D Singer-Lahat

Tel Aviv University, Tell Afif, Tel Aviv, Israel

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Publications (31)121.2 Total impact

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    Dataset: 39

    Full-text · Dataset · Nov 2015
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    Dataset: 39

    Full-text · Dataset · Nov 2015
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    Dataset: 39

    Full-text · Dataset · Nov 2015
  • Source
    Dataset: 39

    Full-text · Dataset · Nov 2015
  • Source
    Dataset: 39

    Full-text · Dataset · Nov 2015
  • Source
    Dataset: 39

    Full-text · Dataset · Nov 2015
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    ABSTRACT: Neuronal M-type K(+) channels heteromers of KCNQ2 and KCNQ3 subunits found in cell bodies, dendrites and the axon initial segment, regulate firing properties of neurons, while presynaptic KCNQ2 homomeric channels directly regulate neurotransmitter release. Previously, we have described a mechanism for gating down-regulation of KCNQ2 homomeric channels by calmodulin and syntaxin1A. Here, we describe a novel mechanism for KCNQ2 channels gating regulation utilized by Src, a non-receptor tyrosine kinase, in which two concurrent distinct structural rearrangements of the cytosolic termini induce two opposing effects, up-regulation of single-channel open probability, mediated by an N-terminal tyrosine, and reduction in functional channels, mediated by a C-terminal tyrosine. In contrast, Src regulation of KCNQ3 homomeric channels, shown before to be mediated by corresponding tyrosines, involves N-terminal tyrosine-mediated down-regulation of the open probability, rather than up-regulation. We argue that the dual bidirectional regulation of KCNQ2 functionality by Src, mediated via two separate sites, renders it modifiable by cellular factors that may specifically interact with either one of the sites, bearing potential significance in the fine-tuning of neurotransmitters release at nerve terminals. © 2015. Published by The Company of Biologists Ltd.
    Full-text · Article · Aug 2015 · Journal of Cell Science
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    ABSTRACT: It is thought that the voltage-dependent potassium channel subunit Kv2.1 (Kv2.1) regulates insulin secretion by controlling beta cell electrical excitability. However, this role of Kv2.1 in human insulin secretion has been questioned. Interestingly, Kv2.1 can also regulate exocytosis through direct interaction of its C-terminus with the soluble NSF attachment receptor (SNARE) protein, syntaxin 1A. We hypothesised that this interaction mediates insulin secretion independently of Kv2.1 electrical function. Wild-type Kv2.1 or mutants lacking electrical function and syntaxin 1A binding were studied in rodent and human beta cells, and in INS-1 cells. Small intracellular fragments of the channel were used to disrupt native Kv2.1-syntaxin 1A complexes. Single-cell exocytosis and ion channel currents were monitored by patch-clamp electrophysiology. Interaction between Kv2.1, syntaxin 1A and other SNARE proteins was probed by immunoprecipitation. Whole-islet Ca(2+)-responses were monitored by ratiometric Fura red fluorescence and insulin secretion was measured. Upregulation of Kv2.1 directly augmented beta cell exocytosis. This happened independently of channel electrical function, but was dependent on the Kv2.1 C-terminal syntaxin 1A-binding domain. Intracellular fragments of the Kv2.1 C-terminus disrupted native Kv2.1-syntaxin 1A interaction and impaired glucose-stimulated insulin secretion. This was not due to altered ion channel activity or impaired Ca(2+)-responses to glucose, but to reduced SNARE complex formation and Ca(2+)-dependent exocytosis. Direct interaction between syntaxin 1A and the Kv2.1 C-terminus is required for efficient insulin exocytosis and glucose-stimulated insulin secretion. This demonstrates that native Kv2.1-syntaxin 1A interaction plays a key role in human insulin secretion, which is separate from the channel's electrical function.
    Full-text · Article · Mar 2012 · Diabetologia
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    Full-text · Article · Feb 2011 · Biophysical Journal
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    ABSTRACT: Regulation of exocytosis by voltage-gated K(+) channels has classically been viewed as inhibition mediated by K(+) fluxes. We recently identified a new role for Kv2.1 in facilitating vesicle release from neuroendocrine cells, which is independent of K(+) flux. Here, we show that Kv2.1-induced facilitation of release is not restricted to neuroendocrine cells, but also occurs in the somatic-vesicle release from dorsal-root-ganglion neurons and is mediated by direct association of Kv2.1 with syntaxin. We further show in adrenal chromaffin cells that facilitation induced by both wild-type and non-conducting mutant Kv2.1 channels in response to long stimulation persists during successive stimulation, and can be attributed to an increased number of exocytotic events and not to changes in single-spike kinetics. Moreover, rigorous analysis of the pools of released vesicles reveals that Kv2.1 enhances the rate of vesicle recruitment during stimulation with high Ca(2+), without affecting the size of the readily releasable vesicle pool. These findings place a voltage-gated K(+) channel among the syntaxin-binding proteins that directly regulate pre-fusion steps in exocytosis.
    Full-text · Article · Jun 2010 · Journal of Cell Science
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    Lori Feinshreiber · Dafna Singer-Lahat · Uri Ashery · Ilana Lotan
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    ABSTRACT: Voltage-gated ion channels are well characterized for their function in excitability signals. Accumulating studies, however, have established an ion-independent function for the major classes of ion channels in cellular signaling. During the last few years we established a novel role for Kv2.1, a voltage-gated potassium (Kv) channel, classically known for its role of repolarizing the membrane potential, in facilitation of exocytosis. Kv2.1 induces facilitation of depolarization-induced release through its direct interaction with syntaxin, a protein component of the exocytotic machinery, independently of the potassium ion flow through the channel's pore. Here, we review our recent studies, further characterize the phenomena (using chromaffin cells and carbon fiber amperometry), and suggest plausible mechanisms that can underlie this facilitation of release.
    Full-text · Article · Feb 2009 · Annals of the New York Academy of Sciences
  • D. Chikvashvili · D. Singer-Lahat · R. Nachman · I. Lotan

    No preview · Article · Aug 2008 · European Neuropsychopharmacology
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    Dafna Singer-Lahat · Dodo Chikvashvili · Ilana Lotan
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    ABSTRACT: K(+) efflux through voltage-gated K(+) (Kv) channels can attenuate the release of neurotransmitters, neuropeptides and hormones by hyperpolarizing the membrane potential and attenuating Ca(2+) influx. Notably, direct interaction between Kv2.1 channels overexpressed in PC12 cells and syntaxin has recently been shown to facilitate dense core vesicle (DCV)-mediated release. Here, we focus on endogenous Kv2.1 channels and show that disruption of their interaction with native syntaxin after ATP-dependent priming of the vesicles by Kv2.1 syntaxin-binding peptides inhibits Ca(2+) -triggered exocytosis of DCVs from cracked PC12 cells in a specific and dose-dependent manner. The inhibition cannot simply be explained by the impairment of the interaction of syntaxin with its SNARE cognates. Thus, direct association between endogenous Kv2.1 and syntaxin enhances exocytosis and in combination with the Kv2.1 inhibitory effect to hyperpolarize the membrane potential, could contribute to the known activity dependence of DCV release in neuroendocrine cells and in dendrites where Kv2.1 commonly expresses and influences release.
    Full-text · Article · Feb 2008 · PLoS ONE
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    ABSTRACT: Kv channels inhibit release indirectly by hyperpolarizing membrane potential, but the significance of Kv channel interaction with the secretory apparatus is not known. The Kv2.1 channel is commonly expressed in the soma and dendrites of neurons, where it could influence the release of neuropeptides and neurotrophins, and in neuroendocrine cells, where it could influence hormone release. Here we show that Kv2.1 channels increase dense-core vesicle (DCV)-mediated release after elevation of cytoplasmic Ca2+. This facilitation occurs even after disruption of pore function and cannot be explained by changes in membrane potential and cytoplasmic Ca2+. However, triggering release increases channel binding to syntaxin, a secretory apparatus protein. Disrupting this interaction with competing peptides or by deleting the syntaxin association domain of the channel at the C terminus blocks facilitation of release. Thus, direct association of Kv2.1 with syntaxin promotes exocytosis. The dual functioning of the Kv channel to influence release, through its pore to hyperpolarize the membrane potential and through its C-terminal association with syntaxin to directly facilitate release, reinforces the requirements for repetitive firing for exocytosis of DCVs in neuroendocrine cells and in dendrites.
    Full-text · Article · Mar 2007 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
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    ABSTRACT: Previously we suggested that interaction between voltage-gated K+ channels and protein components of the exocytotic machinery regulated transmitter release. This study concerns the interaction between the Kv2.1 channel, the prevalent delayed rectifier K+ channel in neuroendocrine and endocrine cells, and syntaxin 1A and SNAP-25. We recently showed in islet beta-cells that the Kv2.1 K+ current is modulated by syntaxin 1A and SNAP-25. Here we demonstrate, using co-immunoprecipitation and immunocytochemistry analyses, the existence of a physical interaction in neuroendocrine cells between Kv2.1 and syntaxin 1A. Furthermore, using concomitant co-immunoprecipitation from plasma membranes and two-electrode voltage clamp analyses in Xenopus oocytes combined with in vitro binding analysis, we characterized the effects of these interactions on the Kv2.1 channel gating pertaining to the assembly/disassembly of the syntaxin 1A/SNAP-25 (target (t)-SNARE) complex. Syntaxin 1A alone binds strongly to Kv2.1 and shifts both activation and inactivation to hyperpolarized potentials. SNAP-25 alone binds weakly to Kv2.1 and probably has no effect by itself. Expression of SNAP-25 together with syntaxin 1A results in the formation of t-SNARE complexes, with consequent elimination of the effects of syntaxin 1A alone on both activation and inactivation. Moreover, inactivation is shifted to the opposite direction, toward depolarized potentials, and its extent and rate are attenuated. Based on these results we suggest that exocytosis in neuroendocrine cells is tuned by the dynamic coupling of the Kv2.1 channel gating to the assembly status of the t-SNARE complex.
    Full-text · Article · Oct 2003 · Journal of Biological Chemistry
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    ABSTRACT: Recently we suggested that direct interactions between voltage-gated K(+) channels and proteins of the exocytotic machinery, such as those observed between the Kv1.1/Kvbeta channel, syntaxin 1A, and SNAP-25 may be involved in neurotransmitter release. Furthermore, we demonstrated that the direct interaction with syntaxin 1A enhances the fast inactivation of Kv1.1/Kvbeta1.1 in oocytes. Here we show that G-protein betagamma subunits play a crucial role in the enhancement of inactivation by syntaxin 1A. The effect caused by overexpression of syntaxin 1A is eliminated in the presence of chelators of endogenous betagamma subunits in the whole cell and at the plasma membrane. Conversely, enhancement of inactivation caused by overexpression of beta(1)gamma(2) subunits is eliminated upon knock-down of endogenous syntaxin or its scavenging at the plasma membrane. We further show that the N terminus of Kv1.1 binds brain synaptosomal and recombinant syntaxin 1A and concomitantly binds beta(1)gamma(2); the binding of beta(1)gamma(2) enhances that of syntaxin 1A. Taken together, we suggest a mechanism whereby syntaxin and G protein betagamma subunits interact concomitantly with a Kv channel to regulate its inactivation.
    Full-text · Article · Oct 2002 · Journal of Biological Chemistry
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    O Fili · I Michaelevski · Y Bledi · D Chikvashvili · D Singer-Lahat · H Boshwitz · M Linial · I Lotan
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    ABSTRACT: Presynaptic voltage-gated K(+) (Kv) channels play a physiological role in the regulation of transmitter release by virtue of their ability to shape presynaptic action potentials. However, the possibility of a direct interaction of these channels with the exocytotic apparatus has never been examined. We report the existence of a physical interaction in brain synaptosomes between Kvalpha1.1 and Kvbeta subunits with syntaxin 1A, occurring, at least partially, within the context of a macromolecular complex containing syntaxin, synaptotagmin, and SNAP-25. The interaction was altered after stimulation of neurotransmitter release. The interaction with syntaxin was further characterized in Xenopus oocytes by both overexpression and antisense knock-down of syntaxin. Direct physical interaction of syntaxin with the channel protein resulted in an increase in the extent of fast inactivation of the Kv1.1/Kvbeta1.1 channel. Syntaxin also affected the channel amplitude in a biphasic manner, depending on its concentration. At low syntaxin concentrations there was a significant increase in amplitudes, with no detectable change in cell-surface channel expression. At higher concentrations, however, the amplitudes decreased, probably because of a concomitant decrease in cell-surface channel expression, consistent with the role of syntaxin in regulation of vesicle trafficking. The observed physical and functional interactions between syntaxin 1A and a Kv channel may play a role in synaptic efficacy and neuronal excitability.
    Full-text · Article · Apr 2001 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
  • D Singer-Lahat · N Dascal · L Mittelman · S Peleg · I Lotan
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    ABSTRACT: We describe the preparation of a Xenopus oocyte plasma membrane patch attached to a cover-slip with its intracellular face exposed to the bath solution. The proteins attached to the plasma membrane were visualized by confocal microscopy after fluorescence labelling. Since cortical microfilament elements were detected in these plasma membrane preparations we termed the patches plasma membrane-cortex patches. The way these patches are formed and the low concentration of proteins needed for cytochemical detection make the membrane-cortex patches similar to electrophysiological membrane patches and therefore allow the cytochemical study of ion channels to be correlated with electrophysiological experiments. Furthermore, the described patch is similar to manually isolated plasma membranes used for biochemical analysis by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Cytochemical analysis of membrane-cortex patches also enables the detection of the two-dimensional pattern of organization of membrane proteins (clustered or non-clustered forms). In addition, patch preparations enable cytochemical study of the relative localization of membrane proteins. The methodology enables integration of electrophysiological, biochemical and cytochemical studies of ion channels, giving a comprehensive perspective on ion channel function.
    No preview · Article · Sep 2000 · Pflügers Archiv - European Journal of Physiology
  • D. Singer-Lahat · N. Dascal · L. Mittelman · S. Peleg · I. Lotan
    [Show abstract] [Hide abstract]
    ABSTRACT: We describe the preparation of a Xenopus oocyte plasma membrane patch attached to a cover-slip with its intracellular face exposed to the bath solution. The proteins attached to the plasma membrane were visualized by confocal microscopy after fluorescence labelling. Since cortical microfilament elements were detected in these plasma membrane preparations we termed the patches plasma membrane-cortex patches. The way these patches are formed and the low concentration of proteins needed for cytochemical detection make the membrane-cortex patches similar to electrophysiological membrane patches and therefore allow the cytochemical study of ion channels to be correlated with electrophysiological experiments. Furthermore, the described patch is similar to manually isolated plasma membranes used for biochemical analysis by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Cytochemical analysis of membrane-cortex patches also enables the detection of the two-dimensional pattern of organization of membrane proteins (clustered or non-clustered forms). In addition, patch preparations enable cytochemical study of the relative localization of membrane proteins. The methodology enables integration of electrophysiological, biochemical and cytochemical studies of ion channels, giving a comprehensive perspective on ion channel function.
    No preview · Article · Aug 2000 · Pflügers Archiv - European Journal of Physiology
  • D Singer-Lahat · N Dascal · I Lotan
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    ABSTRACT: Modulation of fast-inactivating voltage-gated K+ channels can produce plastic changes in neuronal signaling. Previously, we showed that the voltage-dependent K+ channel composed of brain Kv1.1 and Kvbeta1.1 subunits (alpha(beta) channel) gives rise to a current that has a fast-inactivating and a sustained component; the proportion of the fast-inactivating component could be decreased by dephosphorylation of a basally phosphorylated Ser-446 on the alpha subunit. To account for our results we suggested a model that assumes a bimodal gating of the alpha(beta) channel. In this study, using single-channel analysis, we confirm this model. Two modes of gating were identified: (1) an inactivating mode characterized by low open probability and single openings early in the voltage step, and (2) a non-inactivating gating mode with bursts of openings. These two modes were non-randomly distributed, with spontaneous shifts between them. Each mode is characterized by a different set of open time constants (tau) and mean open times (t(0)). The non-inactivating mode is similar to the gating mode of a homomultimeric alpha channel. The phosphorylation-deficient alphaS446Abeta channel has the same two gating modes. Furthermore, alkaline phosphatase promoted the transition to the non-inactivating mode. This is the first report of modal behavior of a fast-inactivating K+ channel; furthermore, it substantiates the notion that direct phosphorylation is one mechanism that regulates the equilibrium between the two modes and thereby regulates the extent of macroscopic fast inactivation of a brain K+ channel.
    No preview · Article · Jan 2000 · Pflügers Archiv - European Journal of Physiology