Transient Membrane Depolarization Research Articles

TRPV1-Expressing Sensory Neuron Stimulation-based Model of Inflammatory Injury Enhances the Excitability of Spinal Neurons Targeting the Periaqueductal Gray

Modeling inflammatory injury via high-intensity stimulation of peripheral afferents at low frequencies (LFS) potentiates excitatory input onto spinal neurons projecting (PNs) to the midbrain periaqueductal gray (PAG), a region critical to pain processing. Therefore, we sought to identify the underlying afferent population driving this potentiation and how this enhanced synaptic drive might affect spino-PAG PN output to supraspinal pain circuits. We used 3–6-week-old male and female mice expressing Cre recombinase driven by expression of the TRPV1 gene, which is expressed in the lineage of virtually all C fibers, and Cre-dependent channelrhodopsin to stimulate C fibers. Spinal PNs were retrogradely labeled from the PAG using diI. We performed whole-cell patch-clamp recordings in spino-PAG PNs using a semi-intact spinal cord preparation. Stimulation of TRPV1-expressing (TRPV1+) peripheral afferents induced burst firing in virtually all spino-PAG PNs sampled. Inflammation increases the firing rates of C fibers to 1-2 Hz; therefore, we used LFS (1 ms, 470 nm LED pulses at 2 Hz for 2 mins) to model inflammatory injury. LFS induced a persistent (≥20 min) increase in the number of action potentials (APs) within a C fiber-induced burst (n=10, p=0.029), rise in the frequency of synaptically evoked after discharge (n=10, p=0.0004), and decrease in intraburst afterhyperpolarization (n=10, p=0.037). Furthermore, LFS induced a transient membrane depolarization (Mean=6.796 mV, n=12, p<0.0001). Additional experiments suggested the enhanced synaptic excitability relied on postsynaptic G protein-coupled signaling, NMDA receptors, and TRPV1+ afferent input. Finally, LFS may increase the intrinsic excitability of spino-PAG PNs—by decreasing AP threshold (n=14, p=0.015) and increasing membrane resistance (n=14, p=0.0003). LFS had no effect on the membrane potential nor the firing frequency of spino-PAG PNs. In summary, this work suggests that LFS of TRPV1+ fibers persistently enhances spino-PAG projection neuron output, likely leading to a lasting increase in the activation of PAG neural circuits. Grant support from F32NS123008 NINDS; Brewer, C. B. T32DA035165 NIDA; Mackey, S. C. R01DA011289 NIDA; Kauer, J. A.

Remodeling of the ryanodine receptor isoform 1 channel regulates the sweet and umami taste perception of Rattus norvegicus

Sweet and umami tastes are elicited by sweet and umami receptors on the tongue and palate epithelium, respectively. However, the molecular machinery allowing the taste reaction remains incompletely understood. Through a phosphoproteomic approach, we identified the key proteins that trigger taste mechanisms based on phosphorylation cascades. Ryanodine receptor isoform 1 (RYR1) was further verified by sensory and behavioral assays. We propose a model of RYR1-mediated sweet/umami signaling in which the RYR1 channel, which mediates Ca2+ release from the endoplasmic reticulum, is closed by dephosphorylation in bud tissue after sweet/umami treatment. The alteration in Ca2+ content in the cytosol induces transient membrane depolarization and generates a cell current for taste signal transduction. We demonstrate that RYR1 is a new channel involved in the regulation of sweet/umami signal transduction and propose a “metabolic clock” notion based on sweet/umami sensing. Our study provides a valuable foundation for a system-level understanding of the taste perception mechanism.

Protons Trigger Mitochondrial Flashes

Emerging evidence indicates that mitochondrial flashes (mitoflashes) are highly conserved elemental mitochondrial signaling events. However, which signal controls their ignition and how they are integrated with other mitochondrial signals and functions remain elusive. In this study, we aimed to further delineate the signal components of the mitoflash and determine the mitoflash trigger mechanism. Using multiple biosensors and chemical probes as well as label-free autofluorescence, we found that the mitoflash reflects chemical and electrical excitation at the single-organelle level, comprising bursting superoxide production, oxidative redox shift, and matrix alkalinization as well as transient membrane depolarization. Both electroneutral H+/K+ or H+/Na+ antiport and matrix proton uncaging elicited immediate and robust mitoflash responses over a broad dynamic range in cardiomyocytes and HeLa cells. However, charge-uncompensated proton transport, which depolarizes mitochondria, caused the opposite effect, and steady matrix acidification mildly inhibited mitoflashes. Based on a numerical simulation, we estimated a mean proton lifetime of 1.42 ns and diffusion distance of 2.06 nm in the matrix. We conclude that nanodomain protons act as a novel, to our knowledge, trigger of mitoflashes in energized mitochondria. This finding suggests that mitoflash genesis is functionally and mechanistically integrated with mitochondrial energy metabolism.

Axonal model for temperature stimulation

Recent studies indicate that a rapid increase in local temperature plays an important role in nerve stimulation by laser. To analyze the temperature effect, our study modified the classical HH axonal model by incorporating a membrane capacitance-temperature relationship. The modified model successfully simulated the generation and propagation of action potentials induced by a rapid increase in local temperature when the Curie temperature of membrane capacitance is below 40°C, while the classical model failed to simulate the axonal excitation by temperature stimulation. The new model predicts that a rapid increase in local temperature produces a rapid increase in membrane capacitance, which causes an inward membrane current across the membrane capacitor strong enough to depolarize the membrane and generate an action potential. If the Curie temperature of membrane capacitance is 31°C, a temperature increase of 6.6-11.2°C within 0.1-2.6ms is required for axonal excitation and the required increase is smaller for a faster increase. The model also predicts that: (1) the temperature increase could be smaller if the global axon temperature is higher; (2) axons of small diameter require a smaller temperature increase than axons of large diameter. Our study indicates that the axonal membrane capacitance-temperature relationship plays a critical role in inducing the transient membrane depolarization by a rapidly increasing temperature, while the effects of temperature on ion channel kinetics cannot induce depolarization. The axonal model developed in this study will be very useful for analyzing the axonal response to local heating induced by pulsed infrared laser.

Satellite microglia show spontaneous electrical activity that is uncorrelated with activity of the attached neuron.

Microglia are innate immune cells of the brain. We have studied a subpopulation of microglia, called satellite microglia. This cell type is defined by a close morphological soma-to-soma association with a neuron, indicative of a direct functional interaction. Indeed, ultrastructural analysis revealed closely attached plasma membranes of satellite microglia and neurons. However, we found no apparent morphological specializations of the contact, and biocytin injection into satellite microglia showed no dye-coupling with the apposed neurons or any other cell. Likewise, evoked local field potentials or action potentials and postsynaptic potentials of the associated neuron did not lead to any transmembrane currents or non-capacitive changes in the membrane potential of the satellite microglia in the cortex and hippocampus. Both satellite and non-satellite microglia, however, showed spontaneous transient membrane depolarizations that were not correlated with neuronal activity. These events could be divided into fast-rising and slow-rising depolarizations, which showed different characteristics in satellite and non-satellite microglia. Fast-rising and slow-rising potentials differed with regard to voltage dependence. The frequency of these events was not affected by the application of tetrodotoxin, but the fast-rising event frequency decreased after application of GABA. We conclude that microglia show spontaneous electrical activity that is uncorrelated with the activity of adjacent neurons.

Reduction of Trial-to-Trial Perceptual Variability by Intracortical Tonic Inhibition.

Variability is a prominent characteristic of cognitive brain function. For instance, different trials of presentation of the same stimulus yield higher variability in its perception: subjects sometimes fail in perceiving the same stimulus. Perceptual variability could be attributable to ongoing-spontaneous fluctuation in neuronal activity prior to sensory stimulation. Simulating a cortical neural network model, we investigated the underlying neuronal mechanism of perceptual variability in relation to variability in ongoing-spontaneous neuronal activity. In the network model, populations of principal cells (cell assemblies) encode information about sensory features. Each cell assembly is sensitive to one particular feature stimulus. Transporters on GABAergic interneurons regulate ambient GABA concentration in a neuronal activity-dependent manner. Ambient GABA molecules activate extrasynaptic GABAa receptors on principal cells and interneurons, and provide them with tonic inhibitory currents. We controlled the variability of ongoing-spontaneous neuronal activity by manipulating the basal level of ambient GABA and assessed the perceptual performance of the network: detection of a feature stimulus. In an erroneous response, stimulus-irrelevant but not stimulus-relevant principal cells were activated, generating trains of action potentials. Perceptual variability, reflected in error rate in detecting the same stimulus that was presented repeatedly to the network, was increased as the variability in ongoing-spontaneous membrane potential among cell assemblies increased. Frequent, transient membrane depolarization below firing threshold was the major cause of the increased neuronal variability, for which a decrease in basal ambient GABA concentration was responsible. We suggest that ambient GABA in the brain may have a role in reducing the variability in ongoing-spontaneous neuronal activity, leading to a decrease in perceptual variability and therefore to reliable sensory perception.

The defence elicitor AsES causes a rapid and transient membrane depolarization, a triphasic oxidative burst and the accumulation of nitric oxide

The newly characterized elicitor AsES obtained from Acremonium strictum induces a strong defence response in strawberry plants and confers plants resistance against the fungal pathogen Colletotricum acutatum the casual agent of anthracnose disease. Previous studies showed that AsES causes the accumulation of reactive oxygen species (ROS) that peaked 4 h post treatment (hpt), but due to the experimental approach used it was not clear whether the accumulation of ROS observed was intracellular or extracellular or took place as a single peak. By using a different experimental setup, a more complex early events associated to the activation of the innate immunity were observed. In this paper we report that strawberry plant cells treated with AsES exhibits a triphasic production of H2O2 and a rapid intracellular accumulation of NO. The first phase consists in a progressive extracellular accumulation of H2O2 that starts immediately after the treatment with AsES and is preceded by a rapid and transient cell membrane depolarization. During this phase takes place also a rapid intracellular accumulation of NO. Microscopic observations of mesophyll cells treated with AsES reveals that NO accumulates at the chloroplast. After the first extracellular H2O2 production phase, two intracellular H2O2 accumulation events occur, the first 2 hpt, and the second 7 hpt. Cells treated with AsES also show a transient increase of ion leakage, and a progressive alkalinization of the extracellular medium.

A positive feedback at the cellular level promotes robustness and modulation at the circuit level.

This article highlights the role of a positive feedback gating mechanism at the cellular level in the robustness and modulation properties of rhythmic activities at the circuit level. The results are presented in the context of half-center oscillators, which are simple rhythmic circuits composed of two reciprocally connected inhibitory neuronal populations. Specifically, we focus on rhythms that rely on a particular excitability property, the postinhibitory rebound, an intrinsic cellular property that elicits transient membrane depolarization when released from hyperpolarization. Two distinct ionic currents can evoke this transient depolarization: a hyperpolarization-activated cation current and a low-threshold T-type calcium current. The presence of a slow activation is specific to the T-type calcium current and provides a slow positive feedback at the cellular level that is absent in the cation current. We show that this slow positive feedback is required to endow the network rhythm with physiological modulation and robustness properties. This study thereby identifies an essential cellular property to be retained at the network level in modeling network robustness and modulation.

Linking oxygen availability with membrane potential maintenance and K+ retention of barley roots: implications for waterlogging stress tolerance.

Oxygen deprivation is a key determinant of root growth and functioning under waterlogging. In this work, changes in net K(+) flux and membrane potential (MP) of root cells were measured from elongation and mature zones of two barley varieties under hypoxia and anoxia conditions in the medium, and as influenced by ability to transport O2 from the shoot. We show that O2 deprivation results in an immediate K(+) loss from roots, in a tissue- and time-specific manner, affecting root K(+) homeostasis. Both anoxia and hypoxia induced transient membrane depolarization; the extent of this depolarization varied depending on severity of O2 stress and was less pronounced in a waterlogging-tolerant variety. Intact roots of barley were capable of maintaining H(+) -pumping activity under hypoxic conditions while disrupting O2 transport from shoot to root resulted in more pronounced membrane depolarization under O2 -limited conditions and in anoxia a rapid loss of the cell viability. It is concluded that the ability of root cells to maintain MP and cytosolic K(+) homeostasis is central to plant performance under waterlogging, and efficient O2 transport from the shoot may enable operation of the plasma membrane H(+) -ATPase in roots even under conditions of severe O2 limitation in the soil solution.

Modulation of cytotoxic and genotoxic effects of nanoparticles in cancer cells by external magnetic field.

Magnetic nanoparticles are well known for anticancer activity by deregulating cellular functions. In the present study, cellular effects of low strength static magnetic field (SMF) were explored. How nanoparticles affect the cellular response in presence and absence of static magnetic field was also studied. Peripheral blood mononuclear cells (PBMC) and human lymphoma monocytic cell line U937 were chosen as representative normal and cancer cells models. The two effects we would like to report in this paper are, DNA damage induced by SMF of the order of 70 mT, and alteration in membrane potential. The other notable aspect was the changes were diametrically opposite in normal and cancer cell types. DNA damage was observed only in cancer cells whereas membrane depolarization was observed in normal cells. Iron oxide nanoparticles (IONP) and gold nanoparticles (AuNP) were also used for cellular response studies in presence and absence of SMF. The effects of the magnetic nanoparticle IONP and also of AuNP were sensitive to presence of SMF. Unlike cancer cells, normal cells showed a transient membrane depolarization sensitive to static magnetic field. This depolarization effect exclusive for normal cells was suggested to have correlations with their higher repair capacity and lesser propensity for DNA damage. The work shows cancer cells and normal cells respond to nanoparticle and static magnetic field in different ways. The static magnetic induced DNA damage observed exclusively in cancer cells may have therapeutic implications. From the conclusions of the present investigation we may infer that static magnetic field enhances the therapeutic potentials of nanoparticles. Such low strength magnetic field seems to be a promising external manoeuvring agent in designing theranostics.Electronic supplementary materialThe online version of this article (doi:10.1186/s12645-014-0002-x) contains supplementary material, which is available to authorized users.

Zn2+ -induced changes at the root level account for the increased tolerance of acclimated tobacco plants.

Evidence suggests that heavy-metal tolerance can be induced in plants following pre-treatment with non-toxic metal concentrations, but the results are still controversial. In the present study, tobacco plants were exposed to increasing Zn2+ concentrations (up to 250 and/or 500 μM ZnSO4) with or without a 1-week acclimation period with 30 μM ZnSO4. Elevated Zn2+ was highly toxic for plants, and after 3 weeks of treatments there was a marked (≥50%) decline in plant growth in non-acclimated plants. Plant acclimation, on the other hand, increased plant dry mass and leaf area up to 1.6-fold compared with non-acclimated ones. In non-acclimated plants, the addition of 250 μM ZnSO4 led to transient membrane depolarization and stomatal closure within 24h from the addition of the stress; by contrast, the acclimation process was associated with an improved stomatal regulation and a superior ability to maintain a negative root membrane potential, with values on average 37% more negative compared with non-acclimated plants. The different response at the plasma-membrane level between acclimated and non-acclimated plants was associated with an enhanced vacuolar Zn2+ sequestration and up to 2-fold higher expression of the tobacco orthologue of the Arabidopsis thaliana MTP1 gene. Thus, the acclimation process elicited specific detoxification mechanisms in roots that enhanced Zn2+ compartmentalization in vacuoles, thereby improving root membrane functionality and stomatal regulation in leaves following elevated Zn2+ stress.

Sonoporation-Induced Depolarization of Plasma Membrane Potential: Analysis of Heterogeneous Impact

Disrupting plasma membrane integrity would inevitably promote anomalous ion fluxes across the membrane and thereby upset the trans-membranous potential. In this article, we report new findings on how sonoporation as a physical membrane perforation strategy would lead to different forms of plasma membrane potential disruption. Our investigation was conducted with a customized fluorescence imaging platform that enabled live monitoring of plasma membrane potential in relation to individual sonoporation events triggered on HeLa cervical cancer cells. Sonovue microbubbles were used as sonoporation agents (added at a 4:3 cell-to-bubble ratio), and they were activated by 1-MHz pulsed ultrasound with 0.35-MPa peak negative pressure, 20-cycle pulse duration, 20-Hz pulse repetition frequency and 1-s total exposure duration. Results indicate that the plasma membrane potential response was heterogeneous among sonoporated cells: (i) membrane potential of irreversibly sonoporated cells was permanently depolarized; (ii) reversibly sonoporated cells exhibited either transient or sustained membrane depolarization; (iii) intact cells adjacent to sonoporated ones underwent transitory membrane depolarization. These findings effectively serve to substantiate the causal relationship between sonoporation and plasma membrane potential.

Proinflammatory Cytokines Stimulate Mitochondrial Superoxide Flashes in Articular Chondrocytes In Vitro and In Situ

ObjectiveMitochondria play important roles in many types of cells. However, little is known about mitochondrial function in chondrocytes. This study was undertaken to explore possible role of mitochondrial oxidative stress in inflammatory response in articular chondrocytes.MethodsChondrocytes and cartilage explants were isolated from wild type or transgenic mice expressing the mitochondrial superoxide biosensor - circularly permuted yellow fluorescent protein (cpYFP). Cultured chondrocytes or cartilage explants were incubated in media containing interleukin-1β (10 ng/ml) or tumor necrosis factor-α (10 ng/ml) to stimulate an inflammatory response. Mitochondrial imaging was carried out by confocal and two-photon microscopy. Mitochondrial oxidative status was evaluated by “superoxide flash” activity recorded with time lapse scanning.ResultsCultured chondrocytes contain abundant mitochondria that show active motility and dynamic morphological changes. In intact cartilage, mitochondrial abundance as well as chondrocyte density declines with distance from the surface. Importantly, sudden, bursting superoxide-producing events or “superoxide flashes” occur at single-mitochondrion level, accompanied by transient mitochondrial swelling and membrane depolarization. The superoxide flash incidence in quiescent chondrocytes was ∼4.5 and ∼0.5 events/1000 µm2*100 s in vitro and in situ, respectively. Interleukin-1β or tumor necrosis factor-α stimulated mitochondrial superoxide flash activity by 2-fold in vitro and 5-fold in situ, without altering individual flash properties except for reduction in spatial size due to mitochondrial fragmentation.ConclusionsThe superoxide flash response to proinflammatory cytokine stimulation in vitro and in situ suggests that chondrocyte mitochondria are a significant source of cellular oxidants and are an important previously under-appreciated mediator in inflammatory cartilage diseases.

Sensitization of gram‐negative bacteria by targeting the membrane potential

Toward generating new tools for fighting multidrug-resistant (MDR) bacteria, we assessed the ability of a membrane-active peptide to sensitize gram-negative bacteria to various antibiotics. The mechanism for affecting inner and/or outer membrane functions was assessed by complementary biophysical methods (SPR, DSC, ITC). The implication of efflux pumps was examined using Acr-AB mutants, as tested with representative antibiotics, host defense peptides, and synthetic mimics. The ability to affect disease course systemically was compared for a single therapy and combination therapy, using the mouse thigh-infection model. The data show that potent antibiotic action can be provoked in vitro and in vivo, by a treatment combining two antibacterial compounds whose individual inefficiency against gram-negative bacteria stems from their efflux. Thus, at subminimal inhibitory concentrations, the lipopeptide-like sequence, N(α)(ω7)dodecenoyl-lysyl-[lysyl-aminododecanoyl-lysyl]-amide (designated C12(ω7)K-β12), has, nonetheless, rapidly achieved a transient membrane depolarization, which deprived bacteria of the proton-motive force required for active efflux. Consequently, bacteria became significantly sensitive to intracellular targeting antibiotics. Collectively, these findings suggest a potentially useful approach for expanding the antibiotics sensitivity spectrum of MDR gram-negative bacteria to include efflux substrates.

Pharmacological Potentiators of TRPM5 Channel Activity

TRPM5 belongs to the ‘melastatin’ transient receptor potential subfamily, fluxes mainly Na+, and is activated by cytosolic Ca2+ increases (&lt; 1 μM). TRPM5 activates and deactivates rapidly even during sustained Ca2+ rises, which leads to transient membrane depolarization kinetics. Only the Ca2+ ionophore ionomycin yielded significant and transient membrane potential (MP) signals in HEK293 cells stably expressing TRPM5. Various agonists for endogenous Gq‐GPCRs induced significant Ca2+ signals, but did not or only weakly activate TRPM5. Five tool potentiators were assessed using two TRPM5‐expressing cell clones, and various pharmacological concentrations of the indirect stimulator ionomycin. For both cell lines, potencies and efficacies of potentiators were best using an EC80 (rather than an EC20) concentration of ionomycin. This indirectly indicates that maximal cytosolic Ca2+ increases lead only to submaximal TRPM5 activation. EC60–80 concentrations of ionomycin yielded acceptable MP assay statistics, and a focused compound library was tested to find further and more potent TRPM5 potentiators. 114 TRPM5 potentiator hits were found (EC50 values: 0.11 –40 μM), and 67 thereof confirmed in a patch clamp assay. 38 of these novel TRPM5 potentiators increased currents over 100%. This work was supported by the Novartis Institutes for BioMedical Research (NIBR).

Transduction of pressure signal to electrical signal upon sudden increase in turgor pressure in Chara corallina

By taking advantage of large cell size of Chara corallina, we analyzed the membrane depolarization induced by decreased turgor pressure (Shimmen in J Plant Res 124:639-644, 2011). In the present study, the response to increased turgor pressure was analyzed. When internodes were incubated in media containing 200 mM dimethyl sulfoxide, their intracellular osmolality gradually increased and reached a steady level after about 3 h. Upon removal of dimethyl sulfoxide, turgor pressure quickly increased. In response to the increase in turgor pressure, the internodes generated a transient membrane depolarization at its nodal end. The refractory period was very long and it took about 2 h for full recovery after the depolarizing response. Involvement of protein synthesis in recovery from refractoriness was suggested, based on experiments using inhibitors.

Extracellular ATP acts on P2Y2 purinergic receptors to facilitate HIV-1 infection

Extracellular adenosine triphosphate (ATP) can activate purinergic receptors of the plasma membrane and modulate multiple cellular functions. We report that ATP is released from HIV-1 target cells through pannexin-1 channels upon interaction between the HIV-1 envelope protein and specific target cell receptors. Extracellular ATP then acts on purinergic receptors, including P2Y2, to activate proline-rich tyrosine kinase 2 (Pyk2) kinase and transient plasma membrane depolarization, which in turn stimulate fusion between Env-expressing membranes and membranes containing CD4 plus appropriate chemokine co-receptors. Inhibition of any of the constituents of this cascade (pannexin-1, ATP, P2Y2, and Pyk2) impairs the replication of HIV-1 mutant viruses that are resistant to conventional antiretroviral agents. Altogether, our results reveal a novel signaling pathway involved in the early steps of HIV-1 infection that may be targeted with new therapeutic approaches.

Thermo-sensitive response based on the membrane fluidity adaptation inParamecium multimicronucleatum

Relationships between the thermo-sensitive response and membrane lipid fluidity were studied using a ciliated protozoan, Paramecium multimicronucleatum. Paramecium elicits a transient membrane depolarization in response to a cooling stimulus (temperature drop). The depolarization amplitude was largest when the cooling stimulus was started from the culture temperature, whilst when cooling started at a temperature more than 5 degrees C higher or lower than the culture temperature, only a small depolarization was induced. Therefore, the cooling-induced response was dependent on the culture temperature and its sensitivity to the cooling stimulus was highest at the culture temperature. Membrane fluidity measurements of living cells using the fluorescent dye 6-lauroyl-2-dimethylaminonaphthalene (laurdan) showed that the fluidity measured at the culture temperature was almost constant irrespective of the temperature at which the cells had been cultured and adapted, which is consistent with homeoviscous adaptation. The constant fluidity at the culture temperature quickly decreased within a few seconds of application of the cooling stimulus, and the decreased fluidity gradually readapted to a constant level at the decreased temperature within 1 h. When the constant fluidity at culture temperature was modified by the addition of procaine or benzyl alcohol, the cooling-induced depolarization was completely abolished. These results suggest the possibility that the adaptation of fluidity to a constant level and its quick decrease below the constant level activate cooling-sensitive channels to elicit the transient depolarization.

CB1 cannabinoid receptor‐dependent and ‐independent inhibition of depolarization‐induced calcium influx in oligodendrocytes

Regulation of Ca(2+) homeostasis plays a critical role in oligodendrocyte function and survival. Cannabinoid CB(1) and CB(2) receptors have been shown to regulate Ca(2+) levels and/or K(+) currents in a variety of cell types. In this study we investigated the effect of cannabinoid compounds on the Ca(2+) influx elicited in cultured oligodendrocytes by transient membrane depolarization with an elevated extracellular K(+) concentration (50 mM). The CB(1) receptor agonist arachidonoyl-chloro-ethanolamide (ACEA) elicited a concentration-dependent inhibition of depolarization-evoked Ca(2+) transients in oligodendroglial somata with a maximal effect (94+/-3)% and an EC(50) of 1.3+/-0.03 microM. This activity was mimicked by the CB(1)/CB(2) agonist CP55,940, as well as by the endocannabinoids N-arachidonoyl-ethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG), whereas the CB(2) receptor selective agonist JWH133 was ineffective. The CB(1) receptor antagonist AM251 (1 microM) also reduced the Ca(2+) response evoked by high extracellular K(+) and did not prevent the inhibition elicited by ACEA (3 microM). Nevertheless, the ability of ACEA and AEA to reduce depolarization-evoked Ca(2+) transients was significantly reduced in oligodendrocytes from CB(1) receptor knockout mice, as well as by pretreatment with pertussis toxin. Bath application of the inwardly rectifying K(+) channels (Kir channels) blockers BaCl(2) (300 microM) and CsCl(2) (1 mM) reduced the size of voltage-induced Ca(2+) influx and partially prevented the inhibitory effect of ACEA. Our results indicate that cannabinoids inhibit depolarization-evoked Ca(2+) transients in oligodendrocytes via CB(1) receptor-independent and -dependent mechanisms that involve the activation of PTX-sensitive G(i/o) proteins and the blockade of Kir channels.

Glutamate receptor subtypes evidenced by differences in desensitization and dependence on the GLR3.3 and GLR3.4 genes.

Ionotropic glutamate (Glu) receptors in the central nervous system of animals are tetrameric ion channels that conduct cations across neuronal membranes upon binding Glu or another agonist. Plants possess homologous molecules encoded by GLR genes. Previous studies of Arabidopsis thaliana root cells showed that the amino acids alanine (Ala), asparagine (Asn), cysteine (Cys), Glu, glycine (Gly), and serine trigger transient Ca(2+) influx and membrane depolarization by a mechanism that depends on the GLR3.3 gene. This study of hypocotyl cells demonstrates that these six effective amino acids are not equivalent agonists. Instead, they grouped into hierarchical classes based on their ability to desensitize the response mechanism. Sequential treatment with two different amino acids separated by a washout phase demonstrated that Glu desensitized the depolarization mechanism to Gly, but Gly did not desensitize the mechanism to Glu. All 36 possible pairs of agonists were tested to characterize the desensitization hierarchy. The results could be explained by a model in which one class of channels contained a subunit that was activated and therefore desensitized only by Glu, while a second class could be activated and desensitized by Ala, Cys, Glu, or Gly. A third class could be activated and desensitized by any of the six effective amino acids. Analysis of knockout mutants indicated that GLR3.3 was a required component of all three classes of channels, while the related GLR3.4 molecule specifically affected only two of the classes. The resulting model is an important step toward understanding the biological roles of these enigmatic ion channels.