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PubMed2004

Activation and modulation of ligand-gated ion channels.

Ligand-gated ionic channels are integral membrane proteins that enable rapid and selective ion fluxes across biological membranes. In excitable cells, their role is crucial for generation and propagation of electrical signals. This survey describes recent results from studies performed in the Department of Cellular Neurophysiology, Institute of Physiology ASCR, aimed at exploring the conformational dynamics of the acetylcholine, glutamate and vanilloid receptors during their activation, inactivation and desensitization. Distinct families of ion channels were selected to illustrate a rich complexity of the functional states and conformational transitions these proteins undergo. Particular attention is focused on structure-function studies and allosteric modulation of their activity. Comprehension of the fundamental principles of mechanisms involved in the operation of ligand-gated ion channels at the cellular and molecular level is an essential prerequisite for gaining an insight into the pathogenesis of many psychiatric and neurological disorders and for efficient development of novel specifically targeted drugs.

Allosteric Regulation↗
PubMed1994

Pf3 coat protein forms voltage-gated ion channels in planar lipid bilayers.

The coat protein of bacteriophage Pf3 forms discrete and stable ion channels of uniform size in planar bilayers of asolectin. Its primary sequence suggests a channel formed by a bundle of transmembrane helices. Since the apparent transmembrane region only consists of strongly hydrophobic residues, it represents a new class of channel-forming proteins. The channel activity is strongly voltage-dependent. The single-channel conductance of 60 pS (at 100 mV) in 0.2 M NaCl is slightly voltage-dependent, indicating conformational changes of the pore upon variation of the transmembrane electric field. The channel is unselective which suggests that the pore is of aqueous character. For the observed conductance, a channel diameter of 3.6 A is consistent with a tetrameric alpha-helix bundle, as calculated from a barrel-stave model. A pronounced dependence of the gating kinetics with increasing voltage arises from two opposing effects: an increase in the number of open channel structures, and a simultaneous, more than 3-fold decrease in the channel lifetime. Thus, a maximum activity is reached around 100 mV, a range which corresponds well with physiological membrane potentials. The channels activate only upon application of a positive voltage on the side of the membrane to which the protein had been added. The slow relaxation of the mean current upon application of sudden voltage jumps indicates a strong activation barrier in the channel gating process, which may result from the membrane translocation of the charged residues of the peptide ends. A channel-mediated import mechanism is suggested for the bacterial infection by phage DNA.

Amino Acid Sequence↗
PubMed2004

Voltage-gated ion channels in nociceptors: modulation by cGMP.

In tissue or nerve injury, proinflammatory mediators are released that can modulate a variety of ion channels found in nociceptors. The changes in channel activity, which primarily occurs through changes in intracellular pathways, may lead to the pathological states of hyperalgesia and allodynia. To understand further the regulatory mechanisms underlying the changes in channel activity, we used whole cell patch-clamp recordings from capsaicin-sensitive nociceptive neurons in rat trigeminal ganglion neurons to examine how the cGMP-dependent pathways may regulate ion channel function. Addition of the 8-(4-chlorophenylthio)-3',5' (CPT)-cGMP, a membrane permeant modulator of ion channels, decreased the number of evoked action potentials by 36% and inhibited the tetrodotoxin-resistant (TTX-R) sodium currents and IA potassium currents by 37 and 32%, respectively. Delayed rectifier potassium (IK) currents were unaffected, suggesting that the effects of CPT-cGMP are unlikely to arise from a nonspecific effect on channel activity as a consequence of the adsorption of amphipathic CPT-cGMP molecules to the membrane's bilayer component. This conclusion was reinforced by the lack of changes in gramicidin A channel function in the presence of CTP-cGMP. In summary, the activation of the cGMP-dependent pathways reduces nociceptor excitability, in part, by decreasing the activity of voltage-gated TTX-R sodium channels. This pathway may be a target for efforts to produce selective analgesics.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

Ligand-gated ion channels opened by 5-HT in molluscan neurones.

1. 5-Hydroxytryptamine (5-HT) activated a fast (70 ms to half maximum) and desensitizing inward current through non-selective channels conducting predominantly monovalent cations in neurons of Helix aspersa. 2. alpha-Methyl-5-HT was equipotent with 5-HT in activating this current, but the known selective agonists at vertebrate 5-HT3 receptors, 2-methyl-5-HT and arylbiguanides were ineffective (< 100 microM). 5-Methoxytryptamine which is inactive on vertebrate 5-HT3 receptors was a very weak agonist. 3. The responses were antagonized by the specific vertebrate 5-HT3 receptor blocker MDL-72222 (IC50 = 1 microM), but were only weakly affected by ondansetron (10 microM). The 5-HT2-type antagonist, ketanserin (< 5 microM) had no effect. The responses were also antagonized by the non-specific antagonists (+)-tubocurarine and strychnine. 4. Unitary currents through channels non-selective for monovalent cations, and with a conductance of 2pS, could be activated repeatedly by 5-HT or alpha-methyl-5-HT in outside-out patches from neurones exhibiting the fast 5-HT-activated current (I[5-HT]fast), even in the presence of 500 microM GDP-[beta S] in the recording pipette. This strongly supports direct-gating of these channels by 5-HT. The properties of these unitary currents resembled those of I[5-HT]fast. 5. The pharmacological properties of this molluscan 5-HT-operated, ligand-gated channel differed sufficiently from known vertebrate 5-HT3-type receptors to suggest that it represents a new class of 5-HT receptor.

Animals↗
PubMed2002

Changes in cardiac myocyte morphology alter the properties of voltage-gated ion channels.

OBJECTIVE: The goal of this study was to determine if the properties of the transient outward potassium (I(to)), TTX-resistant sodium (I(Na)) and L-type calcium (I(Ca)) currents are altered during changes in cardiac cell shape. METHODS: Ventricular myocytes were isolated from 3- to 4-day-old neonatal rats and cultured on either non-aligned or aligned collagen thin gels. In contrast to the flat, stellar-shaped myocytes obtained when the cells are plated on non-aligned collagen gels, myocytes plated on aligned gels display an elongated, rod-like shape. Ion channel expression was measured using the whole-cell arrangement of the patch clamp technique and Western blot analysis. RESULTS: Peak values for I(to), I(Na) and I(Ca) were 9+/-1, 71+/-13 and 7+/-1 pA/pF, respectively, in the flat cells, and increased to 21+/-2, 190+/-26 and 13+/-1 pA/pF, respectively, in the aligned cells. Application of forskolin (2 microM) and 3-isobutyl-1-methylxanthine (100 microM) resulted in a 101+/-18% increase in I(Ca) in the flat cells, but increased the current by only 43+/-9% in the aligned cells. Internal dialysis of the myocytes with cAMP strongly increased the peak I(Ca) in the flat cells, but caused no significant change in the aligned cells. While both basal and forskolin-stimulated levels of cAMP were the same in the two cell morphologies, the expression of the calcium channel alpha(1C) subunit was increased in the aligned cells. CONCLUSIONS: The expression and regulatory properties of voltage-gated calcium channels are modified during changes in neonatal rat myocyte shape.

1-Methyl-3-isobutylxanthine↗
PubMedDate not supplied

The molecular basis of the structure and function of the 5-HT3 receptor: a model ligand-gated ion channel (review).

The ligand-gated ion channel superfamily of neurotransmitter receptors are proteins responsible for rapid transmission of nerve impulses at the synapse and have, therefore, been the subject of intensive research for many years. The cys-loop family, of which the 5-HT3 receptor is a member, includes the nicotinic acetylcholine receptor, the GABAA receptor and the glycine receptor. A diverse range of endogenous and artificial ligands activate these receptors, but, nevertheless, the family shares many similarities of structure and function. Several important questions, however, still remain to be determined, including the mechanism of agonist recognition at the binding site, the nature of the connection between the agonist binding and channel domains, the structure of the transmembrane regions and the mechanism of ion permeation and selectivity. This article reviews recent advances in the characterization of the molecular properties of the 5-HT3 receptor and their role in its function, and assesses its suitability as a model system for the study of the above questions.

Allosteric Regulation↗
PubMed1999

Drug-dependent ion channel gating by application of concentration jumps using U-tube technique.

The rapid application system described has been used to study a variety of ionic channels in several different types of single cells. The system is inexpensive, easy to install, and can be used repeatedly. The consumption of UTS, i.e., drugs or agonists, is low. The time interval between switching the valve and the expected effect is often shorter than 150 ms for cells about 8-15 microns in diameter and is about 20-25 ms for patches positioned in the hole of the U-tube. Time and duration of substance application can be controlled by a computer connected to a digital-analog (D/A) output.

Cells, Cultured↗
PubMed1999

Mutation at the putative GABA(A) ion-channel gate reveals changes in allosteric modulation.

We have mutated a conserved leucine in the putative membrane-spanning domain to serine in human GABA(A) beta2 and investigated the actions of a number of GABA(A) agonists, antagonists and modulators on human alpha1beta2deltaL259Sgamma2s compared to wild type alpha1beta2gamma2s GABA(A) receptors, expressed in Xenopus oocytes. The mutation resulted in smaller maximum currents to gamma-aminobutyric acid (GABA) compared to alpha1beta2gamma2s receptors, and large leak currents resulting from spontaneous channel opening. As reported, this mutation significantly decreased the GABA EC50 (110 fold), and reduced desensitization. Muscimol and the partial agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) and piperidine-4-sulphonic acid (P4S) also displayed a decrease in EC50. In addition to competitively shifting GABA concentration response curves, the antagonists bicuculline and SR95531 both inhibited the spontaneous channel activity on alpha1beta2deltaL259Sgamma2s receptors, with different degrees of maximum inhibition. The effects of a range of allosteric modulators, including benzodiazepines and anaesthetics were examined on a submaximal GABA concentration (EC20). Compared to wild type, none of these modulators potentiated the EC20 response of alpha1beta2deltaL259Sgamma2s receptors, however they all directly activated the receptor in the absence of GABA. To conclude, the above mutation resulted in receptors which exhibit a degree of spontaneous activity, and are more sensitive to agonists. Benzodiazepines and other agents modulate constitutive activity, but positive modulation of GABA is lost. The competitive antagonists bicuculline and SR95531 can also act as allosteric channel modulators through the same GABA binding site.

Allosteric Regulation↗
PubMed2005

Hidden Markov analysis of mechanosensitive ion channel gating.

Patch clamp data from the large conductance mechanosensitive channel (MscL) in E. coli was studied with the aim of developing a strategy for statistical analysis based on hidden Markov models (HMMs) and determining the number of conductance levels of the channel, together with mean current, mean dwell time and equilibrium probability of occupancy for each level. The models incorporated state-dependent white noise and moving average adjustment for filtering, with maximum likelihood parameter estimates obtained using an EM (expectation-maximisation) based iteration. Adjustment for filtering was included as it could be expected that the electronic filter used in recording would have a major effect on obviously brief intermediate conductance level sojourns. Preliminary data analysis revealed that the brevity of intermediate level sojourns caused difficulties in assignment of data points to levels as a result of over-estimation of noise variances. When reasonable constraints were placed on these variances using the better determined noise variances for the closed and fully open levels, idealisation anomalies were eliminated. Nevertheless, simulations suggested that mean sojourn times for the intermediate levels were still considerably over-estimated, and that recording bandwidth was a major limitation; improved results were obtained with higher bandwidth data (10 kHz sampled at 25 kHz). The simplest model consistent with these data had four open conductance levels, intermediate levels being approximately 20%, 51% and 74% of fully open. The mean lifetime at the fully open level was about 1 ms; estimates for the three intermediate levels were 54-92 micros, probably still over-estimates.

Computer Simulation↗
PubMed1995

Adult rat optic nerve oligodendrocyte progenitor cells express a distinct repertoire of voltage- and ligand-gated ion channels.

Cultured oligodendrocyte progenitor cells derived from the developing central nervous system (CNS) express a pattern of ion channels that is distinct from mature oligodendrocytes and other cell types of the CNS. In the present study, we used the whole-cell patch-clamp technique and the fura-2-based Ca++ imaging system to study the ion channel expression of oligodendrocyte progenitor cells derived from the optic nerves of adult rats. We found that the adult oligodendrocyte progenitor cell membrane is dominated by K+ currents, both delayed outward and inward rectifying. The inwardly rectifying K+ currents were often as large as the outward delayed rectifying K+ currents. The delayed rectifying outward currents were partially blocked by 50 mM tetraethylammonium or 1 mM 4-aminopyridine, but not by 2 or 5 mM BaCl2. This suggests that the delayed rectifier channels expressed by adult progenitor cells are different from those expressed by perinatal cells. Most adult oligodendrocyte progenitor cells showed no or only small A-type K+ currents. Both Ca++ and Na+ channels were also detected in these cells. Furthermore, adult progenitor cells responded to the neurotransmitters GABA and kainate and the pharmacology of these responses indicated that these cells express GABAA receptors and kainate receptors that are Ca(++)-permeable. Our study suggests that adult oligodendrocyte progenitor cells are electrophysiologically distinct and that these cells share electrophysiological characteristics with both perinatal progenitor cells and immature oligodendrocytes.

Animals↗
PubMed2004

Role of ligand-gated ion channels in the swimming behaviour of Xenopus tadpoles: experimental data and modelling experiments.

The swimming behaviour of lower vertebrates has been used as a model to study the function of simple neuronal circuits. Good examples are the lamprey and the Xenopus tadpole. In these two cases, glutamate-activated NMDA receptors are involved, and the relative importance of the NMDA and non-NMDA receptors as well as the involvement of other ion channels has been studied using a combination of electrophysiological recordings and modelling experiments, but little attention had been paid to their evolution during development. In the present experiments, which have been performed on Xenopus embryos from stages 31 to 42, we have probed the relative importance of the two categories of receptors using selective blockers [respectively dl-2-amino-5-phosphonovaleric acid (APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)]. The sensitivity of the swimming behaviour to APV was found to increase during development and that to CNQX to decrease. Furthermore, it has been observed that the spike activity recorded from the ventral roots is more complex in late embryonic stages that in early embryos. These modifications are associated with changes of the neuronal circuit, some of which correspond to a lengthening of the axon and an increased complexity of the dendritic tree of the motoneurons. We have incorporated these modifications in a simplified model of the central pattern generator built with Neuron software. The results indicate that at least part of the observed changes can be associated with changes in the length of the dendrites and axons.

2-Amino-5-phosphonovalerate↗
PubMed2000

Electrophysiological evidence for an ATP-gated ion channel in the principal cells of the frog skin epithelium.

In the present study we investigated the effects of adenosine 5'-triphosphate (ATP) on Na+ transport in frog skin epithelium. An experimental set-up was constructed to allow simultaneous measurement of Na+ transport, measured as the amiloride-sensitive short circuit current (Isc), and free cytosolic Ca2+ concentration ([Ca2+]i) measured with the Ca(2+)-sensitive dye fura-2. The cell potential (Vsc) was measured with microelectrodes. Addition of ATP (100 micrM) to the basolateral solution resulted in a fast transient decrease in Isc followed by a slower increase and a transient increase in [Ca2+]i. Microelectrode measurements showed that the primary response, i.e. the decline in Isc was accompanied by transient depolarisation, followed by a return to the control value. The decrease in current was Ca2+ independent; i.e. treatment with thapsigargin in Ca(2+)-free solutions abolished the Ca2+ transient but did not influence the current transient. The secondary response, i.e. the slow increase in current, was accompanied by slow depolarisation of the cell. Measurements of apical Na+ permeability showed that this was due to an opening or activation of apical Na+ channels. These data show that ATP causes a fast initial drop and a secondary, long-lasting increase in Na+ absorption. The ability of ATP to cause the initial decline in current is independent of Ca2+, i.e. it is not caused by secondary effects of the P2Y-type receptors present in the tissue. Measurements of intracellular potential indicate that the initial depolarisation is caused by opening of non-selective cation channels, suggesting that this decrease is due to a transient activation of P2X-type ATP receptors.

Adenosine Triphosphate↗
PubMed2002

Characterization of the interaction between a novel convulsant agent, norbiphen, and GABA(A) and other ligand-gated ion channels.

A hybrid molecule composed of the antimicrobial, norfloxacin, linked to the non-steroidal anti-inflammatory drug (NSAID), biphenylacetic acid, which we have termed norbiphen, is a lethal convulsant in vivo and an antagonist of rodent GABA(A) receptors in vitro. In the present study, the selectivity, molecular site(s) and mechanism of action of this novel convulsant were investigated using electrophysiological techniques. Sub-maximal GABA-evoked currents recorded from rodent hippocampal neurons were reversibly inhibited by norbiphen (1 microM) to 5+/-2% of control whereas glutamate, NMDA and glycine activated responses were little or unaffected. Sub-maximal GABA-evoked currents recorded from oocytes expressing recombinant human alpha1beta2gamma2s or alpha1beta2 GABA(A) receptors were also reversibly inhibited by norbiphen (1-1000 nM) with an IC(50) (+/-s.e.m.) of 5.7+/-1 and 8.8+/-1 nM, respectively. Similarly, GABA currents recorded from alpha1beta1gamma2s, alpha1beta1 and beta2gamma2s receptors were inhibited with IC(50)s of 16.1+/-1, 18.8+/-1 and 4.2+/-1 nM, respectively. In contrast, norbiphen (100 nM) had little or no effect at rho1 GABA(C) homomers. At alpha1beta2gamma2s receptors, norbiphen had no affect on the GABA reversal potential, and inhibition was not voltage-dependent, suggesting that this compound does not act at the ion channel. The GABA concentration response curve was shifted in a competitive-like fashion by norbiphen (10-300 nM) and a Schild analysis of these data yielded a slope of 0.94+/-0.1 and a pA(2) of 7.77. Our data reveal a novel, selective and highly potent antagonist of GABA(A) receptors. Norbiphen should be a valuable agent in future studies of this receptor complex.

Animals↗