Hippocampal ether-à-go-go1 potassium channels blockade: effects in the startle reflex and prepulse inhibition.

Recently, our group described the ether-à-go-go1(Eag1) voltage-gated potassium (K(+)) channel (Kv10.1) expression in the dopaminergic cells indicating that these channels are part of the diversified group of ion channels related to dopaminergic neurons function. The increase of dopamine neurotransmission induces a reduction in the prepulse inhibition (PPI) of the acoustic startle reflex in rodents, which is a reliable index of sensorimotor gating deficits. The PPI response has been reported to be abnormally reduced in schizophrenia patients. The role of Eag1 K(+) channels in the PPI reaction had not been revealed until now, albeit the singular distribution of Eag1 in the dentate gyrus of the hippocampus and the hippocampal regulation of the startle reflex and PPI. The aim of this work was to investigate if Eag1 blockade on hippocampus modifies the PPI-disruptive effects of apomorphine in Wistar rats. Bilateral injection of anti-Eag1 single-chain antibody into the dentate gyrus of hippocampus did not modify apomorphine-disruptive effects in the PPI response. However, Eag1 antibody completely restored the startle amplitude decrease revealed after dentate gyrus surgery. These potentially biological important phenomenon merits further investigation regarding the role of Eag1 K(+) channels, mainly, on startle reflex modulation, since the physiological role of these channels remain obscure.

Approaches targeting K(V)10.1 open a novel window for cancer diagnosis and therapy.

K(V)10.1 has recently become generally accepted as a promising cancer target, as it is ectopically expressed in the majority of solid tumors. Due to its cell-surface accessibility, K(V)10.1 has a strong potential for tumor treatment and diagnosis. Given that its mode of action is likely independent of conventional cancer pathways such as tyrosine kinases, K(V)10.1 opens a novel window for treating cancer. In this review we will give an overview of the current status of data linking K(V)10.1 to cancer, and propose techniques that could exploit K(V)10.1's properties for the management of cancer.

Eag1, Eag2, and SK3 potassium channel expression in the rat hippocampus after global transient brain ischemia.

Transient global brain ischemia causes delayed neuronal death in the hippocampus that has been associated with impairments in hippocampus-dependent brain function, such as mood, learning, and memory. We investigated the expression of voltage-dependent Kcnh1 and Kcnh5, ether à go-go-related Eag1 and Eag2 (K(V) 10.1 and K(V) 10.2), and small-conductance calcium-activated SK3 (K(Ca) 2.3, Kcnn3) K(+) channels in the hippocampus in rats after transient global brain ischemia. We tested whether the expression of these channels is associated with behavioral changes by evaluating the animals in the elevated plus maze and step-down inhibitory avoidance task. Seven or tweny-eight days after transient global brain ischemia, one group of rats had the hippocampus bilaterally dissected, and mRNA levels were determined. Seven days after transient global brain ischemia, the rats exhibited a decrease in anxiety-like behavior and memory impairments. An increase in anxiety levels was detected 28 days after ischemia. Eag2 mRNA downregulation was observed in the hippocampus 7 days after transient global brain ischemia, whereas Eag1 and SK3 mRNA expression remained unaltered. This is the first experimental evidence that transient global brain ischemia temporarily alters Eag2. The number of intact-appearing pyramidal neurons was substantially decreased in CA1 and statistically measurable in CA2, CA3, and CA4 hippocampal subfields compared with sham control animals 7 or 28 days after ischemia. mRNA expression in the rat hippocampus. The present results provide further information for the characterization of the physiological role of Eag2 channels in the central nervous system.

Ether-à-go-go 1 (Eag1) potassium channel expression in dopaminergic neurons of basal ganglia is modulated by 6-hydroxydopamine lesion.

The ether à go-go (Eag) gene encodes the voltage-gated potassium (K(+)) ion channel Kv10.1, whose function still remains unknown. As dopamine may directly affect K(+) channels, we evaluated whether a nigrostriatal dopaminergic lesion induced by the neurotoxin 6-hydroxydopamine (6-OHDA) would alter Eag1-K(+) channel expression in the rat basal ganglia and related brain regions. Male Wistar rats received a microinjection of either saline or 6-OHDA (unilaterally) into the medial forebrain bundle. The extent of the dopaminergic lesion induced by 6-OHDA was evaluated by apomorphine-induced rotational behavior and by tyrosine hydroxylase (TH) immunoreactivity. The 6-OHDA microinjection caused a partial or complete lesion of dopaminergic cells, as well as a reduction of Eag1+ cells in a manner proportional to the extent of the lesion. In addition, we observed a decrease in TH immunoreactivity in the ipsilateral striatum. In conclusion, the expression of the Eag1-K(+)-channel throughout the nigrostriatal pathway in the rat brain, its co-localization with dopaminergic cells and its reduction mirroring the extent of the lesion highlight a physiological circuitry where the functional role of this channel can be investigated. The Eag1-K(+) channel expression in dopaminergic cells suggests that these channels are part of the diversified group of ion channels that generate and maintain the electrophysiological activity pattern of dopaminergic midbrain neurons.

Eag 1, Eag 2 and Kcnn3 gene brain expression of isolated reared rats.

The Eag1 and Eag2, voltage-dependent potassium channels, and the small-conductance calcium-activated potassium channel (Kcnn3) are highly expressed in limbic regions of the brain, where their function is still unknown. Eag1 co-localizes with tyrosine hydroxilase enzyme in the substantia nigra and ventral tegmental area. Kcnn3 deficiency leads to enhanced serotonergic and dopaminergic neurotransmission accompanied by distinct alterations in emotional behaviors. As exposure to stress is able to change the expression and function of several ion channels, suggesting that they might be involved in the consequences of stress, we aimed at investigating Eag 1, Eag2 and Kcnn3 mRNA expression in the brains of rats submitted to isolation rearing. As the long-lasting alterations in emotional and behavioral regulation after stress have been related to changes in serotonergic neurotransmission, expressions of serotonin Htr1a and Htr2a receptors in male Wistar rats' brain were also investigated. Rats were reared in isolation or in groups of five for nine weeks after weaning. Isolated and socially reared rats were tested for exploratory activity in the open field test for 5 min and brains were processed for reverse-transcription coupled to quantitative polymerase chain reaction (qRT-PCR). Isolated reared rats showed decreased exploratory activity in the open field. Compared to socially reared rats, isolated rats showed reduced Htr2a mRNA expression in the striatum and brainstem and reduced Eag2 mRNA expression in all examined regions except cerebellum. To our knowledge, this is the first work to show that isolation rearing can change Eag2 gene expression in the brain. The involvement of this channel in stress-related behaviors is discussed.

Eag1 potassium channel immunohistochemistry in the CNS of adult rat and selected regions of human brain.

Eag1 (K(V)10.1) is the founding member of an evolutionarily conserved superfamily of voltage-gated K(+) channels. In rats and humans Eag1 is preferentially expressed in adult brain but its regional distribution has only been studied at mRNA level and only in the rat at high resolution. The main aim of the present study is to describe the distribution of Eag1 protein in adult rat brain in comparison to selected regions of the human adult brain. The distribution of Eag1 protein was assessed using alkaline-phosphatase based immunohistochemistry. Eag1 immunoreactivity was widespread, although selective, throughout rat brain, especially noticeable in the perinuclear space of cells and proximal regions of the extensions, both in rat and human brain. To relate the results to the relative abundance of Eag1 transcripts in different regions of rat brain a reverse-transcription coupled to quantitative polymerase chain reaction (real time PCR) was performed. This real time PCR analysis showed high Eag1 expression in the olfactory bulb, cerebral cortex, hippocampus, hypothalamus, and cerebellum. The results indicate that Eag1 protein expression greatly overlaps with mRNA distribution in rats and humans. The physiological relevance of potassium channels in the different regions expressing Eag1 protein is discussed.

Role of voltage-gated potassium channels in cancer.

Ion channels are being associated with a growing number of diseases including cancer. This overview summarizes data on voltage-gated potassium channels (VGKCs) that exhibit oncogenic properties: ether-à-go-go type 1 (Eag1). Normally, Eag1 is expressed almost exclusively in tissue of neural origin, but its ectopic expression leads to uncontrolled proliferation, while inhibition of Eag1 expression produces a concomitant reduction in proliferation. Specific monoclonal antibodies against Eag1 recognize an epitope in over 80% of human tumors of diverse origins, endowing it with diagnostic and therapeutic potential. Eag1 also possesses unique electrophysiological properties that simplify its identification. This is particularly important, as specific blockers of Eag1 currents are not available. Molecular imaging of Eag1 in live tumor models has been accomplished with dye-tagged antibodies using 3-D imaging techniques in the near-infrared spectral range.

Super-resolution measurements with evanescent-wave fluorescence excitation using variable beam incidence.

The evanescent wave (EW) elicited by total internal reflection of light selectively excites fluorophores in an optical slice above a reflecting dielectric interface. EW excitation eliminates out-of-focus fluorescence present in epiillumination microscopy, and--close to the coverslip--can offer a fivefold enhancement of axial optical sectioning compared to confocal and two-photon microscopy. The decay length of the evanescent field is a function of the refractive indices and light wavelength involved, and is modulated by the beam angle. EW microscopy was used to study the distribution and concentration of fluorophores at or near the interface in the presence of high concentrations in bulk solution. We modified an upright microscope to accommodate the condenser optics needed for EW excitation. Systematic variations of the angle of incidence were attained using an acousto-optical deflector, telecentric optics, and a hemicylindrical prism. The three-dimensional reconstruction of the fluorophore distribution from angle-resolved image stacks results in topographical information with an axial resolution of tens of nanometers. We applied this technique to study the axial position of dye-labeled subcellular storage organelles ('vesicles') of approximately 300 nm diameter in the "footprint" region of living neuroendocrine cells grown on the interface.

Tracking chromaffin granules on their way through the actin cortex.

Quantitative time-lapse evanescent-wave imaging of individual fluorescently labelled chromaffin granules was used for kinetic analysis of granule trafficking through a approximately 300-nm(1/e2) optical section beneath the plasma membrane. The mean squared displacement (MSD) was used to estimate the three-dimensional diffusion coefficient (D(3)). We calculated the granules' speed, frame-to-frame displacement and direction and their autocorrelation to identify different stages of approach to the membrane. D(3) was about 10,000 times lower than expected for free diffusion. Granules located approximately 60 nm beneath the plasma membrane moved on random tracks (D(3) approximately 10(-10) cm(2)s(-1)) with several reversals in direction before they approached their docking site at angles larger than 45 degrees. Docking was observed as a loss of vesicle mobility by two orders of magnitude within <100 ms. For longer observation times the MSD saturated, as if the granules' movement was confined to a volume only slightly larger than the granule. Rarely, the local random motion was superimposed with a directed movement in a plane beneath the membrane. Stimulation of exocytosis selectively depleted the immobile, near-membrane granule population and caused a recruitment of distant granules to sites at the plasma membrane. Their absolute mobility levels were not significantly altered. Application of latrunculin or jasplakinolide to change F-actin polymerisation caused a change in D(3) of the mobile granule population as well as a reduction of the rate of release, suggesting that granule mobility is constrained by the filamentous actin meshwork and that stimulation-dependent changes in actin viscosity propel granules through the actin cortex.

Cytoskeletal interactions determine the electrophysiological properties of human EAG potassium channels.

The electrophysiological properties of ether a go-go (EAG) potassium channels are modified during the cell cycle when they are expressed in heterologous systems. In Chinese hamster ovary (CHO) mammalian somatic cells we found that the cell-cycle-dependent modulation of human EAG (hEAG) channels occurs during the M phase. This modulation has three components: reduction in current density, increased sensitivity to block by intracellular sodium, and increased selectivity for potassium ions. In this work, these three properties have been used to define the mitotic phenotype of EAG currents. The signaling pathway leading to such changes of channel properties is unknown. We tested the hypothesis that cytoskeletal interactions might affect the electrophysiological changes observed during the cell cycle. The disruption of actin filaments induces a significant increase in current density, without inducing the cell-cycle-related phenotype. In contrast, disturbance of the microtubules, achieved by pharmacological means or by mechanical excision of the membrane patch, does induce the cell-cycle-related phenotype. Our results demonstrate that hEAG channels establish complex interactions with cytoskeletal elements, and that these interactions strongly influence the properties of the channels. We also conclude that the electrophysiological changes observed during the cell cycle are most likely due to reorganization of the cytoskeleton during the G2/M transition.

Oncogenic potential of EAG K(+) channels.

We have investigated the possible implication of the cell cycle-regulated K(+) channel ether à go-go (EAG) in cell proliferation and transformation. We show that transfection of EAG into mammalian cells confers a transformed phenotype. In addition, human EAG mRNA is detected in several somatic cancer cell lines, despite being preferentially expressed in brain among normal tissues. Inhibition of EAG expression in several of these cancer cell lines causes a significant reduction of cell proliferation. Moreover, the expression of EAG favours tumour progression when transfected cells are injected into immune-depressed mice. These data provide evidence for the oncogenic potential of EAG.

Antagonistic properties of the suramin analogue NF023 at heterologously expressed P2X receptors.

The suramin analogue 8,8'-(carbonylbis(imino-3,1-phenylene carbonylimino)bis(1,3,5-naphthalenetrisulfonic acid) (NF023) antagonizes in a competitive fashion P2X receptor-mediated responses in certain vascular and visceral smooth muscles. In the present study, the effect of NF023 on voltage-clamped Xenopus oocytes heterologously expressing homomultimeric P2X1-P2X4 as well as heteromultimeric P2X2/P2X3 receptors has been characterized. P2X1 receptors were most sensitive to inhibition by NF023 with IC50 values of 0.24 and 0.21 microM for the rat and human homologue, respectively. P2X3 receptors have an intermediate sensitivity with IC50 values of 8.5 and 28.9 microM for rat and human subtypes, respectively and P2X2 was the least sensitive subtype (IC50 > 50 microM). P2X4 receptors were insensitive to NF023 at concentrations up to 100 microM. Coexpression of rat P2X3 with rat P2X2 resulted in receptors whose sensitivity to NF023 was identical to that obtained for homomultimeric rat P2X3 receptors (alphabeta meATP as agonist; IC50 = 1.4 and 1.6 microM, respectively). NF023 inhibited P2X1 receptors in a voltage-insensitive manner. In addition, NF023 (5 and 30 microM) caused a shift of the concentration-response curve to the right without affecting the maximal response to ATP (K(B) = 1.1 +/- 0.2 microM). Our results indicate that NF023 is a subtype-selective and surmountable antagonist at P2X1 receptors heterologously expressed in Xenopus oocytes.

Evanescent-wave microscopy: a new tool to gain insight into the control of transmitter release.

Evanescent-wave excitation was used to visualize individual fluorescently labelled vesicles in an optical slice near the plasma membrane of bovine adrenal chromaffin cells. A standard upright microscope was modified to accommodate the optics used for directing a laser beam under a supracritical angle on to the glass-water interface on top of which the cells are grown. Whereas epi-illumination images appeared blurred and structureless, evanescent-wave excitation highlighted acridine orange-labelled vesicles as individual pinpoints. Three-dimensional (3D) trajectories of individual vesicles were obtained from time-resolved image stacks and used to characterize vesicles in terms of their average fluorescence F and mobility, expressed here as the 3D diffusion coefficient D(3). Based on the single-vesicle analysis, two groups of vesicles were identified. Transitions between these states were studied before and after stimulation of exocytosis by repetitive or maintained membrane depolarizations by elevated extracellular [K+]. Findings were interpreted as sequential transitions between the previously characterized pools of vesicles preceding the fusion step. The observed approach of vesicles to their docking sites was not explained in terms of free diffusion: most vesicles moved unidirectionally as if directed to their binding sites at the plasma membrane. Vesicle mobility at the membrane was low, such that the sites of docking and fusion were in close vicinity. Both the rim region and confined areas in the centre of the footprint region were the site of intense vesicle trafficking.

Multiple stimulation-dependent processes regulate the size of the releasable pool of vesicles.

In neuroendocrine cells and neurones, changes in the size of a limited pool of readily releasable vesicles contribute to the plasticity of secretion. We have studied the dynamic alterations in the size of a near-membrane pool of vesicles in living neuroendocrine cells. Using evanescent wave microscopy we monitored the behaviour of individual secretory vesicles at the plasma membrane. Vesicles undergo sequential transitions between several states of differing fluorescence intensity and mobility. The transitions are reversible, except for the fusion step, and even in nonstimulated conditions the vesicles change states in a dynamic equilibrium. Stimulation selectively speeds up the three forward transitions leading towards exocytosis. Vesicles lose mobility in all three dimensions upon approach of the plasma membrane. Their movement is directed and targeted to the docking fusion sites. Sites of vesicle docking and exocytosis are distributed non-uniformly over the studied "footprint" region of the cell. While some areas are the sites of repeated vesicle docking and fusion, others are completely devoid of spots. Vesicular mobility at the membrane is confined, as if the vesicle were imprisoned in a cage or tethered to a binding site.

Cell cycle-related changes in the conducting properties of r-eag K+ channels.

Release from arrest in G2 phase of the cell cycle causes profound changes in rat ether-à-go-go (r-eag) K+ channels heterologously expressed in Xenopus oocytes. The most evident consequence of the onset of maturation is the appearance of rectification in the r-eag current. The trigger for these changes is located downstream of the activation of mitosis-promoting factor (MPF). We demonstrate here that the rectification is due to a voltage-dependent block by intracellular Na+ ions. Manipulation of the intracellular Na+ concentration indicates that the site of Na+ block is located approximately 45% into the electrical distance of the pore and is only present in oocytes undergoing maturation. Since the currents through excised patches from immature oocytes exhibited a fast rundown, we studied CHO-K1 cells permanently transfected with r-eag. These cells displayed currents with a variable degree of block by Na+ and variable permeability to Cs+. Partial synchronization of the cultures in G0/G1 or M phases of the cell cycle greatly reduced the variability. The combined data obtained from mammalian cells and oocytes strongly suggest that the permeability properties of r-eag K+ channels are modulated during cell cycle-related processes.

Structure and function of voltage-gated ion channels.

Voltage-gated ion channels are key molecules for the generation of electrical signals in cells. They are integral membrane proteins which are activated by a depolarized membrane potential resulting in a conformational change, allowing ions to permeate. Voltage-gated ion channels can either be inactivated from this open state by an additional conformational change which leads to a nonconducting state of the channel, or they may be deactivated by a repolarized membrane potential. Following the first successful cloning of voltage-gated ion channels in 1984 the combination of molecular biological and electrophysiological techniques has been very fruitful in the investigation of the structure and function of these membrane proteins. From these studies a molecular picture of the structural elements important for the activity of voltage-gated ion channels has been established. This has assisted in clarifying the molecular basis of the electrical excitability of cells.

ATP binding site of P2X channel proteins: structural similarities with class II aminoacyl-tRNA synthetases.

The extracellular loop of P2X channel proteins contains a sequence stretch (positions 170-330) that exhibits similarities with the catalytic domains of class II aminoacyl-tRNA synthetases as shown by secondary structure predictions and sequence alignments. The arrangement of several conserved cysteines (positions 110-170) shows similarities with metal binding regions of metallothioneins and zinc finger motifs. Thus, for the extracellular part of P2X channel proteins a metal binding domain and an antiparallel six-stranded beta-pleated sheet containing the ATP binding site are very probable. The putative channel forming H5 part (positions 320-340) shows similarities with the enzyme motif 1 responsible for aggregation of subunits to the holoenzyme.