illustrated using experimentally derived parameters describing desensitization of GABA, receptors and modulation of that process by the anesthetic propofol.
Animals↗
Bis[(benzo-15-crown-5)-15-yl methyl] pimelate forms ion channels in planar lipid bilayer: a novel model ion channel.
Some crown ethers translocate cations across the liposomal membrane either by a carrier mechanism or by forming ion channels. We report formation of ion channels in lipid bilayer membranes by bis[(benzo-15-crown-5)-15-yl methyl] pimelate, a crown ether known to form ion inclusion complexes with alkali metal cations. The channels have characteristic long openings lasting several seconds and a low conductance (4 pS in 500 mM KCl and 2.5 pS in 500 mM NaCl). A model of the crown ether channel formed by stacking of four monomers is proposed. A large database of structural information on crown ethers and their ion inclusion complexes as well as large family of crown ethers with a variety of substitutions in the ring are commercially available. Thus the crown ether channel is an attractive model system to study the role of various chemical moieties in ion conduction which may provide deeper insight into understanding the mechanism(s) of selectivity, ion transport, etc. in biological ion channels.
Cations, Monovalent↗