Biomedical subjects

Edwin S Levitan

Publications and source records attributed to Edwin S Levitan.

At least 19 recordsLinked to original sources

PubMed2006

Identification of gamma-aminobutyric acid receptor-interacting factor 1 (TRAK2) as a trafficking factor for the K+ channel Kir2.1.

To identify proteins that regulate potassium channel activity and expression, we performed functional screening of mammalian cDNA libraries in yeast that express the mammalian K(+) channel Kir2.1. Growth of Kir2.1-expressing yeast in media with low K(+) concentration is a function of K(+) uptake via Kir2.1 channels. Therefore, the host strain was transformed with a human cDNA library, and cDNA clones that rescued growth at low K(+) concentration were selected. One of these clones was identical to the protein of unknown function isolated previously as gamma-aminobutyric acid receptor-interacting factor 1 (GRIF-1) (Beck, M., Brickley, K., Wilkinson, H., Sharma, S., Smith, M., Chazot, P., Pollard, S., and Stephenson, F. (2002) J. Biol. Chem. 277, 30079-30090). GRIF-1 specifically enhanced Kir2.1-dependent growth in yeast and Kir2.1-mediated (86)Rb(+) efflux in HEK293 cells. Quantitative microscopy and flow cytometry analysis of immunolabeled surface Kir2.1 channel showed that GRIF-1 significantly increased the number of Kir2.1 channels in the plasma membrane of COS and HEK293 cells. Physical interaction of Kir2.1 channel and GRIF-1 was demonstrated by co-immunoprecipitation from HEK293 lysates and yeast two-hybrid assay. In vivo association of Kir2.1 and GRIF-1 was demonstrated by co-immunoprecipitation from brain lysate. Yeast two-hybrid assays showed that an N-terminal region of GRIF-1 interacts with a C-terminal region of Kir2.1. These results indicate that GRIF-1 binds to Kir2.1 and facilitates trafficking of this channel to the cell surface.

Animals↗

PubMed2006

Voltage-gated k+ channel block by catechol derivatives: defining nonselective and selective pharmacophores.

High-throughput screening led to the identification of a 3-norbornyl derivative of catechol called 48F10 (3-bicyclo[2.2.1]hept-2-yl-benzene-1,2-diol) as a Kv2.1 K(+) channel inhibitor. By virtue of the involvement of Kv2.1 channels in programmed cell death, 48F10 prevents apoptosis in cortical neurons and enterocytes. This uncharged compound acts with an apparent affinity of 1 muM at the tetraethylammonium (TEA) site at the external mouth of the Kv2.1 channel but is ineffective on Kv1.5. Here we investigated the basis of this selectivity with structure-activity studies. We find that catechol (1,2-benzenediol), unlike 48F10, inhibits Kv2.1 currents with a Hill coefficient of 2 and slows channel activation. Furthermore, this inhibition, which requires millimolar concentrations, is unaffected by external TEA or by mutation of the external tyrosine implicated in channel block by TEA and 48F10. In addition, catechol does not distinguish between Kv2.1 and Kv1.5. Thus, catechol acts at conserved sites that are distinct from 48F10. We also tested 11 catechol derivatives based on hydrocarbon adducts including norbornyl substructures, a 48F10 isomer, and a 48F10 diastereomer. These compounds are more potent than catechol, but none replicated the marked selectivity of 48F10 for Kv2.1 over Kv1.5. We conclude that the targeting of 48F10 to the TEA site at the external mouth of the Kv2.1 pore and away from other sites involved in nonselective Kv channel block by catechol requires the norbornyl group in a unique position and orientation on the catechol ring.

Animals↗

PubMed2006

Activity-dependent synaptic capture of transiting peptidergic vesicles.

Synapses require resources synthesized in the neuronal soma, but there are no known mechanisms to overcome delays associated with the synthesis and axonal transport of new proteins generated in response to activity, or to direct resources specifically to active synapses. Here, in vivo imaging of the Drosophila melanogaster neuromuscular junction reveals a cell-biological strategy that addresses these constraints. Peptidergic vesicles continually transit through resting terminals, but retrograde peptidergic vesicle flux is accessed following activity to rapidly boost neuropeptide content in synaptic boutons. The presence of excess transiting vesicles implies that synaptic neuropeptide stores are limited by the capture of peptidergic vesicles at the terminal, rather than by synthesis in the soma or delivery via the axon. Furthermore, activity-dependent capture from a pool of transiting vesicles provides a nerve terminal-based mechanism for directing distally and slowly generated resources quickly to active synapses. Finally, retrograde transport in the nerve terminal is regulated by activity.

Animals↗

PubMed2006

D2 autoreceptors chronically enhance dopamine neuron pacemaker activity.

Activation of D2 autoreceptors on midbrain dopamine neurons has been shown previously to acutely open K+ channels to inhibit intrinsically generated pacemaker activity. Here we report that D2 autoreceptors act chronically to produce an opposite action: to increase the speed and regularity of repetitive action potential firing. Voltage-, current-, and dynamic-clamp experiments, using conventional whole-cell and perforated patch-clamp recording, with cultured rat midbrain dopamine neurons show that a change in the number of functional A-type K+ channels alters firing rate and susceptibility to irregularity produced by other channels. cAMP and protein kinase A mediate the long-term action of D2 receptors in a manner that counters the short-term effect of this signaling pathway on K+ channel gating. We conclude that D2 autoreceptors, in addition to mediating acute negative feedback, are responsible for long-term enhancement of the rate and fidelity of dopamine neuron pacemaker activity.

Action Potentials↗

PubMed2006

Angiotensin II and stretch activate NADPH oxidase to destabilize cardiac Kv4.3 channel mRNA.

Pathological and physiological hypertrophy of the heart is associated with decreased expression of the Kv4.3 transient outward current (Ito) channel. The downregulation of channel mRNA and protein, which may be proarrhythmic, is recapitulated with cultured neonatal rat ventricular myocytes treated with angiotensin II (Ang II). Here we show that the 4.9 kb 3' untranslated region (3' UTR) of the Kv4.3 channel transcript confers Ang II sensitivity to a promoter-reporter construct. In contrast, Kv4.2 and Kv1.5 3'-UTR sequences are insensitive to Ang II. Both Kv4.3 3'-UTR reporter mRNA and activity are decreased in Ang II-treated cardiac myocytes, in accordance with a decrease in mRNA stability. This regulation is mediated by Ang II type 1 (AT1) receptors and abolished by NADPH oxidase inhibitors and dominant negative rac. The Ang II effect is also blocked by expression of superoxide dismutase (SOD), but not catalase, showing that superoxide is required. Dominant negative subunits, enzyme inhibitors and hydrogen peroxide experiments show that the apoptosis signal-regulating kinase 1 (ASK1)-p38 kinase pathway mediates downstream signaling from NADPH oxidase. Mechanical stretch also downregulates Kv4.3 3'-UTR reporter activity and this requires AT1 receptors and NADPH oxidase. Thus, activation of AT1 receptors by Ang II or stretch specifically destabilizes cardiac myocyte Kv4.3 channel mRNA by activating NADPH oxidase. These results link long-term control of cardiac K+ channel gene expression to a physiological reactive oxygen species signaling pathway.

3' Untranslated Regions↗

PubMed2006

Nearly neutral secretory vesicles in Drosophila nerve terminals.

The acidity of mammalian secretory vesicles drives concentration and processing of their contents. Here, pH-sensitive green fluorescent protein (GFP) variants show that the > or =30-fold (H+) difference between secretory vesicles (pH < or = 5.7) and the cytoplasm (pH = 7.2) in mammalian cells is not present in peptidergic and small synaptic vesicles of the Drosophila neuromuscular junction. First, we find that fluorescence from Topaz-tagged atrial natriuretic factor, a peptidergic vesicle pH indicator, is only modestly affected by collapsing the H+ gradient in type III synaptic boutons. Quantitation shows that peptidergic vesicles are nearly neutral (pH = 6.74 +/- 0.05), even when temperature is elevated. Furthermore, small synaptic vesicles in glutamatergic synaptic boutons, studied with synaptophluorin, are as alkaline as peptidergic vesicles. Finally, yellow fluorescent protein measurements show that cytoplasmic pH is only slightly different than in mammals (pH = 7.4). Thus, the marked acidity of mammalian secretory vesicles is not conserved in evolution, and a modest vesicular H+ gradient is sufficient for supporting neurotransmission.

Animals↗

PubMed2006

PDF cycling in the dorsal protocerebrum of the Drosophila brain is not necessary for circadian clock function.

In Drosophila, the neuropeptide pigment-dispersing factor (PDF) is a likely circadian molecule, secreted by central pacemaker neurons (LNvs). PDF is expressed in both small and large LNvs (sLNvs and lLNvs), and there are striking circadian oscillations of PDF staining intensity in the small cell termini, which require a functional molecular clock. This cycling may be relevant to the proposed role of PDF as a synchronizer of the clock system or as an output signal connecting pacemaker cells to locomotor activity centers. In this study, the authors use a generic neuropeptide fusion protein (atrial natriuretic factor-green fluorescent protein [ANF-GFP]) and show that it can be expressed in the same neurons as PDF itself. Yet, ANF-GFP as well as PDF itself does not manifest any cyclical accumulation in sLNv termini in adult transgenic flies. Surprisingly, the absence of detectable PDF cycling is not accompanied by any detectable behavioral pheno-type, since these transgenic flies have normal morning and evening anticipation in a light-dark cycle (LD) and are fully rhythmic in constant darkness (DD). The molecular clock is also not compromised. The results suggest that robust PDF cycling in sLNv termini plays no more than a minor role in the Drosophila circadian system and is apparently not even necessary for clock output function.

Animals↗

PubMed2005

Encoding light intensity by the cone photoreceptor synapse.

How cone synapses encode light intensity determines the precision of information transmission at the first synapse on the visual pathway. Although it is known that cone photoreceptors hyperpolarize to light over 4-5 log units of intensity, the relationship between light intensity and transmitter release at the cone synapse has not been determined. Here, we use two-photon microscopy to visualize release of the synaptic vesicle dye FM1-43 from cone terminals in the intact lizard retina, in response to different stimulus light intensities. We then employ electron microscopy to translate these measurements into vesicle release rates. We find that from darkness to bright light, release decreases from 49 to approximately 2 vesicles per 200 ms; therefore, cones compress their 10,000-fold operating range for phototransduction into a 25-fold range for synaptic vesicle release. Tonic release encodes ten distinguishable intensity levels, skewed to most finely represent bright light, assuming release obeys Poisson statistics.

Animals↗

PubMed2005

Attenuation of apoptosis in enterocytes by blockade of potassium channels.

Apoptosis plays an important role in maintaining the balance between proliferation and cell loss in the intestinal epithelium. Apoptosis rates may increase in intestinal pathologies such as inflammatory bowel disease and necrotizing enterocolitis, suggesting pharmacological prevention of apoptosis as a therapy for these conditions. Here, we explore the feasibility of this approach using the rat epithelial cell line IEC-6 as a model. On the basis of the known role of K+ efflux in apoptosis in various cell types, we hypothesized that K+ efflux is essential for apoptosis in enterocytes and that pharmacological blockade of this efflux would inhibit apoptosis. By probing intracellular [K+] with the K+-sensitive fluorescent dye and measuring the efflux of 86Rb+, we found that apoptosis-inducing treatment with the proteasome inhibitor MG-132 leads to a twofold increase in K+ efflux from IEC-6 cells. Blockade of K+ efflux with tetraethylammonium, 4-aminopyridine, stromatoxin, chromanol 293B, and the recently described K+ channel inhibitor 48F10 prevents DNA fragmentation, caspase activation, release of cytochrome c from mitochondria, and loss of mitochondrial membrane potential. Thus K+ efflux occurs early in the apoptotic program and is required for the execution of later events. Apoptotic K+ efflux critically depends on activation of p38 MAPK. These results demonstrate for the first time the requirement of K+ channel-mediated K+ efflux for progression of apoptosis in enterocytes and suggest the use of K+ channel blockers to prevent apoptotic cell loss occurring in intestinal pathologies.

Animals↗

PubMed2005

Activity-dependent liberation of synaptic neuropeptide vesicles.

Despite the importance of neuropeptide release, which is evoked by long bouts of action potential activity and which regulates behavior, peptidergic vesicle movement has not been examined in living nerve terminals. Previous in vitro studies have found that secretory vesicle motion at many sites of release is constitutive: Ca(2+) does not affect the movement of small synaptic vesicles in nerve terminals or the movement of large dense core vesicles in growth cones and endocrine cells. However, in vivo imaging of a neuropeptide, atrial natriuretic factor, tagged with green fluorescent protein in larval Drosophila melanogaster neuromuscular junctions shows that peptidergic vesicle behavior in nerve terminals is sensitive to activity-induced Ca(2+) influx. Specifically, peptidergic vesicles are immobile in resting synaptic boutons but become mobile after seconds of stimulation. Vesicle movement is undirected, occurs without the use of axonal transport motors or F-actin, and aids in the depletion of undocked neuropeptide vesicles. Peptidergic vesicle mobilization and post-tetanic potentiation of neuropeptide release are sustained for minutes.

Animals↗

PubMed2004

Peroxynitrite-induced neuronal apoptosis is mediated by intracellular zinc release and 12-lipoxygenase activation.

Peroxynitrite toxicity is a major cause of neuronal injury in stroke and neurodegenerative disorders. The mechanisms underlying the neurotoxicity induced by peroxynitrite are still unclear. In this study, we observed that TPEN [N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine], a zinc chelator, protected against neurotoxicity induced by exogenous as well as endogenous (coadministration of NMDA and a nitric oxide donor, diethylenetriamine NONOate) peroxynitrite. Two different approaches to detecting intracellular zinc release demonstrated the liberation of zinc from intracellular stores by peroxynitrite. In addition, we found that peroxynitrite toxicity was blocked by inhibitors of 12-lipoxygenase (12-LOX), p38 mitogen-activated protein kinase (MAPK), and caspase-3 and was associated with mitochondrial membrane depolarization. Inhibition of 12-LOX blocked the activation of p38 MAPK and caspase-3. Zinc itself induced the activation of 12-LOX, generation of reactive oxygen species (ROS), and activation of p38 MAPK and caspase-3. These data suggest a cell death pathway triggered by peroxynitrite in which intracellular zinc release leads to activation of 12-LOX, ROS accumulation, p38 activation, and caspase-3 activation. Therefore, therapies aimed at maintaining intracellular zinc homeostasis or blocking activation of 12-LOX may provide a novel avenue for the treatment of inflammation, stroke, and neurodegenerative diseases in which the formation of peroxynitrite is thought to be one of the important causes of cell death.

Adenosine Triphosphate↗

PubMed2004

Streamlined synaptic vesicle cycle in cone photoreceptor terminals.

Cone photoreceptors tonically release neurotransmitter in the dark through a continuous cycle of exocytosis and endocytosis. Here, using the synaptic vesicle marker FM1-43, we elucidate specialized features of the vesicle cycle. Unlike retinal bipolar cell terminals, where stimulation triggers bulk membrane retrieval, cone terminals appear to exclusively endocytose small vesicles. These retain their integrity until exocytosis, without pooling their membranes in endosomes. Endocytosed vesicles rapidly disperse through the terminal and are reused with no apparent delay. Unlike other synapses where most vesicles are immobilized and held in reserve, only a small fraction (<15%) becomes immobilized in cones. Photobleaching experiments suggest that vesicles move by diffusion and not by molecular motors on the cytoskeleton and that vesicle movement is not rate limiting for release. The huge reservoir of vesicles that move rapidly throughout cone terminals and the lack of a reserve pool are unique features, providing cones with a steady supply for continuous release.

Animals↗

PubMed2004

Using GFP to image peptide hormone and neuropeptide release in vitro and in vivo.

Traditionally, peptide secretion by endocrine cells and neurons was studied by measuring changes in release in response to experimental perturbations. Now it is possible to directly view dense core vesicles (DCVs), secretory apparatus proteins and individual exocytotic events by imaging fluorescent proteins in living cells. Fundamental insights into peptide release by cultured cells have been made with wide field, confocal and total internal reflection (also called evanescent wave) microscopes. Researchers have also used a variety of fluorescent protein constructs that vary in spectra, pH sensitivity, inducibility, and age dependence. Most recently, these approaches have been applied to transgenic animals so that hormone and neuropeptide release can be studied in vivo.

Animals↗

PubMed2004

Novel neuroprotective K+ channel inhibitor identified by high-throughput screening in yeast.

Discovery of K+ channel modulators is limited by low-throughput capacity of existing K+ channel assays. To enable high-throughput screening for novel pharmacological modulators of K+ channels, we developed an assay based on growth of yeast that functionally expresses mammalian Kir2.1 channels. Screening of 10,000 small molecules from a combinatorial chemical library yielded 42 potential Kir2.1 inhibitors. One compound, 3-bicyclo[2.2.1]hept-2-yl-benzene-1,2-diol, was confirmed to inhibit K+ channels in patch-clamp measurements in mammalian cells with EC50 values of 60 and 1 microM for Kir2.1 and Kv2.1 channels, respectively. Inhibition of Kv2.1 channels decreased in the presence of the external pore blocker tetraethylammonium (TEA) and depended on a residue required for extracellular TEA action, suggesting that the identified compound targets the external mouth of the channel. Furthermore, at the nontoxic concentration of 3 microM, the identified compound completely abolished in vitro neuronal apoptosis mediated by Kv2.1 channels. Therefore, yeast-based screening has identified a novel uncharged neuroprotective mammalian K+ channel inhibitor.

Animals↗

PubMed2003

Long-term K+ channel-mediated dampening of dopamine neuron excitability by the antipsychotic drug haloperidol.

Antipsychotic drugs require days of treatment to begin to produce therapeutic effects. We report that in vivo treatment with the antipsychotic drug haloperidol acts with a delay of days to slow spontaneous repetitive firing by isolated midbrain dopamine neurons. The decreased excitability is caused by an increased number of functional A-type K+ channels without any change in gating properties. Upregulation of dopamine neuron Kv4.3 mRNA accounts for this effect, demonstrating a role for channel gene expression in this delayed drug action. The resultant long-term dampening of dopamine neuron excitability may serve to tone down the dopamine system.

Action Potentials↗

PubMed2003

Differential association of the auxiliary subunit Kvbeta2 with Kv1.4 and Kv4.3 K+ channels.

Kvbeta2 subunits associate with Kv1 and Kv4 K+ channels, but the basis of preferential association is not understood. For example, detergent resistance suggests stronger auxiliary subunit association with Kv4.2 than with Kv1.2, but Kvbeta2 preferentially localizes with the latter channels in brain. Here we examine the interaction of Kvbeta2 with two native binding partners in brain: Kv4.3 and Kv1.4. We show that the auxiliary subunit binds more efficiently to Kv1.4 than to Kv4.3 in mammalian cells. However, preexisting Kvbeta2 complexes with Kv1.4 and Kv4.3 have similar detergent sensitivity. Thus, preferential steady state binding may reflect a difference in initial association rather than stability. We also find that that the cytoplasmic C-terminus of Kv4.3 inhibits Kvbeta2 association. Apparently, a region proximal to the Kv4.3 pore contributes to the inefficient auxiliary subunit interaction that produces preferential binding of Kvbeta2 to Kv1 channels.

Binding Sites↗

PubMed2003

Mediation of neuronal apoptosis by Kv2.1-encoded potassium channels.

Cellular K+ efflux is a requisite event in the unfolding of apoptosis programs across many types of cells and death-inducing stimuli; however, the molecular identities of the ion channels mediating this key event have remained undefined. Here, we show that Kv2.1-encoded K+ channels are responsible for the expression of apoptosis in cortical neurons in vitro. Transient expression of two different dominant-negative forms of this subunit in neurons completely eliminated the enhancement of K+ currents that normally accompanies the cell death process. Importantly, neurons deficient in functional Kv2.1-encoded K+ channels were protected from oxidant and staurosporine-induced apoptosis. Finally, Chinese hamster ovary cells, which do not express endogenous voltage-gated K+ channels, became substantially more sensitive to apoptosis after transient expression of wild-type Kv2.1. These results suggest that Kv2.1-encoded K+ channels are necessary for the apoptotic signaling cascade in mammalian cortical neurons in culture and are sufficient for increasing the susceptibility to apoptogens in a nonexcitable cell.

Animals↗

PubMed2003

Unexpected mobility variation among individual secretory vesicles produces an apparent refractory neuropeptide pool.

Most stored neuropeptide cannot be released from nerve terminals suggesting the existence of a refractory pool of dense core vesicles (DCVs). Past fluorescence photobleaching recovery, single particle tracking and release experiments suggested that the refractory neuropeptide pool corresponds to a distinct immobile fraction of cytoplasmic DCVs. However, tracking of hundreds of individual green fluorescent protein-labeled neuropeptidergic vesicles by wide-field or evanescent-wave microscopy shows that a separate immobile fraction is not evident. Instead, the DCV diffusion coefficient (D) distribution is unusually broad and asymmetric. Furthermore, the distribution shifts with a release facilitator. This unexpected variation, which could reflect heterogeneity among vesicles or in their medium, is shown to generate the appearance of a regulated refractory neuropeptide pool.

Animals↗