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At least 937 records · Page 52Linked to original sources
PubMed1999

Sequence of events underlying the allosteric transition of rod cyclic nucleotide-gated channels.

Activation of cyclic nucleotide-gated (CNG) ion channels involves a conformational change in the channel protein referred to as the allosteric transition. The amino terminal region and the carboxyl terminal cyclic nucleotide-binding domain of CNG channels have been shown to be involved in the allosteric transition, but the sequence of molecular events occurring during the allosteric transition is unknown. We recorded single-channel currents from bovine rod CNG channels in which mutations had been introduced in the binding domain at position 604 and/or the rat olfactory CNG channel amino terminal region had been substituted for the bovine rod amino terminal region. Using a hidden Markov modeling approach, we analyzed the kinetics of these channels activated by saturating concentrations of cGMP, cIMP, and cAMP. We used thermodynamic mutant cycles to reveal an interaction during the allosteric transition between the purine ring of the cyclic nucleotides and the amino acid at position 604 in the binding site. We found that mutations at position 604 in the binding domain alter both the opening and closing rate constants for the allosteric transition, indicating that the interactions between the cyclic nucleotide and this amino acid are partially formed at the time of the transition state. In contrast, the amino terminal region affects primarily the closing rate constant for the allosteric transition, suggesting that the state-dependent stabilizing interactions between amino and carboxyl terminal regions are not formed at the time of the transition state for the allosteric transition. We propose that the sequence of events that occurs during the allosteric transition involves the formation of stabilizing interactions between the purine ring of the cyclic nucleotide and the amino acid at position 604 in the binding domain followed by the formation of stabilizing interdomain interactions.

Algorithms↗
PubMed1995

Eye CNG channel is modulated by nicotine.

The cyclic-nucleotide gated channel (CNG channel) of the rod outer segment of the retina (ROS) has its closed conformation stabilized by nicotine. Calcium and cGMP influence the Ki of the channel current to nicotine. Calcium lowers the Ki and cGMP increases it, giving a range of Ki between 10(-11) and 10(-8) M.

Animals↗
PubMed1995

Effects of cysteine modification on the activity of the cGMP-gated channel from retinal rods.

The effect of sulfhydryl reagents on the activity of the cGMP-gated channel from bovine retinal rods was studied by measurements of 8-Br-cGMP-(cGMP)-induced calcium efflux from rod membrane vesicles and records of 8-Br-cGMP-dependent sodium currents through channels incorporated into planar lipid bilayers. N-ethylmaleimide and mersalyl (thiol blockers) as well as diamide (dithiol-disulfide conversion agent) have a dual effect on the channels activity: at low concentration, they increase the apparent affinity for cyclic nucleotide ("activation") at the same time inducing a loss of cooperativity for nucleotide binding; at higher concentration, N-ethylmaleimide and diamide produce a reduction of the amplitude and initial rate of the calcium release at saturating nucleotide concentration, while mersalyl is shown to reduce the activity of the channels in bilayer experiments ("inhibition"). Nitric oxide precursors have no effect. The results suggest that blocking at least 1 of the 3 cytoplasmic cysteine residues situated close to the cGMP-binding site in each channel subunit by N-ethylmaleimide, mersalyl, or diamide (forming a dimer between 2 subunits) increases the affinity for the nucleotide. Inhibition is produced by blocking at least one of the 2 other cytoplasmic sulfhydryl groups (N-ethylmaleimide, mersalyl, oxidized glutathione) or the 2 others (diamide, intrasubunit bridge), and may concern a process of channel inactivation. The 3 cytoplasmic sulfhydryl groups are accessible when the channels are in the open state, but not (or much less) accessible when the channels are in the closed state.

Animals↗
PubMed1993

A new subunit of the cyclic nucleotide-gated cation channel in retinal rods.

Retinal rods respond to light with a membrane hyperpolarization produced by a G-protein-mediated signalling cascade that leads to cyclic GMP hydrolysis and the consequent closure of a cGMP-gated channel that is open in darkness. A protein that forms this channel has recently been purified from bovine retina and molecularly cloned, suggesting that the native cGMP-gated channel might be a homo-oligomer. Here we report the cloning of another protein from human retina which has only about 30% overall identity to the rod channel subunit. This protein, immunocytochemically localized to rod outer segments, does not form functional channels by itself. However, when co-expressed with the cloned human rod channel protein, it introduces rapid flickers to the channel openings that are characteristic of the native channel. The hetero-oligomeric channel is also highly sensitive to the blocker L-cis-diltiazem, like the native channel. This new protein thus seems to be another subunit of the native rod channel. The hetero-oligomeric nature of the rod channel means that it is no exception to a common motif shared by other ligand-gated channels.

Adult↗
PubMed1994

Modulation of ion channels by protein phosphorylation and dephosphorylation.

Modulation of the properties of membrane ion channels is of fundamental importance for the regulation of neuronal electrical activity and of higher neural functions. Among the many potential molecular mechanisms for modulating the activity of membrane proteins such as ion channels, protein phosphorylation has been chosen by cells to play a particularly prominent part. This is not surprising given the central role of protein phosphorylation in a wide variety of cellular, metabolic, and signaling processes (26, 27, 48). As summarized here, regulation by phosphorylation is not restricted to one or another class of ion channel; rather, many, and perhaps all, ion channels are subject to modulation by phosphorylation. Similarly, a number of different protein kinase signaling pathways can participate in the regulation of ion channel properties, and it is not unusual to find that a particular channel is modulated by several different protein kinases, each influencing channel activity in a unique way. Finally, the biophysical mechanisms of modulation also exhibit a striking diversity that ranges from changes in desensitization rates to shifts in the voltage dependence and kinetics of channel activation and inactivation. The convergence of channel molecular biology with patch-clamp technology has been spectacularly productive, even allowing the identification of particular amino acid residues in ion channel proteins that participate in specific modulatory changes in channel biophysical properties. This task is far from complete, and no doubt there remain surprises in store for us, but nevertheless it is appropriate to ask where we go from here. One important direction will be to relate functional modulation, produced by phosphorylation, to changes in the three-dimensional structure of the ion channel protein. Unfortunately, structural studies of membrane proteins are extremely difficult, and to date there is no high resolution structure available for any ion channel protein. A complementary strategy that is more feasible with current technology is to investigate the ways in which channel modulation contributes to the regulation of cellular physiology. Novel computational approaches are being brought to bear on this complex issue, and their combination with channel molecular biology and biophysics should significantly advance our understanding of molecular mechanisms of neuronal plasticity.

Animals↗
PubMed2005

Functional consequences of progressive cone dystrophy-associated mutations in the human cone photoreceptor cyclic nucleotide-gated channel CNGA3 subunit.

Progressive cone dystrophies are a genetically heterogeneous group of disorders characterized by early deterioration of visual acuity and color vision, together with psychophysical and electrophysiological evidence of abnormal cone function and cone degeneration. Recently, three mutations in the gene encoding the CNGA3 subunit of cone photoreceptor cyclic nucleotide-gated (CNG) channels have been linked to progressive cone dystrophy in humans. To investigate the functional consequences of these mutations, we expressed mutant human CNGA3 subunits in Xenopus oocytes, alone or together with human CNGB3, and studied these channels using patch-clamp recording. Compared with wild-type channels, homomeric and heteromeric channels containing CNGA3-N471S or CNGA3-R563H subunits exhibited an increase in apparent affinity for cGMP and an increase in the relative agonist efficacy of cAMP compared with cGMP. In contrast, R277C subunits did not form functional homomeric or heteromeric channels. Cell surface expression levels, determined using confocal microscopy of green fluorescent protein-tagged subunits and patch-clamp recording, were significantly reduced for both R563H and R277C but unchanged for N471S. Overall, these results suggest that the plasma membrane localization and gating properties of cone CNG channels are altered by progressive cone dystrophy-associated mutations, providing evidence that supports the pathogenicity of these mutations.

Animals↗
PubMed2004

Circadian regulation of cGMP-gated channels of vertebrate cone photoreceptors: role of cAMP and Ras.

Circadian oscillators in chicken cone photoreceptors regulate the gating properties of cGMP-gated cationic channels (CNGCs) such that they have a higher apparent affinity for cGMP during the subjective night. Here we show that cAMP, acting through protein kinase A (PKA), Ras, and Erk, is part of the circadian output pathway controlling CNGCs. Endogenous and exogenous cAMP cause activation of Erk and Ras, which are more active at night in cones, and increase the apparent affinity of CNGCs for cGMP. The Ras farnesyl transferase inhibitor manumycin-A, and a dominant-negative form of Ras (RasN17) block the circadian rhythms in CNGC gating, as well as the effects of cAMP. A dominant-negative form of the MEK kinase B-Raf also blocks circadian and cAMP modulation of CNGCs. The circadian output pathway modulating CNGC channels is comprised in part of cAMP --> PKA --> Ras --> B-Raf --> MEK --> Erk --> --> CNGCs. cAMP activation of Ras and Erk occur within minutes, whereas modulation of CNGCs requires >1 hr. However, cAMP protagonists do not alter rhythms in cPer2 mRNA, and their effects on CNGCs cannot be attributed to clock phase-shifting.

Animals↗
PubMed1999

A single hydrophobic residue confers barbiturate sensitivity to gamma-aminobutyric acid type C receptor.

Barbiturate sensitivity was imparted to the human rho1 homooligomeric gamma-aminobutyric acid (GABA) receptor channel by mutation of a tryptophan residue at position 328 (Trp328), which is located within the third transmembrane domain. Substitutions of Trp328 with a spectrum of amino acids revealed that nearly all hydrophobic residues produced receptor channels that were both directly activated and modulated by pentobarbital with similar sensitivities. Previous studies with ligand-gated ion channels (including GABA) have demonstrated that even conservative amino acid substitution within the agonist-dependent activation domain (N-terminal extracellular domain) can markedly impair agonist sensitivity. Thus, the lack of significant variation in pentobarbital sensitivity among the Trp328 mutants attests to an intrinsic difference between pentobarbital- and the GABA-dependent activation domain. Compared with the heterooligomeric alphabetagamma receptor channel, the mode of modulation for homooligomeric Trp328 mutants by pentobarbital was more dependent on the GABA concentration, yielding potentiation only at low concentrations of GABA (fractions of their respective EC50 values), yet causing inhibition at higher concentrations. Agonist-related studies have also demonstrated that residue 328 plays an important role in agonist-dependent activation, suggesting a functional interconnection between the GABA and pentobarbital activation domains.

Amino Acid Sequence↗
PubMed2000

Potassium channels: watching a voltage-sensor tilt and twist.

The fourth transmembrane helix (S4) is the primary voltage-sensor of voltage-gated ion channels. Recent studies have used fluorescence resonance energy transfer as a spectroscopic ruler to determine the nature and magnitude of the voltage-induced movement of S4 that leads to channel opening.

Energy Transfer↗
PubMed1995

Charge dissociation and the rising phase of gating currents.

Gating currents from voltage-sensitive channels are generally attributed to the translocation or redistribution of ionic charge associated with the channel molecule. Such charge moves in the direction of the applied field to produce a decreasing current in the external circuit. An early rising phase for the gating current is observed for a number of channel systems and might be either some special kinetic redistribution of charge or an experimental artifact. A model that produces net charge in the channel through sequential molecular dissociation of a charged channel segment gives a rising phase for the gating current. Translocation of the charged segment produces the decay phase for a biphasic gating current. This kinetic molecular explanation constitutes a physical explanation for the biphasic gating currents that is consistent with present views of channel structure.

Artifacts↗
PubMed2005

The involvement of glutamate-gated channels in negative feedback from horizontal cells to cones.

Photoreceptors are the light sensitive cells in the retina. They project to horizontal cells and bipolar cells via a glutamatergic feed forward pathway. Horizontal cells are strongly electrically coupled and integrate in that way the input from the photoreceptors. Horizontal cells feedback to cones negatively. The combined signal from the photoreceptors and the horizontal cells is sent to the bipolar cells. The feedback pathway from horizontal cells to cones is thought to form the basis for the center/surround organization of bipolar cells. The nature of the feedback pathway is an issue of intense debate. It was thought for a long time that this feedback pathway was GABAergic, because cones have GABA-receptors and horizontal cells release GABA via a GABA-transporter working in the reversed direction. However, recently we showed in goldfish that horizontal cells feed back to cones via an alternative mechanism. In goldfish, negative feedback from horizontal cells to cones shifts the calcium current of the cone to more negative potentials. This feedback pathway is independent of GABA, since feedback cannot be blocked by either saturating concentrations of PTX, the GABA-transporter blocker SKF89976A, or application of GABA. The mechanism of negative feedback from horizontal cells to cones involves hemichannels located at the tips of the invaginating horizontal cells, just opposite to the calcium channels of the cones. Current flowing through these hemichannels changes the extracellular potential deep in the synaptic cleft and in that way modulates the calcium current of the cones. Such a modulation of the extracellular potential is called ephaptic. If negative feedback from horizontal cells to cones is indeed ephaptic, other channels present in the synapse should also be able to act as a current source, i.e., should also be able to change the output of the cone. We showed that glutamate-gated channels present at the tips of the horizontal cell dendrites can also mediate feedback responses. Surprisingly, although the glutamate-gated conductance of the horizontal cells is eight times the hemichannel conductance, glutamate-gated channels are not the major current source in negative feedback from horizontal cells to cones. In this chapter we present evidence that this is due to the more focal localization of the hemichannels, compared to a diffuse and extrasynaptic localization of the glutamate-gated channels.

Animals↗
PubMed2004

Transient receptor potential ion channels and animal sensation: lessons from Drosophila functional research.

Ion channels of the transient receptor potential (TRP) superfamily are non-selective cationic channels with six transmembrane domains. The TRP channel made its first debut as a light-gated Ca2+ channel in Drosophila. Recently, research on animal sensation in Drosophila disclosed other members of the TRP family that are required for touch sensation and hearing as well as the sensation of painful stimuli.

Animals↗
PubMed2000

Odorants suppress voltage-gated currents in retinal horizontal cells in goldfish.

Odorants are known to suppress non-selectively voltage-gated currents in olfactory receptor cells. We found that odorants also suppress voltage-gated currents in neurons of outside of the olfactory system. Under voltage clamp, odorants such as amyl acetate, limonene, and acetophenone suppressed non-selectively voltage-gated currents (a Ca(2+) current, a delayed rectifier K(+) current, a fast transient K(+) current, and an anomalous rectifier K(+) current) in horizontal cells from the goldfish retina. An amyl acetate puff completely and immediately suppressed the Ca(2+) current (I(Ca)) and the delayed rectifier K(+) current induced by repetitive depolarizations, suggesting that amyl acetate is a closed-channel blocker. Odorants did not change significantly the activation curve of I(Ca), but made the slope of inactivation curve of I(Ca) gentler and shifted its half-inactivation voltage toward a negative voltage. These results are similar to the effects of odorants on voltage-gated currents in olfactory receptor cells. This suggests that odorants may suppress the voltage-gated currents in retinal horizontal cells by the same mechanism described in olfactory receptor cells.

Acetophenones↗
PubMed2005

Estimating rate constants from single ion channel currents when the initial distribution is known.

Single ion channel currents can be analysed by hidden or aggregated Markov models. A classical result from Fredkin et al. (Proceedings of the Berkeley conference in honor of Jerzy Neyman and Jack Kiefer, vol I, pp 269-289, 1985) states that the maximum number of identifiable parameters is bounded by 2n(o)n(c), where n(o) and n(c) denote the number of open and closed states, respectively. We show that this bound can be overcome when the probabilities of the initial distribution are known and the data consist of several sweeps.

Biophysics↗
PubMed1993

Positive modulation of human gamma-aminobutyric acid type A and glycine receptors by the inhalation anesthetic isoflurane.

The interactions of the inhalation anesthetic agent isoflurane with ligand-gated chloride channels were studied using transient expression of recombinant human receptors in a mammalian cell line. Isoflurane enhanced gamma-aminobutyric acid (GABA)-activated chloride currents in cells that expressed heteromeric GABAA receptors consisting of combinations of alpha 1 or alpha 2, beta 1, and gamma 2 subunits and in cells that expressed receptors consisting of combinations of only alpha and beta subunits. Receptors consisting of alpha 2 and gamma 2 subunits were poorly expressed but were sensitive to isoflurane. Receptors consisting of beta 1 and gamma 2 subunits were not expressed. Isoflurane also enhanced glycine-activated chloride currents through homomeric alpha glycine receptors but did not enhance GABA currents in cells expressing homomeric rho 1 receptors. These results show that not all ligand-gated chloride channel receptors are sensitive to isoflurane and, therefore, that the anesthetic interacts with specific structural determinants of these ion channel proteins.

Amino Acid Sequence↗
PubMed1998

The nematode degenerin UNC-105 forms ion channels that are activated by degeneration- or hypercontraction-causing mutations.

Nematode degenerins have been implicated in touch sensitivity and other forms of mechanosensation. Certain mutations in several degenerin genes cause the swelling, vacuolation, and death of neurons, and other mutations in the muscle degenerin gene unc-105 cause hypercontraction. Here, we confirm that unc-105 encodes an ion channel and show that it is constitutively active when mutated. These mutations disrupt different regions of the channel and have different effects on its gating. The UNC-105 channels are permeable to small monovalent cations but show voltage-dependent block by Ca2+ and Mg2+. Amiloride also produces voltage-dependent block, consistent with a single binding site 65% into the electric field. Mammalian cells expressing the mutant channels accumulate membranous whorls and multicompartment vacuoles, hallmarks of degenerin-induced cell death across species.

Amiloride↗
PubMed1993

Lipid effects on nicotinic acetylcholine receptor gating and kinetics: a structural-functional correlation.

The nicotinic acetylcholine receptor (AChR) is still the paradigm of rapid ligand-gated ion channels. Since the early finding of a motionally restricted shell of lipids ("annulus") in the immediate perimeter of the membrane-bound AChR, experimental evidence has supported the notion that the interface between the protein moiety and the adjacent lipid molecules is the site of action of a variety of pharmacologically relevant substances, including non-competitive inhibitors of the cholinergic system like some local anesthetics, short-chain alcohols, and steroids. Patch-clamp data on cells expressing the AChR protein add another dimension to this knowledge, enabling correlations to be established between the chemical composition of lipid-modified cells and the functional properties (ligand binding, channel gating) of the receptor protein in situ.

Anesthetics↗