Ca(2+)-activated anion channels and membrane depolarizations induced by blue light and cold in Arabidopsis seedlings.

The activation of an anion channel in the plasma membrane of Arabidopsis thaliana hypocotyls by blue light (BL) is believed to be a signal-transducing event leading to growth inhibition. Here we report that the open probability of this particular anion channel depends on cytoplasmic Ca2+ ([Ca2+]cyt) within the concentration range of 1 to 10 microM, raising the possibility that BL activates the anion channel by increasing [Ca2+]cyt. Arabidopsis seedlings cytoplasmically expressing aequorin were generated to test this possibility. Aequorin luminescence did not increase during or after BL, providing evidence that Ca2+ does not play a second-messenger role in the activation of anion channels. However, cold shock simultaneously triggered a large increase in [Ca2+]cyt and a 110-mV transient depolarization of the plasma membrane. A blocker of the anion channel, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, blocked 61% of the cold-induced depolarization without affecting the increase in [Ca2+]cyt. These data led us to propose that cold shock opens Ca2+ channels at the plasma membrane, allowing an inward, depolarizing Ca2+ current. The resulting large increase in [Ca2+]cyt activates the anion channel, which further depolarizes the membrane. Although an increase in [Ca2+]cyt may activate anion channels in response to cold, it appears that BL does so via a Ca(2+)-independent pathway.     

Early signaling through the Arabidopsis pattern recognition receptors FLS2 and EFR involves Ca2+‐associated opening of plasma membrane anion channels

The perception of microbes by plants involves highly conserved molecular signatures that are absent from the host and that are collectively referred to as microbe‐associated molecular patterns (MAMPs). The Arabidopsis pattern recognition receptors FLAGELLIN‐SENSING 2 (FLS2) and EF‐Tu receptor (EFR) represent genetically well studied paradigms that mediate defense against bacterial pathogens. Stimulation of these receptors through their cognate ligands, bacterial flagellin or bacterial elongation factor Tu, leads to a defense response and ultimately to increased resistance. However, little is known about the early signaling pathway of these receptors. Here, we characterize this early response in situ, using an electrophysiological approach. In line with a release of negatively charged molecules, voltage recordings of microelectrode‐impaled mesophyll cells and root hairs of Col‐0 Arabidopsis plants revealed rapid, dose‐dependent membrane potential depolarizations in response to either flg22 or elf18. Using ion‐selective microelectrodes, pronounced anion currents were recorded upon application of flg22 and elf18, indicating that the signaling cascades initiated by each of the two receptors converge on the same plasma membrane ion channels. Combined calcium imaging and electrophysiological measurements revealed that the depolarization was superimposed by an increase in cytosolic calcium that was indispensable for depolarization. NADPH oxidase mutants were still depolarized upon elicitor stimulation, suggesting a reactive oxygen species‐independent membrane potential response. Furthermore, electrical signaling in response to either flg22 or elf 18 critically depends on the activity of the FLS2‐associated receptor‐like kinase BAK1, suggesting that activation of FLS2 and EFR lead to BAK1‐dependent, calcium‐associated plasma membrane anion channel opening as an initial step in the pathogen defense pathway.     

Activation of K+ Channels in the Plasma Membrane of Arabidopsis by ATP Produced Photosynthetically

Light activates a K+ channel and transiently depolarizes the plasma membrane of Arabidopsis mesophyll cells. Genetically or chemically impairing photosynthesis abolished this electrical response to light. These results indicate that illuminated chloroplasts produce a factor that activated K+ channels in the plasma membrane. By patch clamping at the single-channel level, we have obtained evidence that ATP is one such factor. Application of 0.2 to 2 mM ATP to the cytoplasmic side of excised patches of membrane reversibly activated the type of channel that was activated by light in cell-attached patches. In addition, an outward-rectifying K+ channel and an outward-rectifying nonselective cation channel were similarly activated by ATP, whereas a nonselective stretch-activated channel was unaffected by this treatment. This novel mechanism for controlling the permeability of the plasma membrane to K+ may be important to photosynthetic metabolism.     

Overexpression in Arabidopsis of a plasma membrane-targeting glutamate receptor from small radish increases glutamate-mediated Ca2+ influx and delays fungal infection

Ionotropic glutamate receptors (iGluRs) are ligand-gated nonselective cation channels that mediate fast excitatory neurotransmission. Although homologues of the iGluRs have been identified in higher plants, their roles are largely unknown. In this work we isolated a full-length cDNA clone (RsGluR) encoding a putative glutamate receptor from small radish. An RsGluR: mGFP fusion protein was localized to the plasma membrane. In Arabidopsis thaliana overexpressing the full-length cDNA, glutamate treatment triggered greater Ca2+ influx in the root cells of transgenic seedlings than in those of the wild type. Transgenic plants exhibited multiple morphological changes such as necrosis at their tips and the margins of developing leaves, dwarf stature with multiple secondary inflorescences, and retarded growth, as previously observed in transgenic Arabidopsis overexpressing AtGluR3.2 [Kim et al. (2001)]. Microarray analysis showed that jasmonic acid (JA)-responsive genes including defensins and JA-biosynthetic genes were up-regulated. RsGluR overexpression also inhibited growth of a necrotic fungal pathogen Botrytis cinerea possibly due to up-regulation of the defensins. Based on these results, we suggest that RsGluR is a glutamate-gated Ca2+ channel located in the plasma membrane of higher plants and plays a direct or indirect role in defense against pathogen infection by triggering JA biosynthesis.     

Substrate-dependent gating of anion channels associated with excitatory amino acid transporter 4

EAAT glutamate transporters do not only function as secondary-active glutamate transporters but also as anion channels. EAAT anion channel activity depends on transport substrates. For most isoforms, it is negligible without external Na(+) and increased by external glutamate. We here investigated gating of EAAT4 anion channels with various cations and amino acid substrates using patch clamp experiments on a mammalian cell line. We demonstrate that Li(+) can substitute for Na(+) in supporting substrate-activated anion currents, albeit with changed voltage dependence. Anion currents were recorded in glutamate, aspartate, and cysteine, and distinct time and voltage dependences were observed. For each substrate, gating was different in external Na(+) or Li(+). All features of voltage-dependent and substrate-specific anion channel gating can be described by a simplified nine-state model of the transport cycle in which only amino acid substrate-bound states assume high anion channel open probabilities. The kinetic scheme suggests that the substrate dependence of channel gating is exclusively caused by differences in substrate association and translocation. Moreover, the voltage dependence of anion channel gating arises predominantly from electrogenic cation binding and membrane translocation of the transporter. We conclude that all voltage- and substrate-dependent conformational changes of the EAAT4 anion channel are linked to transitions within the transport cycle.     

Distinct pH regulation of slow and rapid anion channels at the plasma membrane of Arabidopsis thaliana hypocotyl cells

Variations in both intracellular and extracellular pH are known to be involved in a wealth of physiological responses. Using the patch-clamp technique on Arabidopsis hypocotyl cells, it is shown that rapid-type and slow-type anion channels at the plasma membrane are both regulated by pH via distinct mechanisms. Modifications of pH modulate the voltage-dependent gating of the rapid channel. While intracellular alkalinization facilitates channel activation by shifting the voltage gate towards negative potentials, extracellular alkalinization shifts the activation threshold to more positive potentials, away from physiological resting membrane potentials. By contrast, pH modulates slow anion channel activity in a voltage-independent manner. Intracellular acidification and extracellular alkalinization increase slow anion channel currents. The possible role of these distinct modulations in physiological processes involving anion efflux and modulation of extracellular and/or intracellular pH, such as elicitor and ABA signalling, are discussed.     

Activation of a common effector system by different brain neurotransmitter receptors in Xenopus oocytes

Xenopus oocytes possess 'native' muscarinic receptors, which give rise to oscillatory chloride currents; similar responses are elicited by activation of foreign receptors to serotonin, glutamate and noradrenaline, expressed in oocytes after injection of messenger RNA from rat brain. When low concentrations of two agonists are applied together, the combined response is greater than would be expected from the sum of the responses to each agonist applied alone. Potentiation of acetylcholine by serotonin is blocked by the serotonin antagonist methysergide; conversely, the potentiation of serotonin by acetylcholine is blocked by the muscarinic antagonist atropine. This indicates that each agonist acts on a distinct receptor. The interactions between serotonin, acetylcholine and other agonists provide further evidence that the different receptors may all 'link in' to a common receptor-channel coupling system, in which phosphoinositide metabolism and calcium liberation lead to the opening of chloride channels in the oocyte membrane.     

Anion channel activation and proton pumping inhibition involved in the plasma membrane depolarization induced by ABA in Arabidopsis thaliana suspension cells are both ROS dependent

In Arabidopsis thaliana suspension cells, ABA was previously shown to promote the activation of anion channels and the reduction of proton pumping that both contribute to the plasma membrane depolarization. These two ABA responses were shown to induce two successive [Ca(2+)](cyt) spikes. As reactive oxygen species (ROS) have emerged as components of ABA signaling pathways especially by promoting [Ca(2+)](cyt) variations, we studied whether ROS were involved in the regulation of anion channels and proton pumps activities. Here we demonstrated that ABA induced ROS production which triggered the second of the two [Ca(2+)](cyt) increases observed in response to ABA. Blocking ROS generation using diphenyleneiodonium (DPI) impaired the proton pumping reduction, the anion channel activation and the RD29A gene expression in response to ABA. Furthermore, H(2)O(2) was shown to activate anion channels and to inhibit plasma membrane proton pumping, as did ABA. However, ROS partially mimicked ABA's effects since H(2)O(2) treatment elicited anion channel activation but not the subsequent expression of the RD29A gene as did ABA. This suggests that expression of the RD29A gene in response to ABA results from the activation of multiple concomitant signaling pathways: blocking of one of them would impair gene expression whereas stimulating only one would not. We conclude that ROS are a central messenger of ABA in the signaling pathways leading to the plasma membrane depolarization induced by ABA.     

A patch-clamp study on the physiology of aluminum toxicity and aluminum tolerance in maize. Identification and characterization of Al(3+)-induced anion channels

The presence of Al(3+) in the rhizosphere induces citrate efflux from the root apex of the Al-tolerant maize (Zea mays) hybrid South American 3, consequently chelating and reducing the activity of toxic Al(3+) at the root surface. Because citrate is released from root apical cells as the deprotonated anion, we used the patch-clamp technique in protoplasts isolated from the terminal 5 mm of the root to study the plasma membrane ion transporters that could be involved in Al-tolerance and Al-toxicity responses. Acidification of the extracellular environment stimulated inward K(+) currents while inhibiting outward K(+) currents. Addition of extracellular Al(3+) inhibited the remaining K(+) outward currents, blocked the K(+) inward current, and caused the activation of an inward Cl(-) current (anion efflux). Studies with excised membrane patches revealed the existence of Al-dependent anion channels, which were highly selective for anions over cations. Our success in activating this channel with extracellular Al(3+) in membrane patches excised prior to any Al(3+) exposure indicates that the machinery required for Al(3+) activation of this channel, and consequently the whole root Al(3+) response, is localized to the root-cell plasma membrane. This Al(3+)-activated anion channel may also be permeable to organic acids, thus mediating the Al-tolerance response (i.e. Al-induced organic acid exudation) observed in intact maize root apices.     

Glutamate receptors on the somata of dorsal unpaired median neurons in cockroach,Periplaneta americana,thoracic ganglia

Effects of application of glutamate and glutamatergic ligands were studied to characterize the receptors for glutamate present on the soma membrane of the dorsal unpaired median (DUM) neurons in the thoracic ganglia of the cockroach, Periplaneta americana, using the intracellular recording technique. Application of L-glutamate did not block the GABA-response, and application of beta-guanidino-propionic acid, a competitive antagonist for GABA, failed to block the response to L-glutamate. These results indicate that most of L-glutamate action may not be mediated by a GABA-activated channel. To examine glutamate receptor types on the DUM neurons, glutamate receptor agonists were applied. The ionotropic glutamate receptor (iGluR) agonists evoked depolarizations with the following relative rank of order of potency: kainate > AMPA > quisqualate. Metabotropic glutamate receptor (mGluR) agonists also elicited membrane depolarizations or hyperpolarizations associated with an increase in membrane conductance. The mGluR agonists evoked depolarizations or hyperpolarizations with the following relative rank of order: L-CCG-1 > 1S, 3R-ACPD > L-AP4. Depolarization of the same DUM neuron was detected following exposure of kainate and L-CCG-I, suggesting the coexistence of distinct iGluR and mGluR types. A membrane permeable cAMP analog, CPT-cAMP, could not mimic the effect of mGluR agonists. The mGluR selective antagonists, MCCG and MCPG, failed to antagonize the response to mGluR agonists. The involvement of cAMP in the mGluR response was not confirmed in DUM neurons. Although the functional roles of these receptors are unknown, it might be possible then that these extrasynaptic receptors have a modulatory effect on the excitability of the DUM neurons.     

An S-Type Anion Channel SLAC1 Is Involved in Cryptogein-Induced Ion Fluxes and Modulates Hypersensitive Responses in Tobacco BY-2 Cells

Pharmacological evidence suggests that anion channel-mediated plasma membrane anion effluxes are crucial in early defense signaling to induce immune responses and hypersensitive cell death in plants. However, their molecular bases and regulation remain largely unknown. We overexpressed Arabidopsis SLAC1, an S-type anion channel involved in stomatal closure, in cultured tobacco BY-2 cells and analyzed the effect on cryptogein-induced defense responses including fluxes of Cl⁻ and other ions, production of reactive oxygen species (ROS), gene expression and hypersensitive responses. The SLAC1-GFP fusion protein was localized at the plasma membrane in BY-2 cells. Overexpression of SLAC1 enhanced cryptogein-induced Cl⁻ efflux and extracellular alkalinization as well as rapid/transient and slow/prolonged phases of NADPH oxidase-mediated ROS production, which was suppressed by an anion channel inhibitor, DIDS. The overexpressor also showed enhanced sensitivity to cryptogein to induce downstream immune responses, including the induction of defense marker genes and the hypersensitive cell death. These results suggest that SLAC1 expressed in BY-2 cells mediates cryptogein-induced plasma membrane Cl⁻ efflux to positively modulate the elicitor-triggered activation of other ion fluxes, ROS as well as a wide range of defense signaling pathways. These findings shed light on the possible involvement of the SLAC/SLAH family anion channels in cryptogein signaling to trigger the plasma membrane ion channel cascade in the plant defense signal transduction network.     

N-methyl-D-aspartate receptor activation in human cerebral endothelium promotes intracellular oxidant stress

Cerebral endothelial cells in the rat, pig, and, most recently, human have been shown to express several types of receptors specific for glutamate. High levels of glutamate disrupt the cerebral endothelial barrier via activation of N-methyl-d-aspartate (NMDA) receptors. We have previously suggested that this glutamate-induced barrier dysfunction was oxidant dependent. Here, we provide evidence that human cerebral endothelial cells respond to glutamate by generating an intracellular oxidant stress via NMDA receptor activation. Cerebral endothelial cells loaded with the oxidant-sensitive probe dihydrorhodamine were used to measure intracellular reactive oxygen species (ROS) formation in response to glutamate receptor agonists, antagonists, and second message blockers. Glutamate (1 mM) significantly increased ROS formation compared with sham controls (30 min). This ROS response was significantly reduced by 1) MK-801, a noncompetitive NMDA receptor antagonist; 2) 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate, an intracellular Ca(2+) antagonist; 3) LaCl(3), an extracellular Ca(2+) channel blocker; 4) diphenyleiodonium, a heme-ferryl-containing protein inhibitor; 5) itraconazole, a cytochrome P-450 3A4 inhibitor; and 6) cyclosporine A, which prevents mitochondrial membrane pore transition required for mitochondrial-dependent ROS generation. Our results suggest that the cerebral endothelial barrier dysfunction seen in response to glutamate is Ca(2+) dependent and may require several intracellular signaling events mediated by oxidants derived from reduced nicotinamide adenine dinucleotide oxidase, cytochrome P-450, and the mitochondria.     

Characterization of anion channels in the plasma membrane of Arabidopsis epidermal root cells and the identification of a citrate-permeable channel induced by phosphate starvation

Organic-acid secretion from higher plant roots into the rhizosphere plays an important role in nutrient acquisition and metal detoxification. In this study we report the electrophysiological characterization of anion channels in Arabidopsis (Arabidopsis thaliana) root epidermal cells and show that anion channels represent a pathway for citrate efflux to the soil solution. Plants were grown in nutrient-replete conditions and the patch clamp technique was applied to protoplasts isolated from the root epidermal cells of the elongation zone and young root hairs. Using SO4(2-) as the dominant anion in the pipette, voltage-dependent whole-cell inward currents were activated at membrane potentials positive of -180 mV exhibiting a maximum peak inward current (I(peak)) at approximately -130 mV. These currents reversed at potentials close to the equilibrium potential for SO4(2-), indicating that the inward currents represented SO4(2-) efflux. Replacing intracellular SO4(2-) with Cl- or NO3(-) resulted in inward currents exhibiting similar properties to the SO4(2-) efflux currents, suggesting that these channels were also permeable to a range of inorganic anions; however when intracellular SO4(2-) was replaced with citrate or malate, no inward currents were ever observed. Outside-out patches were used to characterize a 12.4-picoSiemens channel responsible for these whole-cell currents. Citrate efflux from Arabidopsis roots is induced by phosphate starvation. Thus, we investigated anion channel activity from root epidermal protoplasts isolated from Arabidopsis plants deprived of phosphate for up to 7 d after being grown for 10 d on phosphate-replete media (1.25 mm). In contrast to phosphate-replete plants, protoplasts from phosphate-starved roots exhibited depolarization-activated voltage-dependent citrate and malate efflux currents. Furthermore, phosphate starvation did not regulate inorganic anion efflux, suggesting that citrate efflux is probably mediated by novel anion channel activity, which could have a role in phosphate acquisition.     

Hippocampal astrocytes exhibit Ca2+-elevating muscarinic cholinergic and histaminergic receptors in situ

Recent findings suggest that astrocytes respond to neuronally released neurotransmitters with Ca2+ elevations. These Ca2+ elevations may trigger astrocytes to release glutamate, affecting neuronal activity. Neuronal activity is also affected by modulatory neurotransmitters that stimulate G protein-coupled receptors. These neurotransmitters, including acetylcholine and histamine, might affect neuronal activity by triggering Ca2+-dependent release of neurotransmitters from astrocytes. However, there is no physiological evidence for histaminergic or cholinergic receptors on astrocytes in situ. We asked whether astrocytes have these receptors by imaging Ca2+-sensitive dyes sequestered by astrocytes in hippocampal slices. Our results show that immunocytochemically identified astrocytes respond to carbachol and histamine with increases in intracellular free Ca2+ concentration. The H1 histamine receptor antagonist chlorpheniramine inhibited responses to histamine. Similarly, atropine and the M1-selective muscarinic receptor antagonist pirenzepine inhibited carbachol-elicited responses. Astrocyte responses to histamine and carbachol were compared with responses elicited by alpha1-adrenergic and metabotropic glutamate receptor agonists. Individual astrocytes responded to different subsets of receptor agonists. Ca2+ oscillations were the prevalent response pattern only with metabotropic glutamate receptor stimulation. Finally, functional alpha1-adrenergic receptors and muscarinic receptors were not detected before postnatal day 8. Our data show that astrocytes have acetylcholine and histamine receptors coupled to Ca2+. Given that Ca2+ elevations in astrocytes trigger neurotransmitter release, it is possible that these astrocyte receptors modulate neuronal activity.     

Signalling mechanisms underlying the morphological responses of the root system to nitrogen in Arabidopsis thaliana

Plants display considerable developmental plasticity in response to changing environmental conditions. The adaptations of the root system to variations in N supply are an excellent example of such developmental plasticity. In Arabidopsis, four morphological adaptations to the N supply have been characterized: (i) a localized stimulatory effect of external nitrate on lateral root elongation; (ii) a systemic inhibitory effect of high tissue nitrate concentrations on the activation of lateral root meristems; (iii) a suppression of lateral root initiation by high C:N ratios, and (iv) an inhibition of primary root growth and stimulation of root branching by external L-glutamate. These responses have provided valuable experimental systems for the study of N signalling in plants. This article will highlight some recent progress made in this direction from studies using the Arabidopsis root system. One recent development of note has been the emerging evidence of a regulatory role of nitrate transporters in some of the responses. It has been reported that the AtNRT1.1 (CHL1) dual-affinity nitrate transporter acts upstream of the ANR1 MADS box gene in mediating the stimulatory effect of a localized nitrate supply on lateral root proliferation. The AtNRT2.1 high-affinity nitrate transporter seems to be involved in the repression of lateral root initiation by high C:N ratios. The systemic inhibitory effect of high nitrate supply on lateral root development, which is mediated by abscisic acid (ABA), may be linked to the recently identified ABA receptor, FCA. The newly discovered root architectural response to external L-glutamate potentially offers a valuable experimental tool for studying the biological function of plant glutamate receptors and amino acid signalling.     

Structural organization of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptor of the electric ray Torpedo is the most comprehensively characterized neurotransmitter receptor. It consists of five subunits (alpha2beta gammadelta) amino acid sequences of which were determined by cDNA cloning and sequencing. The shape and size of the receptor were determined by electron cryomicroscopy. It has two agonist/competitive antagonist binding sites which are located between subunits near the membrane surface. The receptor ion channel is formed by five transmembrane helices (M2) of all five subunits. The position of the binding site for noncompetitive ion channel blockers was found by photoaffinity labelling and site-directed mutagenesis. The intrinsic feature of the receptor structure is the position of the agonist/competitive antagonist binding sites in close vicinity to the ion channel spanning the bilayer membrane. This peculiarity may substantially enhance allosteric transitions transforming the ligand binding into the channel opening and physiological response. Muscle nicotinic acetylcholine receptors from birds and mammals are also pentaoligomers consisting of four different subunits (alpha2beta gammadelta or alpha2beta epsilondelta) with high homology to the Torpedo receptor. Apparently, the pentaoligomeric structure is the main feature of all nicotinic, both muscle and neuronal, receptors. However, the neuronal receptors are formed only by two subunit types (alpha and beta) or are even pentahomomers (alpha7 neuronal receptors). All nicotinic receptors are ligand-gated ion channel, the properties of the channels being essentially determined by amino acid residues forming M2 transmembrane fragments.     

Noninvasive imaging of 5-HT3 receptor trafficking in live cells: from biosynthesis to endocytosis

Sequential stages in the life cycle of the ionotropic 5-HT(3) receptor (5-HT(3)R) were resolved temporally and spatially in live cells by multicolor fluorescence confocal microscopy. The insertion of the enhanced cyan fluorescent protein into the large intracellular loop delivered a fluorescent 5-HT(3)R fully functional in terms of ligand binding specificity and channel activity, which allowed for the first time a complete real-time visualization and documentation of intracellular biogenesis, membrane targeting, and ligand-mediated internalization of a receptor belonging to the ligand-gated ion channel superfamily. Fluorescence signals of newly expressed receptors were detectable in the endoplasmic reticulum about 3 h after transfection onset. At this stage receptor subunits assembled to form active ligand binding sites as demonstrated in situ by binding of a fluorescent 5-HT(3)R-specific antagonist. After novel protein synthesis was chemically blocked, the 5-HT(3) R populations in the endoplasmic reticulum and Golgi cisternae moved virtually quantitatively to the cell surface, indicating efficient receptor folding and assembly. Intracellular 5-HT(3) receptors were trafficking in vesicle-like structures along microtubules to the cell surface at a velocity generally below 1 mum/s and were inserted into the plasma membrane in a characteristic cluster distribution overlapping with actin-rich domains. Internalization of cell surface 5-HT(3) receptors was observed within minutes after exposure to an extracellular agonist. Our orchestrated use of spectrally distinguishable fluorescent labels for the receptor, its cognate ligand, and specific organelle markers can be regarded as a general approach allowing subcellular insights into dynamic processes of membrane receptor trafficking.     

Single channel recordings from synaptosomal AMPA receptors

Synaptic glutamate receptors play a prominent role in the excitatory neurotransmission in the vertebrate central nervous system. Although elucidation of the functional properties of glutamate receptors using electrophysiologic analyses has yielded important information, methodological and technological limitations have prevented direct measurement of single channel properties of synaptic receptors. Here, we have isolated murine mossy fiber synaptosomes and reconstituted them into small artificial lipid bilayers to characterize the single-channel properties of synaptic alpha amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-subtype glutamate receptors. The reconstituted synaptosomal receptors were activated by nanomolar concentrations of AMPA and blocked by a potent AMPA receptor antagonist. The synaptosomal AMPA receptors exhibited channel conductances of 14-56 pS and linear current-voltage relationship. The open and closed dwell time distributions of single channel currents were best described by three exponentials. These channels frequently exhibited burst behavior with long burst duration of approx 60 ms. Experiments with multichannel recordings revealed that steady state probabilities could not be fitted using a binomial distribution, indicating a cooperative channel gating behavior that would account for larger membrane currents. Our findings suggest that isolation, reconstitution into lipid bilayers, and subsequent single channel analysis of synaptosomal receptors is a useful method for investigation of synaptic AMPA receptors.     

Genes for calcium-permeable channels in the plasma membrane of plant root cells

In plant cells, Ca(2+) is required for both structural and biophysical roles. In addition, changes in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) orchestrate responses to developmental and environmental signals. In many instances, [Ca(2+)](cyt) is increased by Ca(2+) influx across the plasma membrane through ion channels. Although the electrophysiological and biochemical characteristics of Ca(2+)-permeable channels in the plasma membrane of plant cells are well known, genes encoding putative Ca(2+)-permeable channels have only recently been identified. By comparing the tissue expression patterns and electrophysiology of Ca(2+)-permeable channels in the plasma membrane of root cells with those of genes encoding candidate plasma membrane Ca(2+) channels, the genetic counterparts of specific Ca(2+)-permeable channels can be deduced. Sequence homologies and the physiology of transgenic antisense plants suggest that the Arabidopsis AtTPC1 gene encodes a depolarisation-activated Ca(2+) channel. Members of the annexin gene family are likely to encode hyperpolarisation-activated Ca(2+) channels, based on their corresponding occurrence in secretory or elongating root cells, their inhibition by La(3+) and nifedipine, and their increased activity as [Ca(2+)](cyt) is raised. Based on their electrophysiology and tissue expression patterns, AtSKOR encodes a depolarisation-activated outward-rectifying (Ca(2+)-permeable) K(+) channel (KORC) in stelar cells and AtGORK is likely to encode a KORC in the plasma membrane of other Arabidopsis root cells. Two candidate gene families, of cyclic-nucleotide gated channels (CNGC) and ionotropic glutamate receptor (GLR) homologues, are proposed as the genetic correlates of voltage-independent cation (VIC) channels.