Abstract: Chronic exposure to Delta9-tetrahydrocannabinol (THC) induces tolerance to cannabinoid-induced locomotor effects, which are mediated by cannabinoid receptors (CB1Rs) located in motor control regions, including the cerebellum. There is substantial evidence of cerebellar CB1R molecular adaptation and modifications in receptor signaling after prolonged cannabinoid exposure. However, very little is known about the effects of chronic cannabinoid administration on cerebellar synaptic plasticity, which may contribute to the development of cannabinoid behavioral tolerance. In the cerebellar cortex, activation of CB1R inhibits excitatory synaptic transmission at parallel fiber (PF)-Purkinje cell (PC) synapses by decreasing neurotransmitter release. Our study aimed to investigate the neurophysiological adaptive responses occurring at cerebellar PF-PC cell synapses after repeated THC exposure. In THC-tolerant mice, an increase of the basal release probability was found at PF-PC synapses, in parallel with a facilitation of slow mGluR1 (metabotropic glutamate receptor type 1)-mediated excitatory postsynaptic currents and a reduced sensitivity to the inhibitory effects of the CB1R agonist CP55,940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol]. Additionally, after repeated THC exposures, presynaptic PF-PC long-term potentiation was blocked by A1R (adenosine receptor-1) activation. Inhibition of the extracellular signal regulated kinase (ERK) pathway prevented these alterations of cerebellar synaptic transmission and plasticity. In summary, we provide evidence for ERK-dependent modulatory mechanisms at PF-PC synapses after chronic THC administration. This contributes to generation of forms of pathological synaptic plasticity that might play a role in cannabinoid dependence.

Abstract: A large body of evidence indicates that ligand-gated channels may open spontaneously, exhibiting a basal activity in the absence of the neurotransmitter. In the present work, we were interested in studying the Ca(2+)-induced modulation of the basal channel activity of unliganded human L248Talpha7 receptors expressed in Xenopus oocytes. While the basal channel activity was blocked by either the nicotinic antagonist methyllycaconitine or the superfusion with a Ca(2+)-free medium, it was enhanced by increasing external Ca2+ concentrations. External Ca2+ significantly influenced the channel properties lengthening the channel duration and reducing the channel conductance, in a dose dependent manner. Furthermore, the basal channel activity in standard medium was blocked by N,N,N',N'-tetrakis-2-pyridylmethyl-ethylenediamine, the chelator of divalent cations with very high affinity for Zn2+, and was induced by Zn2+ when Ca2+ was present in the external medium. We conclude that basal activity of alpha7 mutant receptor-channels is caused by divalent cation contaminants present in the external medium, namely Zn2+; is positively modulated by the external Ca2+; and is inhibited when Ca2+ is absent from the medium. The patho-physiological consequences of these findings are discussed.

Abstract: Most proteins adopt a well defined three-dimensional structure; however, it is increasingly recognized that some proteins can exist with at least two stable conformations. Recently, a class of intracellular chloride ion channel proteins (CLICs) has been shown to exist in both soluble and integral membrane forms. The structure of the soluble form of CLIC1 is typical of a soluble glutathione S-transferase superfamily protein but contains a glutaredoxin-like active site. In this study we show that on oxidation CLIC1 undergoes a reversible transition from a monomeric to a non-covalent dimeric state due to the formation of an intramolecular disulfide bond (Cys-24-Cys-59). We have determined the crystal structure of this oxidized state and show that a major structural transition has occurred, exposing a large hydrophobic surface, which forms the dimer interface. The oxidized CLIC1 dimer maintains its ability to form chloride ion channels in artificial bilayers and vesicles, whereas a reducing environment prevents the formation of ion channels by CLIC1. Mutational studies show that both Cys-24 and Cys-59 are required for channel activity.

Abstract: It is widely believed that the inflammatory events mediated by microglial activation contribute to several neurodegenerative processes. Alzheimer's disease, for example, is characterized by an accumulation of beta-amyloid protein (Abeta) in neuritic plaques that are infiltrated by reactive microglia and astrocytes. Although Abeta and its fragment 25-35 exert a direct toxic effect on neurons, they also activate microglia. Microglial activation is accompanied by morphological changes, cell proliferation, and release of various cytokines and growth factors. A number of scientific reports suggest that the increased proliferation of microglial cells is dependent on ionic membrane currents and in particular on chloride conductances. An unusual chloride ion channel known to be associated with macrophage activation is the chloride intracellular channel-1 (CLIC1). Here we show that Abeta stimulation of neonatal rat microglia specifically leads to the increase in CLIC1 protein and to the functional expression of CLIC1 chloride conductance, both barely detectable on the plasma membrane of quiescent cells. CLIC1 protein expression in microglia increases after 24 hr of incubation with Abeta, simultaneously with the production of reactive nitrogen intermediates and of tumor necrosis factor-alpha (TNF-alpha). We demonstrate that reducing CLIC1 chloride conductance by a specific blocker [IAA-94 (R(+)-[(6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5yl)-oxy] acetic acid)] prevents neuronal apoptosis in neurons cocultured with Abeta-treated microglia. Furthermore, we show that small interfering RNAs used to knock down CLIC1 expression prevent TNF-alpha release induced by Abeta stimulation. These results provide a direct link between Abeta-induced microglial activation and CLIC1 functional expression.

Abstract: The beta-amyloid(1-42) peptide (Abeta(1-42)), a major constituent of the Alzheimer's disease amyloid plaque, specifically binds to the neuronal alpha-bungarotoxin (alpha-BuTx)-sensitive alpha7 nicotinic acetylcholine receptor (alpha7 nAChR). Accordingly, Abeta1-42 interferes with the function of alpha7 nAChRs in chick and rodent neurons. To gain insights into the human disease, we studied the action of Abeta(1-42) on human alpha7 nAChRs expressed in Xenopus oocytes. In voltage-clamped oocytes expressing the wild-type receptor, Abeta(1-42) blocked ACh-evoked currents. The block was non-competitive, required over 100 s to develop and was partially reversible. In oocytes expressing the mutant L248T receptor, Abeta(1-42) activated methyllycaconitine-sensitive currents in a dose-dependent manner. Peptide-evoked unitary events, recorded in outside-out patches, showed single-channel conductances and open duration comparable to ACh-evoked events. Abeta(1-42) had no effect on the currents evoked by glutamate, GABA or glycine in oocytes expressing human or mouse receptors for these transmitters. Muscle nAChRs are also alpha-BuTx-sensitive and we therefore investigated whether they respond to Abeta(1-42). In human kidney BOSC 23 cells expressing the fetal or adult mouse muscle nAChRs, Abeta(1-42) blocked ACh-evoked whole-cell currents, accelerating their decay. Outside-out single-channel recordings showed that the block was due to a reduced channel open probability and enhanced block upon ACh application. We also report that the inverse peptide Abeta(42-1), but not Abeta(40-1), partially mimicked the effects of the physiological Abeta(1-42) peptide. Possible implications for degenerative neuronal and muscular diseases are discussed.

Abstract: We have shown previously that mutating to threonine the leucine residue in the M2 domain of the alpha7 nicotinic acetylcholine receptor (human L248T, L248T; chick L247T, L247T) converts bicuculline (BIC) from an antagonist into an agonist. In this work we studied the functional properties of the BIC-activated channels and report that, in Xenopus oocytes injected with L248T subunit cDNA, BIC activates single-channel currents that have similar conductances, but shorter mean burst duration, than the channels activated by ACh. In contrast, both the conductance and kinetics of the channels activated by either ACh or BIC are substantially the same in oocytes expressing L247T receptors. We have also shown previously that if Cys 189 and 190, which are thought to be at or near the transmitter binding site, are additionally mutated to Ser, the new mutant receptor (L247T-C189S-C190S) has a reduced affinity for ACh. We now find that the EC(50) in the BIC dose-current response relation, as well the characteristics of the channels activated by BIC, are similar in oocytes expressing either L247T or L247T-C189S-C190S receptors. On the other hand, ACh activation of L247T-C189S-C190S receptors gates channels whose mean open time and burst duration are much shorter than those of ACh-gated L247T-channels. Therefore, the gating kinetics of both L248T and L247R-C189S-C190S receptor-channels change when BIC is replaced by ACh; and we conclude that both ACh and BIC activate mutant alpha7 receptors with different patterns of activation.

Abstract: CLIC1 (NCC27) is an unusual, largely intracellular, ion channel that exists in both soluble and membrane-associated forms. The soluble recombinant protein can be expressed in Escherichia coli, a property that has made possible both detailed electrophysiological studies in lipid bilayers and an examination of the mechanism of membrane integration. Soluble E. coli-derived CLIC1 moves from solution into artificial bilayers and forms chloride-selective ion channels with essentially identical conductance, pharmacology, and opening and closing kinetics to those observed in CLIC1-transfected Chinese hamster ovary cells. The process of membrane integration of CLIC1 is pH-dependent. Following addition of protein to the trans solution, small conductance channels with slow kinetics (SCSK) appear in the bilayer. These SCSK modules then appear to undergo a transition to form a high conductance channel with fast kinetics. This has four times the conductance of the SCSK and fast kinetics that characterize the native channel. This suggests that the CLIC1 ion channel is likely to consist of a tetrameric assembly of subunits and indicates that despite its size and unusual properties, it is able to form a completely functional ion channel in the absence of any other ancillary proteins.

Abstract: In this study, we investigated the role of Ras and the mitogen-activated protein kinase (MAPK) pathway in the modulation of the inward rectifier potassium channel IRK1. We show that although expression of IRK1 in HEK 293 cells leads to the appearance of a potassium current with strong inward rectifying properties, coexpression of the constitutively active form of Ras (Ras-L61) results in a significant reduction of the mean current density without altering the biophysical properties of the channel. The inhibitory effect of Ras-L61 is not due to a decreased expression of IRK1 since Northern analysis indicates that IRK1 mRNA level is not affected by Ras-L61 co-expression. Moreover, the inhibition can be relieved by treatment with the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor PD98059. Confocal microscopy analysis of cells transfected with the fusion construct green fluorescent protein-IRK1 shows that the channel is mainly localized at the plasma membrane. Coexpression of Ras-L61 delocalizes fluorescence to the cytoplasm, whereas treatment with PD98059 partially restores the membrane localization. In conclusion, our data indicate that the Ras-MAPK pathway modulates IRK1 current by affecting the subcellular localization of the channel. This suggests a role for Ras signaling in regulating the intracellular trafficking of this channel.

Abstract: CLIC1 (NCC27) is a member of the highly conserved class of chloride ion channels that exists in both soluble and integral membrane forms. Purified CLIC1 can integrate into synthetic lipid bilayers forming a chloride channel with similar properties to those observed in vivo. The structure of the soluble form of CLIC1 has been determined at 1.4-A resolution. The protein is monomeric and structurally homologous to the glutathione S-transferase superfamily, and it has a redox-active site resembling glutaredoxin. The structure of the complex of CLIC1 with glutathione shows that glutathione occupies the redox-active site, which is adjacent to an open, elongated slot lined by basic residues. Integration of CLIC1 into the membrane is likely to require a major structural rearrangement, probably of the N-domain (residues 1-90), with the putative transmembrane helix arising from residues in the vicinity of the redox-active site. The structure indicates that CLIC1 is likely to be controlled by redox-dependent processes.

Abstract: Despite growing concern about electromagnetic radiation, the interaction between 50- to 60-Hz fields and biological structures remains obscure. Epidemiological studies have failed to prove a significantly correlation between exposure to radiation fields and particular pathologies. We demonstrate that a 50- to 60-Hz magnetic field interacts with cell differentiation through two opposing mechanisms: it antagonizes the shift in cell membrane surface charges that occur during the early phases of differentiation and it modulates hyperpolarizing K channels by increasing intracellular Ca. The simultaneous onset of both mechanisms prevents alterations in cell differentiation. We propose that cells are normally protected against electromagnetic insult. Pathologies may arise, however, if intracellular Ca regulation or K channel activation malfunctions.

Abstract: Ras-GRF1 is a neuron-specific guanine nucleotide exchange factor for Ras proteins. Mice lacking Ras-GRF1 (-/-) are severely impaired in amygdala-dependent long-term synaptic plasticity and show higher basal synaptic activity at both amygdala and hippocampal synapses (Brambilla et al., 1997). In the present study we investigated the effects of Ras-GRF1 deletion on hippocampal neuronal excitability. Electrophysiological analysis of both primary cultured neurons and adult hippocampal slices indicated that Ras-GRF1-/- mice displayed neuronal hyperexcitability. Ras-GRF1-/- hippocampal neurons showed increased spontaneous activity and depolarized resting membrane potential, together with a higher firing rate in response to injected current. Changes in the intrinsic excitability of Ras-GRF1-/- neurons can entail these phenomena, suggesting that Ras-GRF1 deficiency might alter the balance between ionic conductances. In addition, we showed that mice lacking Ras-GRF1 displayed a higher seizure susceptibility following acute administration of convulsant drugs. Taken together, these results demonstrated a role for Ras-GRF1 in neuronal excitability.

Abstract: NCC27 is a nuclear chloride ion channel, identified in the PMA-activated U937 human monocyte cell line. NCC27 mRNA is expressed in virtually all cells and tissues and the gene encoding NCC27 is also highly conserved. Because of these factors, we have examined the hypothesis that NCC27 is involved in cell cycle regulation. Electrophysiological studies in Chinese hamster ovary (CHO-K1) cells indicated that NCC27 chloride conductance varied according to the stage of the cell cycle, being expressed only on the plasma membrane of cells in G2/M phase. We also demonstrate that Cl- ion channel blockers known to block NCC27 led to arrest of CHO-K1 cells in the G2/M stage of the cell cycle, the same stage at which this ion channel is selectively expressed on the plasma membrane. These data strongly support the hypothesis that NCC27 is involved, in some as yet undetermined manner, in regulation of the cell cycle.

Abstract: NCC27 belongs to a family of small, highly conserved, organellar ion channel proteins. It is constitutively expressed by native CHO-K1 and dominantly localized to the nucleus and nuclear membrane. When CHO-K1 cells are transfected with NCC27-expressing constructs, synthesized proteins spill over into the cytoplasm and ion channel activity can then be detected on the plasma as well as nuclear membrane. This provided a unique opportunity to directly compare electrophysiological characteristics of the one cloned channel, both on the nuclear and cytoplasmic membranes. At the same time, as NCC27 is unusually small for an ion channel protein, we wished to directly determine whether it is a membrane-resident channel in its own right. In CHO-K1 cells transfected with epitope-tagged NCC27 constructs, we have demonstrated that the NCC27 conductance is chloride dependent and that the electrophysiological characteristics of the channels are essentially identical whether expressed on plasma or nuclear membranes. In addition, we show that a monoclonal antibody directed at an epitope tag added to NCC27 rapidly inhibits the ability of the expressed protein to conduct chloride, but only when the antibody has access to the tag epitope. By selectively tagging either the amino or carboxyl terminus of NCC27 and varying the side of the membrane from which we record channel activity, we have demonstrated conclusively that NCC27 is a transmembrane protein that directly forms part of the ion channel and, further, that the amino terminus projects outward and the carboxyl terminus inward. We conclude that despite its relatively small size, NCC27 must form an integral part of an ion channel complex.

Abstract: Ras-GRF, a neuron-specific Ras exchange factor of the central nervous system, was transfected in the SK-N-BE neuroblastoma cell line and stable clones were obtained. When exposed to retinoic acid, these clones showed a remarkable enhancement of Ras-GRF expression with a concomitant high increase in the level of active (GTP-bound) Ras already after 24 h of treatment. In the presence of retinoic acid, the transfected cells stopped growing and acquired a differentiated neuronal-like phenotype more rapidly than the parental ones. Cells expressing Ras-GRF also exhibited a more hyperpolarized membrane potential. Moreover, treatment with retinoic acid led to the appearance of an inward rectifying potassium channel with electrophysiological properties similar to IRK1. This current was present in a large number of cells expressing Ras-GRF, while only a small percentage of parental cells exhibited this current. However, Northern analysis with a murine cDNA probe indicated that IRK1 mRNA was induced by retinoic acid at a similar level in both kinds of cells. Brief treatment with a specific inhibitor of the mitogen-activated protein kinase (MAPK) pathway reduced the number of transfected cells showing IRK1 activity. These findings suggest that activation of the Ras pathway accelerates neuronal differentiation of this cell line. In addition, our results suggest that Ras-GRF and/or Ras-pathway may have a modulatory effect on IRK1 channel activity.

Abstract: Several types of ionic channels on the outer membrane of the nuclear envelope communicate with the nuclear cisternae. These are distinct from nucleocytoplasmic pathways, the nuclear pores that span the double membrane of the envelope and are the route for RNA and protein traffic in the nucleus. Recent data indicate that the nuclear pores may also function as ion channels. The most probable candidate for nucleocytoplasmic ion flux is a 300-400 pS pathway observed in many nuclear preparations. Morphological and functional studies of nuclear envelope suggest a tight relationship between the large conductance channel and the pore complex. However, there is no direct evidence for gating of the nuclear pore or its ability to open and close as a conventional channel. This study shows that in liver nuclei isolated from newborn mouse, there is a substantial correspondence between the number of pores and the number of channels recorded during patch-clamp. This is not the case for adult nuclei. Although pore density is comparable, some nuclear cytoskeletal components, such as actin and nonmuscle myosin, show a significant increase in the adult preparation. Previous studies demonstrate the presence of these two proteins in association with the pore complex. Here we show that by using actin filament disrupter, we were able to increase the number of active channels in adult isolated nuclei. We suggest that a functional interaction between actin filaments and the nuclear pore complex could regulate nucleocytoplasmic permeability.

Abstract: Nuclear patch clamp is an emerging research field that aims to disclose the electrical phenomena underlying macromolecular transport across the nuclear envelope (NE), its properties as an ion barrier and its function as an intracellular calcium store. The authors combined the patch clamp technique with atomic force microscopy (AFM) to investigate the structure-function relationship of NE. In principle, patch clamp currents, recorded from the NE can indicate the activity of the nuclear pore complexes (NPCs) and/or of ion channels in the two biomembranes that compose the NE. However, the role of the NPCs is still nuclear because the observed NE current in patch clamp experiments is lower than expected from the known density of the NPCs. Therefore, AFM was applied to link patch clamp currents to structure. The membrane patch was excised from the nuclear envelope and, after electrical evaluation, transferred from the patch pipette to a substrate. We could identify the native nuclear membrane patches with AFM at a lateral and a vertical resolution of 3 nm and 0.1 nm, respectively. It was shown that complete NE together with NPCs can be excised from the nucleus after their functional identification in patch clamp experiments. However, we also show that membranes of the endoplasmic reticulum can contaminate the tip of the patch pipette during nuclear patch clamp experiments. This possibility must be considered carefully in nuclear patch clamp experiments.