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PubMed2003

Presynaptic cyclic nucleotide-gated ion channels modulate neurotransmission in the mammalian olfactory bulb.

Cyclic nucleotide-gated channels (CNGCs) on the dendritic cilia of olfactory receptor neurons (ORNs) are critical for sensory transduction in the olfactory system. Do CNGCs also play a role in the axons and/or nerve terminals of ORNs? We find that the cyclic nucleotides cAMP and cGMP can both facilitate and depress synaptic transmission between olfactory nerve fibers and their targets in olfactory bulb glomeruli. Cyclic nucleotides increase intracellular Ca(2+) in ORN terminals and enhance spontaneous transmitter release; at higher concentrations, cyclic nucleotides depress evoked transmission by altering olfactory nerve excitability. Cyclic nucleotides have no effect on transmission or nerve excitability, however, in mice lacking olfactory CNGCs. Taken together, our results identify a novel role for presynaptic CNGCs in modulating neurotransmission.

Animals↗
PubMed2006

A prokaryotic proton-gated ion channel from the nicotinic acetylcholine receptor family.

Ligand-gated ion channels (LGICs) mediate excitatory and inhibitory transmission in the nervous system. Among them, the pentameric or 'Cys-loop' receptors (pLGICs) compose a family that until recently was found in only eukaryotes. Yet a recent genome search identified putative homologues of these proteins in several bacterial species. Here we report the cloning, expression and functional identification of one of these putative homologues from the cyanobacterium Gloeobacter violaceus. It was expressed as a homo-oligomer in HEK 293 cells and Xenopus oocytes, generating a transmembrane cationic channel that is opened by extracellular protons and shows slow kinetics of activation, no desensitization and a single channel conductance of 8 pS. Electron microscopy and cross-linking experiments of the protein fused to the maltose-binding protein and expressed in Escherichia coli are consistent with a homo-pentameric organization. Sequence comparison shows that it possesses a compact structure, with the absence of the amino-terminal helix, the canonical disulphide bridge and the large cytoplasmic domain found in eukaryotic pLGICs. Therefore it embodies a minimal structure required for signal transduction. These data establish the prokaryotic origin of the family. Because Gloeobacter violaceus carries out photosynthesis and proton transport at the cytoplasmic membrane, this new proton-gated ion channel might contribute to adaptation to pH change.

Amino Acid Sequence↗
PubMed2005

Crystallization and preliminary X-ray diffraction analyses of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels.

Cyclic nucleotide-gated (CNG) ion channels play pivotal roles in sensory transduction of retinal and olfactory neurons. The elapid snake toxins pseudechetoxin (PsTx) and pseudecin (Pdc) are the only known protein blockers of CNG channels. These toxins are structurally classified as cysteine-rich secretory proteins and exhibit structural features that are quite distinct from those of other known small peptidic channel blockers. This article describes the crystallization and preliminary X-ray diffraction analyses of these toxins. Crystals of PsTx belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 60.30, b = 61.59, c = 251.69 A, and diffraction data were collected to 2.25 A resolution. Crystals of Pdc also belonged to space group P2(1)2(1)2(1), with similar unit-cell parameters a = 60.71, b = 61.67, c = 251.22 A, and diffraction data were collected to 1.90 A resolution.

Animals↗
PubMed1997

Antisense oligonucleotides induce functional deletion of ligand gated ion channels in cultured neurons and brain explants.

The in situ application of the antisense technique for the study of ligand gated channels is discussed here. Using antisense oligodeoxynucleotides to downregulate a gene of interest means being confronted with a number of choices that will determine the success. These include choosing a target sequence, considering chemical modifications of the oligo as well as its length and estimation of the turnover of the target protein in order to set up the treatment schedule. In this paper a short overview of technical aspects of the antisense approach on primary cultured neurons and brain slice cultures is presented. In addition, the effects of antisense oligos on the expression of neuronal nicotinic acetylcholine receptors and GABA(A) receptors are discussed: Patch-clamp recordings of neurons treated with specific antisense oligos targeted at individual subunits showed a clear downregulation of the expression of native ligand gated channels. Moreover, in a number of experiments novel channel types with altered properties were observed following antisense treatment. Thus, non-targeted channel subunits that remain expressed after antisense deletion, may aggregate to form novel channel types that are normally not present. Alternatively, the translational arrest of a protein may be accompanied by compensatory changes in the synthesis and/or targeting of other channel subunits to the cell surface. The antisense technique enables identification of the functional contribution of individual channel subunits to endogenous channel activity in the central nervous system. As such it paves the way to the elucidation of in vivo channel-subunit composition and channel functions, of post- as well as pre-synaptic ligand gated channel receptors.

Brain↗
PubMed2004

On the fate of skeletal myoblasts in a cardiac environment: down-regulation of voltage-gated ion channels.

We have analysed the voltage-gated ion channels and fusion competence of skeletal muscle myoblasts labelled with green fluorescent protein (GFP) and the membrane dye PKH transplanted into the infarcted myocardium of syngenic rats. After cell transplantation the animals were killed and GFP(+)-PKH(+) myoblasts enzymatically isolated for subsequent studies of ionic currents through voltage-gated sodium, calcium and potassium channels. A down-regulation of all three types of ion channels after engraftment was observed. The fraction of cells with calcium (68%) and sodium channels (65%) declined to zero within 24 h and 1 week, respectively. Down-regulation of potassium currents (90% in control) occurred within 2 weeks to about 30%. Before injection myoblasts expressed predominantly transient outward potassium channels whereas after isolation from the myocardium exclusively rapid delayed rectifier channels. The currents recovered completely between 1 and 6 weeks under cell culture conditions. The down-regulation of ion channels and changes in potassium current kinetics suggest that the environment provided by infarcted myocardium affects expression of voltage-gated ion channels of skeletal myoblasts.

Animals↗
PubMed1994

New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor.

The adenosine-5'-triphosphate (ATP) molecule is an extracellular messenger in neural and non-neural tissues, where it activates several cell-surface-receptor subtypes, including G-protein-coupled receptors and ligand-gated ion channels. ATP-gated channels (termed P2x receptors) have been characterized on smooth muscle cells and autonomic and sensory neurons, where they mediate membrane depolarization and, in some cases, Ca2+ entry. P2x receptors are functionally heterogeneous, but resemble acetylcholine- and serotonin-gated channels with respect to ion selectivity and kinetic parameters of channel gating. We report here that despite such close functional similarities, the deduced sequence of a cloned P2x receptor predicts an unusual subunit structure resembling voltage-insensitive cation channels. Thus, the P2x receptor provides a striking example of convergent evolution, whereby proteins have been fashioned with similar functional properties from subunits having very different structural characteristics. There is sequence similarity between the ATP receptor and RP-2, a gene activated in thymocytes undergoing programmed cell death. RP-2 may encode a receptor for ATP or another metabolite released during apoptosis.

Adenosine Triphosphate↗
PubMed2000

Effects of gaseous anesthetics nitrous oxide and xenon on ligand-gated ion channels. Comparison with isoflurane and ethanol.

BACKGROUND: Ligand-gated ion channels are considered to be potential general anesthetic targets. Although most general anesthetics potentiate the function of gamma-aminobutyric acid receptor type A (GABAA), the gaseous anesthetics nitrous oxide and xenon are reported to have little effect on GABAA receptors but inhibit N-methyl-d-aspartate (NMDA) receptors. To define the spectrum of effects of nitrous oxide and xenon on receptors thought to be important in anesthesia, the authors tested these anesthetics on a variety of recombinant brain receptors. METHODS: The glycine, GABAA, GABA receptor type C (GABAC), NMDA, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate, 5-hydroxytryptamine3 (5-HT3), and nicotinic acetylcholine (nACh) receptors were expressed in Xenopus oocytes and effects of nitrous oxide and xenon, and as equipotent concentrations of isoflurane and ethanol, were studied using the two-electrode voltage clamp. RESULTS: Nitrous oxide (0.58 atmosphere [atm]) and xenon (0.46 atm) exhibited similar effects on various receptors. Glycine and GABAA receptors were potentiated by gaseous anesthetics much less than by isoflurane, whereas nitrous oxide inhibited GABAC receptors. Glutamate receptors were inhibited by gaseous anesthetics more markedly than by isoflurane, but less than by ethanol. NMDA receptors were the most sensitive among glutamate receptors and were inhibited by nitrous oxide by 31%. 5-HT3 receptors were slightly inhibited by nitrous oxide. The nACh receptors were inhibited by gaseous and volatile anesthetics, but ethanol potentiated them. The sensitivity was different between alpha4beta2 and alpha4beta4 nACh receptors; alpha4beta2 receptors were inhibited by nitrous oxide by 39%, whereas alpha4beta4 receptors were inhibited by 7%. The inhibition of NMDA and nACh receptors by nitrous oxide was noncompetitive and was slightly different depending on membrane potentials for NMDA receptors, but not for nACh receptors. CONCLUSIONS: Nitrous oxide and xenon displayed a similar spectrum of receptor actions, but this spectrum is distinct from that of isoflurane or ethanol. These results suggest that NMDA receptors and nACh receptors composed of beta2 subunits are likely targets for nitrous oxide and xenon.

Anesthetics, Inhalation↗
PubMed1995

Modulation of the cGMP-gated ion channel in frog rods by calmodulin and an endogenous inhibitory factor.

1. Outer segment patches excised in the light were used to investigate the effects of exogenous calmodulin and an endogenous inhibitory factor on the cGMP-gated channel of frog rods. 2. Calmodulin shifted to the right the dose-response relation for activation of the channels by 8-Br-cGMP, but did not change the maximum current or the form of the relation. Reversal of this effect by removal of calmodulin was accelerated by brief exposure to saturating [8-Br-cGMP]. Inhibition by calmodulin required calcium and gave as much as a 5-fold decrease in current for an [8-Br-cGMP] functionally comparable to the presumed physiological [cGMP]. 3. Exposure to low [Ca2+]i (tens of nanomolar) appeared to irreversibly remove or inactivate an endogenous channel inhibitory factor from the patches, increasing the current at low [8-Br-cGMP]. Like calmodulin, this factor slowed the voltage-dependent channel-gating kinetics and did not change the maximum current. However, unlike calmodulin, the endogenous factor remained stably associated with the patches at high [Ca2+]i (1 microM), even with exposure to saturating [8-Br-cGMP]. 4. After the low-Ca2+ treatment increased the current, calmodulin reduced the current to about the same level as it had before the low-Ca2+ treatment, giving a larger fractional suppression. Furthermore, patches with high initial sensitivity to 8-Br-cGMP had small low-Ca2+ effects and large calmodulin effects, while the reverse was true for patches with low initial agonist sensitivity. 5. Application of trypsin to the intracellular surface of the patch prevented the responses to calmodulin and to low [Ca2+]i, suggesting involvement of a cytoplasmic portion of the channel. However, trypsin also reduced the total agonist-induced patch current. 6. Our results are consistent with a model in which calmodulin and an endogenous calcium-binding protein compete for the same site, inhibiting channel opening or cGMP binding. The tight association of the endogenous factor with the channel even at relatively low [Ca2+]i suggests that in the transducing rod it may inhibit the channels most of the time in darkness and in dim light, preventing any potential inhibitory effects of calmodulin. The endogenous factor would be expected to leave the channel only in bright or prolonged light, when the [Ca2+]i is thought to be very low.

Animals↗
PubMed1995

Permeability and interaction of Ca2+ with cGMP-gated ion channels differ in retinal rod and cone photoreceptors.

We studied the ionic permeability of cGMP-dependent currents in membrane patches detached from the outer segment of retinal cone and rod photoreceptors. Reversal potentials measured in membranes exposed to symmetric Na+ but with varying cytoplasmic Ca2+ concentrations reveal that the permeability ratio, PCa/PNa, is higher in the cGMP-gated channels of cones (7.6 +/- 0.8) than in those of rods (3.1 +/- 1.0). Ca2+ blocks both channels in a voltage-dependent manner. At any Ca2+ concentration, the channel block is maximal near the ionic reversal potential. The maximal block is essentially identical in channels of cones and rods with respect to its extent and voltage and Ca2+ dependence. The Ca2+ block is relieved by voltage, but the features of this relief differ markedly between rods and cones. Whereas the Boltzmann distribution function describes the relief of block by hyperpolarizing voltages, any given voltage is more effective in relieving the Ca2+ block in cones than in rods. Similarly, depolarizing voltages more effectively relieve Ca2+ block in cones than in rods. Our results suggest that channels contain two binding sites for Ca2+, one of which is similar in the two receptor types. The second site either interacts more strongly with Ca2+ than the first one or it is located differently in the membrane, so as to be less sensitive to membrane voltage. The channels in rods and cones differ in the features of this second site. The difference in Ca2+ permeability between the channels is likely to result in light-dependent changes in cytoplasmic Ca2+ concentration that are larger and faster in cones than in rods. The functional differences between channels, therefore, may be critically important in explaining the differences in the phototransduction signal of the two photoreceptor types.

Ambystoma↗
PubMed2000

Modification of cyclic nucleotide-gated ion channels by ultraviolet light.

We irradiated cyclic nucleotide-gated ion channels in situ with ultraviolet light to probe the role of aromatic residues in ion channel function. UV light reduced the current through excised membrane patches from Xenopus oocytes expressing the alpha subunit of bovine retinal cyclic nucleotide-gated channels irreversibly, a result consistent with permanent covalent modification of channel amino acids by UV light. The magnitude of the current reduction depended only on the total photon dose delivered to the patches, and not on the intensity of the exciting light, indicating that the functionally important photochemical modification(s) occurred from an excited state reached by a one-photon absorption process. The wavelength dependence of the channels' UV light sensitivity (the action spectrum) was quantitatively consistent with the absorption spectrum of tryptophan, with a small component at long wavelengths, possibly due to cystine absorption. This spectral analysis suggests that UV light reduced the currents at most wavelengths studied by modifying one or more "target" tryptophans in the channels. Comparison of the channels' action spectrum to the absorption spectrum of tryptophan in various solvents suggests that the UV light targets are in a water-like chemical environment. Experiments on mutant channels indicated that the UV light sensitivity of wild-type channels was not conferred exclusively by any one of the 10 tryptophan residues in a subunit. The similarity in the dose dependences of channel current reduction and tryptophan photolysis in solution suggests that photochemical modification of a small number of tryptophan targets in the channels is sufficient to decrease the currents.

Animals↗
PubMed2004

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↗
PubMed1996

P2X receptors: a third major class of ligand-gated ion channels.

Three classes of ligand-gated ion channels are defined by their molecular architecture. The first embraces nicotinic, 5-HT3, glycine and GABA receptors. The second class contains the glutamate receptors-AMPA, kainate and NMDA types. The third class is the P2X receptors for ATP. Current knowledge of the structure of these channels is reviewed, and set beside what is known of their basic functional properties. The aim of this paper is to consider how our more complete understanding of the first two classes of channels might be helpful in forming a molecular picture of P2X receptor function.

Adenosine Triphosphate↗
PubMed2001

ATP-gated ion channel P2X(3) is increased in human inflammatory bowel disease.

P2X(3) is a novel ATP-gated cation channel that is selectively expressed by small-diameter sensory neurones in rodents, and may play a role in nociception by binding ATP released from damaged or inflamed tissues. We have studied, for the first time, P2X(3) immunoreactivity in human inflammatory bowel disease, using Western blotting and immunohistochemistry. A major 66-kDa specific protein was found by Western blotting in all colon extracts. In the inflamed group there was a significant two-fold increase in the relative optical density of the 66-kDa band (21.2 +/- 3.1; n=8) compared to controls (11.4 +/- 3.7; n=8; P=0.009). In the control colon, P2X(3)-immunoreactive neurones were scattered throughout the myenteric and submucosal plexuses, with some neurones showing immunopositive axons/dendrites. The pattern of immunostaining was similar to the neuronal marker peripherin. In general, the intensity of the staining was greater in myenteric than submucosal neurones. The number of P2X(3)-immunoreactive neurones was significantly increased in the myenteric plexus of inflamed colon compared to controls (n=13; P=0.01). In humans, unlike rodents, P2X(3) is thus not restricted to sensory neurones. Increased P2X(3) in inflamed intestine suggests a potential role in dysmotility and pain, for which it represents a new therapeutic target.

Adenosine Triphosphate↗
PubMed2006

A K+-selective cGMP-gated ion channel controls chemosensation of sperm.

Eggs attract sperm by chemical factors, a process called chemotaxis. Sperm from marine invertebrates use cGMP signalling to transduce incident chemoattractants into changes in the Ca2+ concentration in the flagellum, which control the swimming behaviour during chemotaxis. The signalling pathway downstream of the synthesis of cGMP by a guanylyl cyclase is ill-defined. In particular, the ion channels that are involved in Ca2+ influx and their mechanisms of gating are not known. Using rapid voltage-sensitive dyes and kinetic techniques, we record the voltage response that is evoked by the chemoattractant in sperm from the sea urchin Arbacia punctulata. We show that the chemoattractant evokes a brief hyperpolarization followed by a sustained depolarization. The hyperpolarization is caused by the opening of K+-selective cyclic-nucleotide-gated (CNG) channels in the flagellum. Ca2+ influx commences at the onset of recovery from hyperpolarization. The voltage threshold of Ca2+ entry indicates the involvement of low-voltage-activated Ca(v) channels. These results establish a model of chemosensory transduction in sperm whereby a cGMP-induced hyperpolarization opens Ca(v) channels by a 'recovery-from-inactivation' mechanism and unveil an evolutionary kinship between transduction mechanisms in sperm and photoreceptors.

Animals↗
PubMed2005

Charge selectivity of the Cys-loop family of ligand-gated ion channels.

The determinants of charge selectivity of the Cys-loop family of ligand-gated ion channels have been studied for more than a decade. The investigations have mainly covered homomeric receptors e.g. the nicotinic acetylcholine receptor alpha7, the glycine receptor alpha1 and the serotonin receptor 5-HT(3A). Only recently, the determinants of charge selectivity of heteromeric receptors have been addressed for the GABA(A) receptor alpha2beta3gamma2. For all receptor subtypes, the selectivity determinants have been located to an intracellular linker between transmembrane domains M1 and M2. Two features of the M1-M2 linker appear to control ion selectivity. A central role for charged amino acid residues in selectivity has been almost universally observed. Furthermore, recent studies point to an important role of the size of the narrowest constriction in the pore. In the present review, these determinants of charge selectivity of the Cys-loop family of ligand-gated ion channels will be discussed in detail.

Animals↗
PubMed2003

International Union of Pharmacology. XLIII. Compendium of voltage-gated ion channels: transient receptor potential channels.

The transient receptor potential (TRP) proteins are six transmembrane-containing subunits that combine to form cation-selective ion channels. TRP channels are present in yeast, Drosophila, Caenorhabditis elegans, and mammals. They are widely distributed and sense local changes in stimuli ranging from light to temperature and osmolarity. Mammals contain at least 22 distinct genes encoding these ion channels. This summary article presents an overview of the molecular relationships among the TRP channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels. The complete Compendium, including data tables for each member of the TRP channel family, can be found at http://www.iuphar-db.org/iuphar-ic/.

Animals↗
PubMed2005

Effects of hydrophobic mismatch and spontaneous curvature on ion channel gating with a hinge.

We analyze the energetics of an ion-channel gating, focusing on effects of hydrophobic mismatch between the channel protein and the nearby lipid molecules, spontaneous curvature of monolayers, and thickness change of membranes. For the analysis we consider recently proposed open and closed conformations of a potassium channel which has a gating hinge, using the elastic continuum model of membranes. Gating energy, defined as the difference of deformation free energies for open and closed conformations, is quantitatively evaluated for various values of moduli related to the deformation of membranes and spontaneous curvature of monolayer imposing a strong hydrophobic boundary condition. We show that the gating mechanism with a hinge can work successfully even in a continuum model that considers hydrophobic mismatch and spontaneous curvature. When the energy cost for the thickness change of the membrane is neglected, the surface tension is not necessarily strong enough to open the channel. Otherwise, a relatively strong surface tension is required to open the channel.

Animals↗
PubMed1997

Properties of glutamate-gated ion channels in horizontal cells of the perch retina.

The effect of two different concentrations of L-glutamate and kainate on the gating kinetics of amino acid-sensitive non-NMDA channels were studied in cultured teleost retinal horizontal cells by single-channel recording and by noise analysis of whole-cell currents. When the glutamate agonist kainate was applied clearly parabolic mean-variance relations of whole-cell membrane currents (up to 3000 pA) indicated that this agonist was acting on one type of channels with a conductance of 5-10 pS. The cells were less sensitive when L-glutamate was used as the agonist and in most cases whole-cell currents amounted to less than 200 pA. The mean-variance relation of glutamate induced currents was complex, indicating that more than one type of channel opening could be involved. Power spectra of whole-cell currents were fitted with two Lorentzians with time constants of approx. 1 and 5-20 msec. Effects on amplitudes and time constants of agonist concentrations are demonstrated. Two categories of unitary events with mean open times of approx. 1 and 7 msec and conductances of approx. 7 and 12 pS, respectively, were obtained in single-channel recordings from cell-attached patches at different concentrations of glutamate in the pipette.

Animals↗