SEARCH · Search PubMed

Results for “Ion Channel Gating”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 307 records · Page 17Linked to original sources

PubMedDate not supplied

Expression of ligand-gated ion channels with the Semliki Forest virus expression system.

Two types of ligand-gated ion channels were expressed with the Semliki Forest virus (SFV) expression system. The cDNAs for mouse serotonin 5-HT3 receptor and rat and human purinoreceptor P2x subtypes were introduced into the pSFV1 vector. In vitro transcribed RNAs were coelectroporated with pSFV-Helper2 RNA into BHK cells, where in vivo packaging resulted in high titer SFV-5-HT3 and SFV-P2x virus stocks. Infection of BHK, CHO and RIN cells resulted in high-level expression of recombinant receptors. Saturation binding analysis indicated the presence of more than 3 x 10(6) 5-HT3 receptors per cell. Binding studies on isolated membranes yielded from 10 to 60 pmol of either 5-HT3 or P2x receptor per mg protein. Functional responses to the P2x receptors were demonstrated in SFV-infected CHO cells by Ca2+ mobilization or by 45Ca2+ influx. High amplitude electrophysiological responses were also detected for both SFV-5-HT3 and SFV-P2x infected CHO cells in whole-cell patch clamp recordings. To facilitate the purification procedure of SFV-expressed recombinant receptors a histidine tag was introduced at the C-terminus of the 5-HT3 receptor. This 5-HT3His receptor showed high levels of expression, specific binding and high amplitude electrophysiological responses. For large scale expression the BHK cells were adapted to suspension culture and were efficiently infected in a 11.5 liter fermentor culture with SFV-5-HT3His resulting in high-level expression, 52 pmol receptor per mg protein corresponding to 3.2 x 10(6) receptors per cell.

Adenosine Triphosphate↗

PubMed2001

Capsaicin receptor VR1 and ATP-gated ion channel P2X3 in human urinary bladder.

OBJECTIVES: To determine the presence, distribution and molecular forms of the vanilloid receptor VR1, and confirm the presence and distribution of the ATP-gated ion channel P2X3 in the human urinary bladder. Materials and methods Normal urinary bladder tissues were obtained at postmortem from four subjects. Eight urinary bladder biopsies were also taken from patients with detrusor hyper-reflexia treated with intravesical resiniferatoxin. The specimens were studied using affinity-purified specific antibodies to VR1 and P2X3 by Western blotting and immunocytochemistry, and compared with immunostaining using antibodies to the pan-neuronal marker PGP 9.5 and Schwann cell marker S-100. RESULTS: VR1- and P2X3-immunoreactive fine nerve fibres were scattered throughout the suburothelium of the normal bladder and cystoscopic biopsies, and traversed the muscle layer. They had a similar distribution to PGP 9.5-immunoreactive fibres, but there were fewer, suggesting localization in subsets of axons. Western blot studies showed an expected 100-kDa VR1 protein and a P2X3-immunoreactive 66-kDa protein. Conclusion VR1 and P2X3 are present in the human urinary bladder and may contribute to distinct pathophysiological states of bladder overactivity, in accord with their differential expression in sensory neurones. Intravesical vanilloids act via VR1 and are effective in the treatment of detrusor hyper-reflexia. P2X3 may represent a selective therapeutic target for other causes of overactive bladder.

Adenosine Triphosphate↗

PubMed2006

Electrostatic couplings in OmpA ion-channel gating suggest a mechanism for pore opening.

The molecular forces that drive structural transitions between the open and closed states of channels and transporters are not well understood. The gate of the OmpA channel is formed by the central Glu52-Arg138 salt bridge, which can open to form alternate ion pairs with Lys82 and Glu128. To gain deeper insight into the channel-opening mechanism, we measured interaction energies between the relevant side chains by double-mutant cycle analysis and correlated these with the channel activities of corresponding point mutants. The closed central salt bridge has a strong interaction energy of -5.6 kcal mol(-1), which can be broken by forming the open-state salt bridge Glu52-Lys82 (DeltaDeltaG(Inter) = -3.5 kcal mol(-1)) and a weak interaction between Arg138 and Glu128 (DeltaDeltaG(Inter) = -0.6 kcal mol(-1)). A covalent disulfide bond in place of the central salt bridge completely blocks the channel. Growth assays indicate that this gating mechanism could physiologically contribute to the osmoprotection of Escherichia coli cells from environmental stress.

Amino Acids↗

PubMed1998

Approaches to proving there are general anesthetic sites on ligand gated ion channels.

(1) There are at least two broad classes of general anesthetic action on the anesthetic-sensitive ligand gated superfamily of ion channels. (2) First, some channels may be inhibited upon opening. Pharmacology, kinetics and site directed mutagenesis all suggest that inhibition is mediated by a site on the acetylcholine receptor probably located in the channel lumen. (3) Second, the agonist's concentration response curve may be shifted to the left without affecting the maximum response. (4) This effect does not saturate with anesthetic concentration and might involve partial occupancy of many low affinity sites, mechanism consistent with the observation that the conformation changes accompanying channel gating involve most structural features of the receptor and its surrounding environment.

Anesthetics, General↗

PubMed1991

Modulation of a gated ion channel admittance in lipid bilayer membranes.

A future class of amperometric biosensors may utilize gated ion channels such as acetylcholine and glutamate receptors as chemical detection components. In this study, bilayer lipid membranes containing voltage-dependent anion channels (VDAC) were used to model an ion-channel-based biosensor which could continuously monitor AC amperometric changes resulting from induced changes in channel conductance. The in-phase and quadrature components of the induced alternating membrane current were monitored as a function of the applied DC offset voltage which was superimposed on the sinusoidal test voltage. The accuracy and sensitivity of the AC-measured VDAC response was dependent on the magnitude of the AC test voltage relative to the DC offset necessary for channel closure. The VDAC channel appears to be a suitable model protein for AC impedance-based biosensor fabrication.

Biosensing Techniques↗

PubMed1995

Mutagenic analysis of double-stranded RNA adenosine deaminase, a candidate enzyme for RNA editing of glutamate-gated ion channel transcripts.

Mutagenic analysis of the substrate binding and catalytic domains of double-stranded RNA (dsRNA) adenosine deaminase (DRADA) was carried out. This nuclear enzyme is likely to be involved in the RNA editing of glutamate-gated ion channels that are essential for fast excitatory neurotransmission in mammalian brain. The deletion of the first or the third of the three dsRNA binding motifs within the substrate binding domain dramatically decreases enzyme activity, whereas the second motif seems to be dispensable. The results indicate that the three motifs are not functionally equivalent in the catalytic action of DRADA. Mutation of the putative zinc-coordinating residues, His910, Cys966, and Cys1036, abolished the DRADA activity. Similarly, the Glu912 residue, predicted to be involved in the proton transfer functions of the enzyme, was found to be indispensable. Our results reinforce the previous proposal that the hydrolytic deamination mechanism of DRADA may be more similar to that of the cytidine deaminases than of adenosine deaminases.

Adenosine Deaminase↗

PubMed2002

Mechanism of activation and selectivity in a ligand-gated ion channel: structural and functional studies of GluR2 and quisqualate.

Glutamate is the major excitatory neurotransmitter in the mammalian brain. The (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazole)propionic acid (AMPA)-subtype glutamate receptor, a ligand-gated ion channel, mediates most of the fast excitatory synaptic transmission in the mammalian central nervous system. Here we present electrophysiological, biochemical, and crystallographic data on the interactions between quisqualate and the GluR2 receptor ion channel and its corresponding ligand binding core. Quisqualate is a high-affinity, full agonist which like AMPA and glutamate elicits maximum peak current responses, and stabilizes the ligand binding core in a fully closed conformation, reinforcing the concept that full agonists produce similar conformational changes [Armstrong, N., and Gouaux, E. (2000) Neuron 28, 165-181]. Nevertheless, the mechanism of quisqualate binding is different from that of AMPA but similar to that of glutamate, illustrating that quisqualate is a faithful glutamate analogue. A detailed comparison of the three agonist complexes reveals distinct binding mechanisms, particularly in the region of a hydrophobic pocket that is proximal to the anionic gamma-substituents, and demonstrates the importance of agonist-water-receptor interactions. The hydrophobic pocket, which is predicted to vary in chemical character between receptor subtypes, probably plays an important role in determining receptor subtype specificity.

Animals↗

PubMed1990

Modeling of agonist binding to the ligand-gated ion channel superfamily of receptors.

A generalized model is presented of agonist binding to ligand-gated ion channels (LGICs). Broad similarity in the structure of agonists suggests that the binding sites of LGICs may have evolved from a protobinding site. Aligned sequence data identified as a candidate for such a site a highly conserved 15 residue stretch of primary structure in the N-terminal extracellular region of all known LGIC subunits. We modeled this subregion, termed the cys-loop, as a rigid, amphiphilic beta-hairpin and propose that it may form a major determinant of a conserved structural binding cleft. In the model of the binding complex (1) an invariant aspartate residue at position 11 of the cys-loop is the anionic site interacting with the positively charged amine group of agonists, (2) a local dipole within the pi-electron system of agonists is favorably oriented in the electrostatic field of the invariant aspartate, (3) the epsilon ring-proton of a conserved aromatic residue at the turn of the cys-loop interacts orthogonally with the agonist pi-electron density at its electronegative center, and (4) selective recognition is partly a result of the type of amino acid residue at position 6 of the cys-loop. Additionally, formation of a hydrogen bond between the electronegative atom of the pi-electron system of agonist and a complementary group in the receptor may be important in the high-affinity binding of agonists.

Amino Acid Sequence↗

PubMed2003

Evolution of vertebrate voltage-gated ion channel alpha chains by sequential gene duplication.

Phylogenetic analysis of alpha chains of voltage-gated ion channels revealed that extensive gene duplication has occurred among both Ca(2+) and Na(+)-channels since the origin of vertebrates. Rather than showing a pattern of gene duplication consistent with the hypothesis of polyploidization early in vertebrate history, both Ca(2+) and Na(+) channels showed patterns of sequential gene duplication associated with specialization of the gene products. In the case of Na(+) channels, the phylogeny supported the hypothesis that the ancestral vertebrate gene had an expression pattern including both central and peripheral nervous system cells and that duplication of vertebrate Na(+) channel genes has repeatedly been followed by specialization for the central nervous system, the peripheral nervous system, or muscle cells. Thus, cephalization in vertebrate evolution has been accompanied by specialization of this important family of neuromuscular proteins along the central-peripheral axis.

Animals↗

PubMed2005

Cis-trans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel.

5-hydroxytryptamine type 3 (5-HT3) receptors are members of the Cys-loop receptor superfamily. Neurotransmitter binding in these proteins triggers the opening (gating) of an ion channel by means of an as-yet-uncharacterized conformational change. Here we show that a specific proline (Pro 8*), located at the apex of the loop between the second and third transmembrane helices (M2-M3), can link binding to gating through a cis-trans isomerization of the protein backbone. Using unnatural amino acid mutagenesis, a series of proline analogues with varying preference for the cis conformer was incorporated at the 8* position. Proline analogues that strongly favour the trans conformer produced non-functional channels. Among the functional mutants there was a strong correlation between the intrinsic cis-trans energy gap of the proline analogue and the activation of the channel, suggesting that cis-trans isomerization of this single proline provides the switch that interconverts the open and closed states of the channel. Consistent with this proposal, nuclear magnetic resonance studies on an M2-M3 loop peptide reveal two distinct, structured forms. Our results thus confirm the structure of the M2-M3 loop and the critical role of Pro 8* in the 5-HT3 receptor. In addition, they suggest that a molecular rearrangement at Pro 8* is the structural mechanism that opens the receptor pore.

Animals↗

PubMed2006

The role of cystic fibrosis transmembrane conductance regulator phenylalanine 508 side chain in ion channel gating.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel employing the ABC transporter structural motif. Deletion of a single residue (Phe508) in the first nucleotide-binding domain (NBD1), which occurs in most patients with cystic fibrosis, impairs both maturation and function of the protein. However, substitution of the Phe508 with small uncharged amino acids, including cysteine, is permissive for maturation. To explore the possible role of the phenylalanine aromatic side chain in channel gating we introduced a cysteine at this position in cysless CFTR, enabling its selective chemical modification by sulfhydryl reagents. Both cysless and wild-type CFTR ion channels have identical mean open times when activated by different nucleotide ligands. Moreover, both channels could be locked in an open state by introducing an ATPase inhibiting mutation (E1371S). However, the introduction of a single cysteine (F508C) prevented the cysless E1371S channel from maintaining the permanently open state, allowing closing to occur. Chemical modification of cysless E1371S/F508C by sulfhydryl reagents was used to probe the role of the side chain in ion channel function. Specifically, benzyl-methanethiosulphonate modification of this variant restored the gating behaviour to that of cysless E1371S containing the wild-type phenylalanine at position 508. This provides the first direct evidence that a specific interaction of the Phe508 aromatic side chain plays a role in determining the residency time in the closed state. Thus, despite the fact that this aromatic side chain is not essential for CFTR folding, it is important in the ion channel function.

Action Potentials↗

PubMed1999

ATP-gated ion channel expression in primary auditory neurones.

Extracellular ATP acts via ionotropic P2X receptors to mediate fast neurotransmission in the central and autonomic nervous systems. Recent data, including identification of P2X2 receptor mRNA expression by spiral ganglion neurones, suggests that purinergic signalling may influence auditory neurotransmission via ATP-gated ion channels assembled from these subunits. Expression of the P2X2 receptor was localized to the region of the spiral ganglion neurone synapses with the inner hair cells using a P2X2 receptor specific antiserum. Whole-cell patch clamping of neurones cultured from post-natal day 3-5 spiral ganglia demonstrated a heterogeneity of ATP-activated conductances, consistent with the functional expression of P2X2 receptor subunit isoforms along with possible co-expression of additional P2X receptor subunits. These data provide substantive support for a purinergic transmission element at the peripheral auditory synapse.

Adenosine Triphosphate↗

PubMed1998

Stoichiometry and arrangement of heteromeric olfactory cyclic nucleotide-gated ion channels.

Native cylic nucleotide-gated (CNG) channels are composed of alpha and beta subunits. Olfactory CNG channels were expressed from rat cDNA clones in Xenopus oocytes and studied in inside-out patches. Using tandem dimers composed of linked subunits, we investigated the stoichiometry and arrangement of the alpha and beta subunits. Dimers contained three subunit types: alphawt, betawt, and alpham. The alpham subunit lacks an amino-terminal domain that greatly influences gating, decreasing the apparent affinity of the channel for ligand by 9-fold, making it a reporter for inclusion in the tetramer. Homomeric channels from injection of alphawtalphawt dimers and from alphawt monomers were indistinguishable. Channels from injection of alphawtalpham dimers had apparent affinities 3-fold lower than alphawt homomultimers, suggesting a channel with two alphawt and two alpham subunits. Channels from coinjection of alphawtalphawt and betabeta dimers were indistinguishable from those composed of alpha and beta monomers and shared all of the characteristics of the alpha+beta phenotype of heteromeric channels. Coinjection of alphawtalpham and beta beta dimers yielded channels also of the alpha+beta phenotype but with an apparent affinity 3-fold lower, indicating the presence of alpham in the tetramer and that alpha+beta channels have adjacent alpha-subunits. To distinguish between an alpha-alpha-alpha-beta and an alpha-alpha-beta-beta arrangement, we compared apparent affinities for channels from coinjection of alphawtalphawt and betaalphawt or alphawtalphawt and betaalpham dimers. These channels were indistinguishable. To further argue against an alpha-alpha-alpha-beta arrangement, we quantitatively compared dose-response data for channels from coinjection of alphawtalpham and beta beta dimers to those from alpha and beta monomers. Taken together, our results are most consistent with an alpha-alpha-beta-beta arrangement for the heteromeric olfactory CNG channel.

Amino Acid Sequence↗

PubMed2002

The anesthetic mechanism of urethane: the effects on neurotransmitter-gated ion channels.

UNLABELLED: Urethane is widely used as an anesthetic for animal studies because of its minimal effects on cardiovascular and respiratory systems and maintenance of spinal reflexes. Despite its usefulness in animal research, there are no reports concerning its molecular actions. We designed this study to determine whether urethane affects neurotransmitter-gated ion channels. We examined the effects of urethane on recombinant gamma-aminobutyric acid(A), glycine, N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, and neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes. Urethane potentiated the functions of neuronal nicotinic acetylcholine, gamma-aminobutyric acid(A), and glycine receptors, and it inhibited N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors in a concentration-dependent manner. At concentrations close to anesthetic 50% effective concentration, urethane had modest effects on all channels tested, suggesting the lack of a single predominant target for its action. This may account for its usefulness as a veterinary anesthetic. However, a large concentration of urethane exerts marked effects on all channels. These findings not only give insight into the molecular mechanism of anesthetics but also caution that neurophysiologic measurements from animals anesthetized with urethane may be complicated by the effects of urethane on multiple neurotransmitter systems. Our results also suggest that small changes in multiple receptor systems can produce anesthesia. IMPLICATIONS: Urethane modestly affects multiple neurotransmitter systems at an anesthetic concentration. Our findings suggest that these degenerate effects of urethane can produce anesthesia and that urethane has a potential to influence neuronal measurements made in in vivo preparations.

Anesthetics, Intravenous↗

PubMed1988

Diffusion models of ion-channel gating and the origin of power-law distributions from single-channel recording.

The lifetimes of the unitary currents from ion channels, as revealed from single-channel recording, are traditionally thought to follow exponential or multiexponential distributions. The interpretation of these event-time distributions is that the gating process follows Markov kinetics among a small number of states. There is recent evidence, however, that certain systems exhibit distributions that follow power laws or functions related to power laws. Likewise, it has been suggested that data sets that appear to be multiexponential may be fit to simple power laws as well. In this paper we propose a different view of ion-channel-gating kinetics that is consistent with these recent experimental observations. We retain the Markovian nature of the kinetics, but, in contrast to the traditional models, we suggest that ion-channel proteins have a very large number of states all of similar energy. Gating, therefore, resembles a diffusion process. We show that our simplest one-dimensional model exhibits single-channel distributions that follow power laws of the form t-a, where 1/2 less than or equal to a less than or equal to 3/2. Exponents determined from recent experiments approximately fall within this range. We believe that this model is consistent with modern views of protein dynamics and, thus, may provide a key to the molecular details of the gating process.

Computer Simulation↗

PubMed2003

International Union of Pharmacology. XLI. Compendium of voltage-gated ion channels: potassium channels.

This summary article presents an overview of the molecular relationships among the voltage-gated potassium 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 potassium channel family can be found at http://www.iuphar-db.org/iuphar-ic/.

Phylogeny↗

PubMed1999

Voltage-gated ion channels and hereditary disease.

By the introduction of technological advancement in methods of structural analysis, electronics, and recombinant DNA techniques, research in physiology has become molecular. Additionally, focus of interest has been moving away from classical physiology to become increasingly centered on mechanisms of disease. A wonderful example for this development, as evident by this review, is the field of ion channel research which would not be nearly as advanced had it not been for human diseases to clarify. It is for this reason that structure-function relationships and ion channel electrophysiology cannot be separated from the genetic and clinical description of ion channelopathies. Unique among reviews of this topic is that all known human hereditary diseases of voltage-gated ion channels are described covering various fields of medicine such as neurology (nocturnal frontal lobe epilepsy, benign neonatal convulsions, episodic ataxia, hemiplegic migraine, deafness, stationary night blindness), nephrology (X-linked recessive nephrolithiasis, Bartter), myology (hypokalemic and hyperkalemic periodic paralysis, myotonia congenita, paramyotonia, malignant hyperthermia), cardiology (LQT syndrome), and interesting parallels in mechanisms of disease emphasized. Likewise, all types of voltage-gated ion channels for cations (sodium, calcium, and potassium channels) and anions (chloride channels) are described together with all knowledge about pharmacology, structure, expression, isoforms, and encoding genes.

Animals↗

PubMed2004

In vitro inhibition of recombinant ligand-gated ion channels by high concentrations of milnacipran.

RATIONALE: Tricyclic antidepressants are known to inhibit various ligand-gated ion-channel (LGIC) receptors, and some of their clinical features may be associated with this activity. The effects of milnacipran, a selective inhibitor of the reuptake of serotonin and noradrenaline, on LGIC receptors have not yet been investigated on such ion-channel receptors. OBJECTIVES: To determine the in vitro effect of milnacipran on four recombinant LGIC receptors, nicotinic acetylcholine, N-methyl-D-aspartate, gamma-amino butyric acid (GABA) and 5-hydroxytryptamine3A receptors. METHODS: Receptors were expressed in Xenopus oocytes and LGIC activity measured using a two-voltage clamp technique. RESULTS: There was no interaction at the GABA receptor. The results at the other LGIC receptors showed that they could be inhibited by high concentrations of milnacipran. CONCLUSIONS: At high concentrations, milnacipran can inhibit certain LGICs. It is, however, unlikely that these interactions have any clinical consequence under normal therapeutic conditions, since the concentrations required are considerably higher than those achieved in plasma of treated patients.

Animals↗