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At least 109 records · Page 6Linked to original sources
PubMed1992

Glutamate-gated ion channels in the brain. Genetic mechanism for generating molecular and functional diversity.

L-glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system. Most cells are responsive to glutamate which activates cation channels with different pharmacological, kinetic, and ion permeability properties. These channels play important roles in neurotransmission, memory acquisition as well as acute and chronic disorders of the brain. The present report summarizes recent knowledge on AMPA (a-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid) receptors which mediate fast synaptic neurotransmission.

Animals↗
PubMed1995

Platelet-derived growth factor receptor is a novel modulator of type A gamma-aminobutyric acid-gated ion channels.

Platelet-derived growth factor (PDGF) and PDGF receptors (PDGFRs) are ubiquitously expressed in the mammalian central nervous system, where they exert trophic actions on both neuronal and glial cells. However, the acute actions of PDGF on synaptic transmission are unknown. We report a novel regulatory action of PDGF/PDGFR. Activation of PDGFRs inhibited the function of native type A gamma-aminobutyric acid (GABAA) receptors (GABAA-RS) in rat hippocampal CA1 pyramidal neurons and mouse brain membrane vesicles. The mechanism of this inhibition was studied with a panel of mutant PDGFRS-beta coexpressed with cloned human GABAA-Rs in Xenopus oocytes. These experiments revealed that phospholipase C-gamma is the protein that relays the inhibitory signal from PDGFRS to GABAA-Rs. Experiments with microinjected EGTA and inositol-1, 3, 4-triphosphate demonstrated that inhibition of GABAA-Rs depended on a phospholipase C-gamma-mediated increase in intracellular Ca(2+)-levels. The PDGFR-induced inhibitory effect was independent of the subunit composition of GABAA-RS. Moreover, GABAA-RS composed of alpha 1 beta 1 S409A subunits, which do not contain any known protein kinase C phosphorylation sites, were inhibited by PDGF to the same extent as wild-type GABAA-RS. Inhibitors of protein kinase C, CA2+/calmodulin-dependent protein kinase II, calcineurin, and tyrosine phosphatases did not affect the modulatory actions of PDGFR. In conclusion, our results suggest that PDGFRs exert potent modulatory actions on GABAA-R-dependent inhibitory synaptic transmission. These regulatory actions of PDGF could play important roles in the function of the mammalian central nervous system during physiological and pathophysiological conditions.

Animals↗
PubMed1997

Ligand-gated ion channel subunit partnerships: GABAA receptor alpha6 subunit gene inactivation inhibits delta subunit expression.

Cerebellar granule cells express six GABAA receptor subunits abundantly (alpha1, alpha6, beta2, beta3, gamma2, and delta) and assemble various pentameric receptor subtypes with unknown subunit compositions; however, the rules guiding receptor subunit assembly are unclear. Here, removal of intact alpha6 protein from cerebellar granule cells allowed perturbations in other subunit levels to be studied. Exon 8 of the mouse alpha6 subunit gene was disrupted by homologous recombination. In alpha6 -/- granule cells, the delta subunit was selectively degraded as seen by immunoprecipitation, immunocytochemistry, and immunoblot analysis with delta subunit-specific antibodies. The delta subunit mRNA was present at wild-type levels in the mutant granule cells, indicating a post-translational loss of the delta subunit. These results provide genetic evidence for a specific association between the alpha6 and delta subunits. Because in alpha6 -/- neurons the remaining alpha1, beta2/3, and gamma2 subunits cannot rescue the delta subunit, certain potential subunit combinations may not be found in wild-type cells.

Animals↗
PubMed1997

Drosophila serotonin transporters have voltage-dependent uptake coupled to a serotonin-gated ion channel.

Serotonin (5HT) transporters (SERTs) couple to existing ion gradients to transport 5HT into presynaptic terminals. In mammalian SERTs, the transport cycle is reported as electroneutral, with a translocation of zero net charge, and 5HT uptake is independent of membrane voltage. Yet mammalian SERTs exhibit 5HT-induced currents, and Drosophila SERTs (dSERTs) show voltage-dependent uptake. Thus, the relationship between uptake and current remains controversial; furthermore, the number of 5HT molecules translocated per ion channel event is unknown. To investigate this, we have used heterologous expression of cloned dSERTs to measure 5HT flux and dSERT currents concurrently under voltage clamp, and we have used fluctuation analysis to measure the size of the elementary ionic events in the same cells. RNA-injected Xenopus oocytes accumulate 5HT, and paroxetine or desipramine inhibit this uptake. RNA-injected oocytes also display paroxetine-sensitive 5HT-induced currents and 5HT-independent leak currents. Na replacement decreases the uptake and the induced currents. 5HT-induced current and 5HT uptake both increase at negative potentials, where 5HT carries approximately 5% of the induced current. Recently, several groups have reported similar phenomena for other transporters, in which transmitter-induced currents exceed the predictions of coupled transport. We now provide evidence that in dSERT, approximately 500 5HT molecules are translocated per channel opening, which, at -20 mV, carries approximately 10,000 electronic charges. These data support a model in which 500 SERT cycles occur for each 5HT-induced channel opening or a model in which 500 5HT molecules and 10,000 electronic charges pass through a common pore.

Animals↗
PubMed2003

Water, proton transfer, and hydrogen bonding in ion channel gating.

Several types of ion channels, the proteins responsible for the transport of ions across cell membranes, are described. Those of most interest are responsible for the functioning of nerve cells, and are voltage gated. Here, we propose a model for voltage gating that depends on proton transport. There are also channels that are proton-gated, of which some are bacterial. For one, a structure is known in the closed state, the KcsA channel (1). The proton gating of this channel suggests a part of the overall gating model we propose. Other bacterial channel structures are also known, but none that are relevant here, at least in one case because it appears to be in the open state. Voltage-gated channels of eukaryotes open in response to the depolarization of the membrane. It appears that there is some analogy in the structure of the voltage-gated channels to the structure of the smaller bacterial channels, including the one that is proton-gated. There is also significant experimental work in the literature on the nature of the gating current, a capacitative current that precedes the opening of the channel. The model we provide is based on the known properties of channels; in this model, voltage gating consists of three stages: first, the tunneling of a proton as depolarization begins, to initiate the sequence; second, proton transport along a sequence of (mostly) arginines, which is postulated to bring a proton to a critical gating region, where, third, a strong, short, hydrogen bond is weakened by the added proton, allowing the four domains to separate. The separation of the domains allows ions to pass through, and thus constitutes the opening of the channel. An analogy to the behavior of ferroelectrics is also described.

Animals↗
PubMed2006

Engineering light-gated ion channels.

Ion channels are gated by a variety of stimuli, including ligands, voltage, membrane tension, temperature, and even light. Natural gates can be altered and augmented using synthetic chemistry and molecular biology to develop channels with completely new functional properties. Light-sensitive channels are particularly attractive because optical manipulation offers a high degree of spatial and temporal control. Over the last few decades, several channels have been successfully rendered responsive to light, including the nicotinic acetylcholine receptor, gramicidin A, a voltage-gated potassium channel, an ionotropic glutamate receptor, alpha-hemolysin, and a mechanosensitive channel. Very recently, naturally occurring light-gated cation channels have been discovered. This review covers the molecular principles that guide the engineering of light-gated ion channels for applications in biology and medicine.

Animals↗
PubMed1996

Subunit interactions in the activation of cyclic nucleotide-gated ion channels.

Cyclic nucleotide-gated (CNG) ion channels of retinal photoreceptors and olfactory neurons are multimeric proteins of unknown stoichiometry. To investigate the subunit interactions that occur during CNG channel activation, we have used tandem cDNA constructs of the rod CNG channel to generate heteromultimeric channels composed of wild-type and mutant subunits. We introduced point mutations that affect channel activation: 1) D604M, which alters the relative ability of agonists to promote the allosteric conformational change(s) associated with channel opening, and 2) T560A, which primarily affects the initial binding affinity for cGMP, and to a lesser extent, the allosteric transition. At saturating concentrations of agonist, heteromultimeric channels were intermediate between wild-type and mutant homomultimers in agonist efficacy and apparent affinity for cGMP, cIMP, and cAMP, consistent with a model for the allosteric transition involving a concerted conformational change in all of the channel subunits. Results were also consistent with a model involving independent transitions in two or three, but not one or four, of the channel subunits. The behavior of the heterodimers implies that the channel stoichiometry is some multiple of 2 and is consistent with a tetrameric quaternary structure for the functional channel complex. Steady-state dose-response relations for homomultimeric and heteromultimeric channels were well fit by a Monod, Wyman, and Changeux model with a concerted allosteric opening transition stabilized by binding of agonist.

Allosteric Site↗
PubMed2006

Identification of two critical residues within the Cys-loop sequence that determine fast-gating kinetics in a pentameric ligand-gated ion channel.

Pentameric ligand-gated ion channels (LGICs) are fast-gating receptors, represented by cationic nicotinic acetylcholine (nAChR) and serotonin (5HT3R) receptors, and by anionic GABA and glycine (GlyR) receptors. Because of a highly conserved sequence of 13 amino acids flanked by two canonical cysteine residues shared by all members of the family, these receptors are also known as the Cys-loop family. These receptors are allosteric transmembrane proteins made of five identical (or not) subunits arranged (pseudo) symmetrically around a central ion pore in the membrane. In nAChR, upon ACh binding, the receptor interconverts into discrete allosteric states, with each state corresponding to a different physiological state: resting (closed), active (open), and desensitized (closed).

Amino Acid Sequence↗
PubMed1995

Actinobacillus actinomycetemcomitans leukotoxin forms large conductance, voltage-gated ion channels when incorporated into planar lipid bilayers.

Actinobacillus actinomycetemcomitans leukotoxin is a member of the bacterial RTX (repeats in toxin) toxin family, produced by a diverse group of Gram-negative pathogens. Members of this group of toxins, although similar in sequence, differ in target cell specificity with Actinobacillus actinomycetemcomitans leukotoxin demonstrating a unique species- and cell-type specificity. Purified A. actinomycetemcomitans leukotoxin added to pre-formed POPE/POPS lipid bilayers showed no spontaneous incorporation (to concentrations of 250 ng/ml). Reproducible channel activity was seen when the bilayer was reformed from lipid monolayers in the presence of toxin (50 ng/ml) in one of the aqueous chambers. Control experiments with heat-inactivated toxin did not display channel activity under the same experimental conditions. The channel behavior showed a complex pattern of multiple conductance levels of 118, 262 and 406 pS in solutions containing 0.140 M NaCl. The first two states showed voltage-dependent channel gating with approximately equal but opposite apparent gating charges of 1.4 electrons. A model accounting for the multiple conducting states and gating properties is presented.

Aggregatibacter actinomycetemcomitans↗
PubMed2005

Voltage-gated ion channels.

Voltage-dependent ion channels are membrane proteins that conduct ions at high rates regulated by the voltage across the membrane. They play a fundamental role in the generation and propagation of the nerve impulse and in cell homeostasis. The voltage sensor is a region of the protein bearing charged amino acids that relocate upon changes in the membrane electric field. The movement of the sensor initiates a conformational change in the gate of the conducting pathway thus controlling the flow of ions. Major advances in molecular biology, spectroscopy, and structural techniques are delineating the main features and possible structural changes that account for the function of voltage-dependent channels.

Animals↗
PubMed1995

Family of cyclic nucleotide gated ion channels.

A variety of different cyclic nucleotide gated ion channels have recently been identified using molecular cloning and electrophysiological techniques. Current research is focussed on the specific molecular determinants that endow these channels with their distinctive character of gating, selectivity and modulation. In some cases, it has been possible to identify the specific physiological roles of different cyclic nucleotide gated channels. Their interactions with Ca2+ and calmodulin are particularly important, and determine the specific functions these channels subserve in distinct cells.

Animals↗
PubMed1996

Cyclic nucleotide-gated ion channels: an extended family with diverse functions.

An ion channel directly activated by cGMP was first discovered about ten years ago. Since then, a number of ion channels with the same property (cyclic nucleotide-activated channels) have been reported that are involved in a variety of cell functions. In addition, other channels have been found that are not primarily controlled by cyclic nucleotides but are modulated by them (cyclic nucleotide-modulated channels). These channels likewise have diverse functions and tissue distributions. Both channel classes are reviewed here. Coverage includes the cyclic-nucleotide binding site on these channels, ion permeation, pharmacological blockers, channel gating and modulation, and physiological functions of the channels.

Amino Acid Sequence↗
PubMed1995

Phosphorylation controls conductance of 5-HT3 receptor ligand-gated ion channels.

Single 5-HT3 receptor-gated ion channels were recorded in cell-attached and excised membrane patches of mouse N1E-115 neuroblastoma cells. In cell-attached patches 5-HT3 receptor-gated ion channels show distinct conductance levels of 27, 18, 11 and 6 pS. Patch excision, buffering of intracellular Ca2+ by BAPTA and inhibition of protein kinase activity by staurosporine increase the probability of occurrence of the 6 pS level and decrease that of the 27 pS level. Conversely, patch cramming and stimulation of protein kinase activity by the phorbol ester PMA enhance the probability of occurrence of the 27 pS level and decrease that of low conductance levels. We conclude that phosphorylation controls the conductance of 5-HT3 receptor ligand-gated ion channels. Control of channel conductance may prove an additional mechanism in the modulation of synaptic efficacy.

Animals↗
PubMed1998

Co-expression of P2X1 and P2X5 receptor subunits reveals a novel ATP-gated ion channel.

P2X receptors are a family of ion channels gated by extracellular ATP. Each member of the family can form functional homomeric channels, but only P2X2 and P2X3 have been shown to combine to form a unique heteromeric channel. Data from in situ hybridization studies suggest that P2X1 and P2X5 may also co-assemble. In this study, we tested this hypothesis by expressing recombinant P2X1 and P2X5 receptor subunits either individually or together in human embryonic kidney 293 cells. In cells expressing the homomeric P2X1 receptor, 30 microM alpha,beta-methylene ATP (alpha,beta-me-ATP) evoked robust currents that completely desensitized in less than 1 sec, whereas alpha,beta-me-ATP failed to evoke current in cells expressing the homomeric P2X5 receptor. By contrast, alpha, beta-me-ATP evoked biphasic currents with a pronounced nondesensitizing plateau phase in cells that co-expressed both subunits. Further, the EC50 for alpha,beta-me-ATP was greater in cells expressing both P2X1 and P2X5 than in cells expressing P2X1 alone (5 and 1.6 microM, respectively). Heteromeric assembly was confirmed using a co-immunoprecipitation assay of epitope-tagged P2X1 and P2X5 subunits. In summary, this study provides biochemical and functional evidence of a novel channel formed by P2X subunit heteropolymerization. This finding suggests that heteromeric subunit assembly constitutes an important mechanism for generating functional diversity of ATP-mediated responses.

Adenosine Triphosphate↗
PubMed2000

Effects of mutation at a conserved N-glycosylation site in the bovine retinal cyclic nucleotide-gated ion channel.

Bovine retinal cyclic nucleotide-gated (CNG) ion channel contains an evolutionary conserved N-glycosylation site in the external loop between the fifth transmembrane segment and the pore-forming region. The effect of tunicamycin treatment and the site-specific mutation suggested that the channel is glycosylated when expressed in Xenopus oocytes. To test the role of glycosylation in this channel, N-glycosylation was abolished by mutation, and the detailed permeation and the gating characteristics of the mutant channel were investigated. The charge contribution turned out to be detectable, although the mutation of the N-glycosylation site did not affect expression and functionality of the CNG channel in oocytes.

Amino Acid Sequence↗
PubMed1999

Molecular cloning of a putative cyclic nucleotide-gated ion channel cDNA from Limulus polyphemus.

Cyclic nucleotide-gated channels have been proposed to mediate the electrical response to light in the ventral photoreceptor cells of the horseshoe crab, Limulus polyphemus. However, a cyclic nucleotide-gated channel has not been identified from Limulus. We have cloned a putative full-length cyclic nucleotide-gated channel cDNA by screening cDNA libraries constructed from Limulus brain using a probe developed from Limulus ventral eye nerves. The putative full-length cDNA was derived from two overlapping partial cDNA clones. The open reading frame encodes 905 amino acids; the sequence shows 44% identity to that of the alpha subunit of the bovine rod cyclic GMP-gated channel over the region containing the transmembrane domains and the cyclic nucleotide binding domain. This Limulus channel has a novel C-terminal region of approximately 200 amino acids, containing three putative Src homology domain 3 binding motifs and a putative coiled-coil domain. The possibility that this cloned channel is the same as that detected previously in excised patches from the photoreceptive membrane of Limulus ventral photoreceptors is discussed in terms of its sequence and its expression in the ventral eye nerves.

Animals↗
PubMed2002

Water as a structural element in a channel: gating in the Kcsa channel, and implications for voltage-gated ion channels.

Water is becoming understood as a structural element in proteins. Here we are concerned with one particular type of protein, ion channels. The S. Lividans KcsA K(+) channel, the X-ray structure of which is known, is gated by protons (i.e, by a drop in pH). Ab initio calculations suggest that an H(5)O(2) group, partially charged, connects the E118 residues in the gating region, when the four residues have a -2 net charge, but that the hydrogen bonding is not strong enough to do this when the charge becomes -1. The H(5)O(2) group would block the channel, in the -2 state, and prevent motion of the four transmembrane (TM) segments of the protein, by binding them. With the weaker bond in the -1 state, the TM segments would be able to separate (as they have been found to do experimentally, opening the channel. Voltage gated channels have four additional TM segments for each of the four domains of the channel protein. These appear to allow motion of protons; in fact there is evidence that the initial step in gating must be the transfer of a proton. We have earlier shown that the transfer of a single proton between two methylamines under the influence of a field is possible, as proton tunneling. Subsequent steps are hypothesized to result from four proton transfer cascades of about three protons each, triggered by the initial proton transfer. We suggest that the extra 4 TM segments of the voltage gated channel act as a voltage to proton-current transducer. Water, held by hydrogen bonds, is also suggested as the source of the accessibility data found with MTS reagents, based largely on simulations, our earlier Monte Carlo simulations as well as molecular dynamics studies reported by others. These waters may also play a structural role in the protein.

Bacterial Proteins↗
PubMed2006

Conserved motifs in voltage-sensing and pore-forming modules of voltage-gated ion channel proteins.

Voltage-gated ion channels (VGCs) mediate selective diffusion of ions across cell membranes to enable many vital cellular processes. Three-dimensional structure data are lacking for VGC proteins; hence, to better understand their function, there is a need to identify the conserved motifs using sequence analysis methods. In this study, we have used a profile-to-profile alignment method to identify several new conserved motifs specific to each transmembrane segment (TMS) of the voltage-sensing and the pore-forming modules of Ca2+, Na+, and K+ channel subfamilies. For Ca2+ and Na+, the functional theme of motif conservation is similar in all segments while they differ with those of the K+ channel proteins. Nevertheless, the conservation is strikingly similar in the S4 segment of the voltage-sensing module across all subfamilies. In each subfamily and for each TMS, we have identified conserved motifs/residues and correlated their functional significance and disease associations in human, using mutational data from the literature.

Amino Acid Motifs↗