Morphing structures and signal transduction in Mimosa pudica L. induced by localized thermal stress

Leaf movements in Mimosa pudica, are in response to thermal stress, touch, and light or darkness, appear to be regulated by electrical, hydrodynamical, and chemical signal transduction. The pulvinus of the M. pudica shows elastic properties. We have found that the movements of the petiole, or pinnules, are accompanied by a change of the pulvinus morphing structures. After brief flaming of a pinna, the volume of the lower part of the pulvinus decreases and the volume of the upper part increases due to the redistribution of electrolytes between these parts of the pulvinus; as a result of these changes the petiole falls. During the relaxation of the petiole, the process goes in the opposite direction. Ion and water channel blockers, uncouplers as well as anesthetic agents diethyl ether or chloroform decrease the speed of alert wave propagation along the plant. Brief flaming of a pinna induces bidirectional propagation of electrical signal in pulvini. Transduction of electrical signals along a pulvinus induces generation of an action potential in perpendicular direction between extensor and flexor sides of a pulvinus. Inhibition of signal transduction and mechanical responses in M. pudica by volatile anesthetic agents chloroform or by blockers of voltage gated ion channels shows that the generation and propagation of electrical signals is a primary effect responsible for turgor change and propagation of an excitation. There is an electrical coupling in a pulvinus similar to the electrical synapse in the animal nerves.     

Brain GABAA receptors studied with subunit-specific antibodies

Brain GABA_A/benzodiazepine receptors are highly heterogeneous. This heterogeneity is largely derived from the existence of many pentameric combinations of at least 16 different subunits that are differentially expressed in various brain regions and cell types. This molecular heterogeneity leads to binding differences for various ligands, such as GABA agonists and antagonists, benzodiazepine agonists, antagonists, and inverse agonists, steroids, barbiturates, ethanol, and Cl⁻ channel blockers. Different subunit composition also leads to heterogeneity in the properties of the Cl⁻ channel (such as conductance and open time); the allosteric interactions among subunits; and signal transduction efficacy between ligand binding and Cl⁻ channel opening. The study of recombinant receptors expressed in heterologous systems has been very useful for understanding the functional roles of the different GABA_A receptor subunits and the relationships between subunit composition, ligand binding, and Cl⁻ channel properties. Nevertheless, little is known about the complete subunit composition of the native GABA_A receptors expressed in various brain regions and cell types. Several laboratories, including ours, are using subunit-specific antibodies for dissecting the heterogeneity and subunit composition of native (not reconstituted) brain GABA_A receptors and for revealing the cellular and subcellular distribution of these subunits in the nervous system. These studies are also aimed at understanding the ligand-binding, transduction mechanisms, and channel properties of the various brain GABA_A receptors in relation to synaptic mechanisms and brain function. These studies could be relevant for the discovery and design of new drugs that are selective for some GABA_A receptors and that have fewer side effects.     

Contractile Proteins in Chemical Signal Transduction in Plant Microspores

Involvement of contractile components in chemical signal transduction from the cell surface to the organelles was studied using unicellular systems. Neurotransmitters dopamine and serotonin as well as active oxygen species hydrogen peroxide and tert-butyl peroxide were used as chemical signals. Experiments were carried out on vegetative microspores of field horsetail Equisetum arvense and generative microspores (pollen) of knight’s star Hippeastrum hybridum treated with cytochalasin B (an inhibitor of actin polymerization in microfilaments), colchicine, and vinblastine (inhibitors of tubulin polymerization in microtubules). Both types of the treated microspores demonstrated suppressed development, particularly, after cytochalasin B treatment. At the same time, increased blue fluorescence was observed in certain cell regions (along the cell wall and around nuclei and chloroplasts) where the corresponding contractile proteins could be localized. In contrast to anticontractile agents, dopamine, serotonin B, and peroxides stimulated microspore germination. Microspore pretreatment with cytochalasin B and colchicine followed by the treatment with serotonin, dopamine, or peroxides decreased the germination rate. The involvement of actin and tubulin in chemical signal transduction from the cell surface to the nucleus is proposed.__________Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 3, 2005, pp. 281–286.Original Russian Text Copyright © 2005 by Roshchina.     

Fluorescence of plant microspores as biosensors

Possibilities of fluorescent microscopic single-cell analysis on plant microspores as biosensors for study of chemosignaling involving neurotransmitters and mechanisms of action of fluorescent medicinal compounds—antagonists of neurotransmitters were studied on some examples. By methods of luminescence microscopy, microspectrofluorimetry and laser scanning confocal microscopy using as an example field horsetail Equisetum arvense microspores, the penetration of these compounds into the cell and associate it with individual compartments (estimated as the changes in their autofluorescence) has been analyzed. Fluorescent in blue antagonists of neurotransmitters d-tubocurarine, yohimbine and azulene (blockers of cholinoreceptor, adrenoreceptor, and histamine receptor, respectively) decreased the number of the E. arvense cells with red fluorescence. Tubocurarine and yohimbine bound to the cellular surface and did not penetrate into the cells. Azulene was found both on the cell surface and inside cells, demonstrating blue (excitation 360–380 nm) or green (excitation 420 nm) fluorescence of DNA-containing organelles. The effects of lipophilic (lecithin and amphotericin B) and proteinous (albumin, enzyme cholinesterase, cytoskeleton proteins actin, myosin, and titin) compounds on the manifestation of the effects of the neurotransmitters and antagonist d-tubocurarine have been shown. The intensity of the red light at 680 nm, has evolved in many variants. Most notable was the decline of the emission in the presence of albumin and cholinesterase as compared with the action of dopamine itself. After the addition of the cytoskeleton proteins and cholinesterase to the medium, the decrease of red fluorescence intensity, usually induced by d-tubocurarine, was not observed.     

Muscarinic acetylcholine receptor compounds alter net Ca<Superscript>2+</Superscript> flux and contractility in an invertebrate smooth muscle

Responses of a holothurian smooth muscle to a range of muscarinic (M₁ to M₅) acetylcholine receptor (mAChR) agonists and antagonists were surveyed using calcium (Ca²⁺)-selective electrodes and a mechanical recording technique. Most of the mAChR agonists and antagonists tested increased both contractility and net Ca²⁺ efflux, with M₁-specific agents like oxotremorine M being the most potent in their action. To investigate the possible sources of Ca²⁺ used during mAChR activation, agents that disrupt intracellular Ca²⁺ ion sequestration [cyclopiazonic acid (CPA), caffeine, ryanodine], the phosphoinositide signaling pathway [lithium chloride (LiCl)], and L-type Ca²⁺ channels (diltiazem and verapamil) were used to challenge contractions induced by oxotremorine M. These contractions were blocked by treatment with CPA, caffeine, LiCl, and by channel blockers, diltiazem and verapamil, but were unaltered by ryanodine. Our data suggest that this smooth muscle had an M_1,3,5-like receptor that was associated with the phosphoinositide signaling pathway that relied on intracellular Ca²⁺ stores, but secondarily used extracellular Ca²⁺ via the opening of L-type channels.     

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca²⁺]_cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.     

Second messengers and ion channels in acetylcholine-induced chloride secretion in hen trachea

1. Hen tracheal epithelium can be stimulated by serosal application of acetylcholine (ACh) to secrete Cl⁻ equal to ~ 60–90 μA/cm².2. Radio-ligand-displacement for IP₃, cAMP and cGMP and ion channel selective drugs in voltage clamp setups were employed to characterize second messengers and Cl⁻, K⁺ and Ca²⁺ channels involved in the ACh response.3. ACh induced a significant rise in IP, in isolated tracheocytes, while ACh did not influence the production of cAMP in whole tissue, isolated tracheocytes or basolateral cell membrane vesicles. Further ACh desensitization did not effect cAMP level in tracheocytes. In addition neither ACh stimulation nor desensitization interfered with cAMP production in presence of 4.5 μM forskolin in tracheocytes, a level of forskolin rising base level cAMP by around five fold.4. Around 35% of ACh Cl⁻ secretion depends on Ca²⁺ mobilization from internal stores and about 65% on Ca²⁺ influx over basolateral membrane. The activated Ca²⁺ channel is insensitive to class I, II, III and IV Ca²⁺ antagonists.5. A 23187 can mimic the ACh effect although 30% is indomethacin-sensitive demonstrating a prostaglandin activated adenylyl cyclase.6. Two K⁺ channels are involved in ACh secretion, one sensitive to Ba²⁺ and quinine and both insensitive to 4-aminopyridine, apamin, charybdotoxin and TEA.7. Flufenamate and triaminopyrimidine block a non-selective ion channel likely involved in the ACh response. An ACh activated apical Cl⁻ channel is NPPB-sensitive.     

K+ Channels and the Intracellular Calcium Signal in Human Melanoma Cell Proliferation

K⁺ channels, membrane voltage, and intracellular free Ca²⁺ are involved in regulating proliferation in a human melanoma cell line (SK MEL 28). Using patch-clamp techniques, we found an inwardly rectifying K⁺ channel and a calcium-activated K⁺ channel. The inwardly rectifying K⁺ channel was calcium independent, insensitive to charybdotoxin, and carried the major part of the whole-cell current. The K⁺ channel blockers quinidine, tetraethylammonium chloride and Ba²⁺ and elevated extracellular K⁺ caused a dose-dependent membrane depolarization. This depolarization was correlated to an inhibition of cell proliferation. Charybdotoxin affected neither membrane voltage nor proliferation. Basic fibroblast growth factor and fetal calf serum induced a transient peak in intracellular Ca²⁺ followed by a long-lasting Ca²⁺ influx. Depolarization by voltage clamp decreased and hyperpolarization increased intracellular Ca²⁺, illustrating a transmembrane flux of Ca²⁺ following its electrochemical gradient. We conclude that K⁺ channel blockers inhibit cell-cycle progression by membrane depolarization. This in turn reduces the driving force for the influx of Ca²⁺, a messenger in the mitogenic signal cascade of human melanoma cells. Received: 9 May 1995/Revised: 30 January 1996     

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K⁺ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue^1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (G_i and G_o) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K⁺ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders³. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations³.Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K⁺ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K⁺ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry⁴ or radiotracer analysis⁵, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.     

Ion channels in the immune system as targets for immunosuppression

The discovery of a diverse and unique subset of ion channels in T lymphocytes has led to a rapidly growing body of knowledge about their functional roles in the immune system. Potent and specific blockers have provided molecules tools to probe channel structure—function relations and to elucidate the involvement of K⁺, Ca²⁺, and Cl⁻ channels in T-cell activation and cell volume regulation. Recent advances in analyzing Kv1.3 channel structure—function relationships have defined binding sites for channel blockers, which have now been shown to be effective in suppressing T-cell function in vivo. Ion channels may provide excellent pharmaceutical targets for modulating immune system function.     

High throughput chemical screening supports the involvement of Ca2+ in cyclic nucleotide-gated ion channel-mediated programmed cell death in Arabidopsis

Recently, we reported the role of Arabidopsis cyclic nucleotide-gated ion channel (AtCNGC) 11 and 12 in Ca²⁺-dependent physiological responses. AtCNGC11 and 12 have been reported to be involved in plant immunity, but whether these channels play additional physiological roles was not clear before. Using single and double knockout mutants, we have found that these channels play significant roles in Ca²⁺ signaling, which mediates several physiological processes, such as gravitropic bending and senescence. Here, we conducted a high throughput, non-biased chemical screen using the gain-of-function mutant of AtCNGC11 and 12, cpr22. Our data presented here indicates that Ca²⁺ but not K⁺ channel blockers suppress AtCNGC11/12-induced lethality. Our data further suggest that AtCNGC11 and 12 are involved in Ca²⁺-dependent, but not K⁺-dependent physiological responses in planta.     

Heat shock proteins and p53 play a critical role in K+ channel-mediated tumor cell proliferation and apoptosis

Plasma membrane potassium (K⁺) channels are required for tumor cell proliferation and apoptosis. However, the signal transduction mechanisms underlying K⁺ channel-dependent tumor cell proliferation or apoptosis remains elusive. Using HeLa and A2780 cells as study models, we tested the hypothesis that apoptotic proteins are linked with K⁺ channel-dependent tumor cell cycle and apoptosis. The patch-clamping study using the whole-cell mode revealed two components of voltage-gated outward K⁺ currents: one is sensitive to either tetraethylammonium (TEA) or tetrandrine (Tet), a maxi-conductance Ca²⁺-activated K⁺ (BK) channel blocker, and the other is sensitive to 4-aminopyridine (4-AP), a delayed rectifier K⁺ channel blocker. MTT and flow cytometry assays showed that TEA, Tet, or iberiotoxin (Ibtx), a selective BK channel blocker, inhibited HeLa and A2780 cell proliferation in a dose-dependent manner with G₁ phase arrest. Pretreatment with TEA or Tet also induced apoptosis in HeLa and A2780 cells. However, glibenclamide (Gli), an ATP-sensitive K⁺ channel blocker, did not influence K⁺ currents, proliferation or apoptosis. Western blot analyses showed that while pretreatment of TEA and Tet produced an increase in expressions of p53, p21, and Bax, pretreatment of these two agents led to a decrease in expressions of heat shock protein (hsp)90α, hsp90β, and hsp70. Our results indicate that the blockade of BK channels results in tumor cell apoptosis and cycle arrest at G₁ phase, and the transduction pathway underlying the anti-proliferative effects is linked to the increased expression of apoptotic protein p53 and the decreased expression of its chaperone proteins hsp.     

Distribution of ion channels on taste cells and its relationship to chemosensory transduction

Summary The presence and regional localization of voltagegated ion channels on taste cells inNecturus maculosus were studied. Lingual epithelium was dissected from the animal and placed in a modified Ussing chamber such that individual taste cells could be impaled with intracellular microelectrodes and the chemical environment of the apical and basolateral membranes of cells could be strictly controlled. That is, solutions bathing the the mucosal and serosal surfaces of the epithelium could be exchanged independently and the effects of pharmacological agents could be tested selectively on the apical or basolateral membranes of taste cells. In the presence of amphibian physiological saline, action potentials were elicited by passing brief depolarizing current pulses through the recording electrode. Action potentials provided a convenient assay of voltage-gated ion channels. As in other excitable tissues, blocking current through Na⁺, K⁺, or Ca²⁺ channels had predictable and consistent effects on the shape and magnitude of the action potential. A series of experiments was conducted in which the shape and duration of regenerative action potentials were monitored when the ionic composition was altered and/or pharmacological blocking agents were added to the mucosal or to the serosal chamber. We have found the following: (1) voltage-gated K⁺ channels (delayed rectifier) are found predominately, if not exclusively, on the chemoreceptive apical membrane; (ii) voltage-gated Na⁺ and Ca²⁺ channels are found on the apical (chemoreceptive) and basolateral (synaptic) membrane; (iii) there is a K⁺ leak channel on the basolateral membrane which appears to vary seasonally in its sensitivity to TEA. The nonuniform distribution of voltage-gated K⁺ channels and their predominance on the apical membrane may be important in taste transduction: alterations in apical K⁺ conductance may underlie receptor potentials ellicted by rapid stimuli.     

Functional role of the N-terminus of Na,K-ATPase α-subunit as an inactivation gate of palytoxin-induced pump channel

The N-terminus of the Na⁺,K⁺-ATPase α-subunit shows some homology to that of Shaker-B K⁺ channels; the latter has been shown to mediate the N-type channel inactivation in a ball-and-chain mechanism. When the Torpedo Na⁺,K⁺-ATPase is expressed in Xenopus oocytes and the pump is transformed into an ion channel with palytoxin (PTX), the channel exhibits a time-dependent inactivation gating at positive potentials. The inactivation gating is eliminated when the N-terminus is truncated by deleting the first 35 amino acids after the initial methionine. The inactivation gating is restored when a synthetic N-terminal peptide is applied to the truncated pumps at the intracellular surface. Truncated pumps generate no electrogenic current and exhibit an altered stoichiometry for active transport. Thus, the N-terminus of the α-subunit appears to act like an inactivation gate and performs a critical step in the Na⁺,K⁺-ATPase pumping function.     

Involvement of Ca2+ Channel Signalling in Sclerotial Formation of Polyporus umbellatus

Growth and morphogenesis transformation in Polyporus umbellatus were examined in the presence of various pharmacological compounds, to investigate signal transduction pathways that influence the development of sclerotia. Both the calcium channel blocker nifedipine and the calcium ionophor A23187 reduced sclerotial production in P. umbellatus; four classes of Ca²⁺ signal agent—including calcium chelators, calcium channel blockers, calcium ionophors and calmodulin inhibitors—were further studied. Among them, EGTA and BAPTA, as calcium chelators, exhibited a complete inhibitory effect on sclerotial formation, among the levels tested. Calcium channel blockers and calcium ionophors at the concentrations used in this study could not eliminate sclerotia formation completely, but did greatly reduce sclerotial production. Notoginsenoside in dosages >250 μg/ml produced a significant negative effect on mycelial growth, and it prevented sclerotial formation entirely at a dosage of 500 μg/ml; no other drug influenced vegetative growth at all. The calcium ionophor A23187 did not decrease sclerotial mean weight at low doses (20 nM); at higher doses (200 nM), however, sclerotial development was significantly reduced, albeit not completely halted. The CaM inhibitors (W-7 and chlorpromazine) could each completely stop sclerotial formation. Using Fluo-3/AM as the indicator of cytosolic free calcium, the Ca²⁺ content in the cytoplasm was found to have decreased significantly when hyphae were treated with different drugs, and there was no active Ca²⁺ signal in the sclerotial mycelium. In general, the results suggest that Ca²⁺ signal transduction may play an important role in sclerotial formation in P. umbellatus.     

Molecular electronics in pinnae of Mimosa pudica

Bioelectrochemical circuits operate in all plants including the sensitive plant Mimosa pudica Linn. The activation of biologically closed circuits with voltage gated ion channels can lead to various mechanical, hydrodynamical, physiological, biochemical and biophysical responses. Here the biologically closed electrochemical circuit in pinnae of Mimosa pudica is analyzed using the charged capacitor method for electrostimulation at different voltages. Also the equivalent electrical scheme of electrical signal transduction inside the plant''s pinna is evaluated. These circuits remain linear at small potentials not exceeding 0.5 V. At higher potentials the circuits become strongly non-linear pointing to the opening of ion channels in plant tissues. Changing the polarity of electrodes leads to a strong rectification effect and to different kinetics of a capacitor. These effects can be caused by a redistribution of K⁺, Cl⁻, Ca²⁺ and H⁺ ions through voltage gated ion channels. The electrical properties of Mimosa pudica were investigated and equivalent electrical circuits within the pinnae were proposed to explain the experimental data.Key words: electrophysiology, plant cell electrostimulation, charged capacitor method, electrical circuits, electrical signaling, Mimosa pudica     

The Permeability of the Sodium Channel to Metal Cations in Myelinated Nerve

The relative permeability of sodium channels to eight metal cations is studied in myelinated nerve fibers. Ionic currents under voltage-clamp conditions are measured in Na-free solutions containing the test ion. Measured reversal potentials and the Goldman equation are used to calculate the permeability sequence: Na⁺ ≈ Li⁺ > Tl⁺ > K⁺. The ratio P_K/P_Na is 1/12. The permeabilities to Rb⁺, Cs⁺, Ca⁺⁺, and Mg⁺⁺ are too small to measure. The permeability ratios agree with observations on the squid giant axon and show that the reversal potential E_Na differs significantly from the Nernst potential for Na⁺ in normal axons. Opening and closing rates for sodium channels are relatively insensitive to the ionic composition of the bathing medium, implying that gating is a structural property of the channel rather than a result of the movement or accumulation of particular ions around the channel. A previously proposed pore model of the channel accommodates the permeant metal cations in a partly hydrated form. The observed sequence of permeabilities follows the order expected for binding to a high field strength anion in Eisenman's theory of ion exchange equilibria.     

Imaging of Calcium Channels During Polarity Induction in Plant Cells

Understanding the molecular basis of polarity induction in plant cells is a research aspect that extends from signal perception and transduction to morphogenesis. A gradient of cytoplasmic ion fluxes generated through ion channels plays a crucial role in subsequent events leading to polar growth. Convincing evidence is now available implicating temporal and spatial distribution of Ca²⁺ in cytoplasm, generated by localized activity of calcium channels, as the early biochemical events associated with polarity induction. Ion channel antagonists are common tools for studying ion channel structure and function. Coupled with a fluorescent dyes, calcium channel antagonists (phenylalkylamine and dihydropyridine), have been used to localize L-type calcium channels. Additionally, the advent of Confocal Laser Scanning Microscopy has made possible the visualization of Ca²⁺ channels in plant cells. Persisting problems of dye loading and their cellular compartmentation have been addressed by developing a variety of experimental protocols. Present article highlights the current state of our understanding of these concepts, methodologies and their applications in different aspects of plant development.     

Comparison of the inhibitory effects of brain extract,high K+ and forskolin on juvenile hormone synthesis by Diploptera punctata corpora allata

We compared the effects of three treatments (brain extracts, high K⁺, forskolin) which inhibit JH synthesis by corpora allata of the cockroach Diploptera punctata incubated in vitro. Corpora allata showed a similar developmental sensitivity to all three treatments, with a high level of inhibition in glands of low activity. Inhibition of JH III synthesis by forskolin, high K⁺ or brain extract was antagonized by mevalonate and farnesoic acid, thus implicating an early biosynthetic step as the target of inhibition. All three treatments were also antagonized by Mn²⁺, suggesting that the inhibitory mode of action may be dependent upon Ca²⁺. Inhibition for forskolin and by high K⁺ was not only reversible, but caused a stimulation of JH synthesis upon removal of the inhibitor, suggesting that more than one regulatory mechanism is affected by these treatments.     

Ion channels of glutamate receptors: structural modeling

Ionotropic glutamate receptors belong to the superfamily of P-loop channels as well as K⁺, Na⁺, and Ca²⁺ channels. However, the structural similarity between ion channels of the glutamate receptors and K⁺ channels is a matter of discussion. The aim of this study was to analyze differences between the structures of K⁺ channels and glutamate receptor channels. For this purpose, homology models of NMDA and AMPA receptor channels (M2 and M3 segments) were built using X-ray structures of K⁺ channels as templates. The models were optimized and used to reproduce specific data on the structure of glutamate receptor channels. Particular attention was paid to the data of the binding of channel blockers and to the results of scanning mutagenesis. The modeling demonstrates that properties of glutamate receptor channel can be reproduced assuming only local structural deformations of the K⁺ channel templates. The most valuable differences were found in the selectivity-filter region, whereas helical parts of M2 and M3 segments could have similar spatial organization with homologous segments in K⁺ channels. It is concluded that the current experimental data on glutamate receptor channels does not reveal global structural differences with K⁺ channels.