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ABSTRACT: During development of the central nervous system, precise synaptic connections between presynaptic and postsynaptic neurons are formed. While significant progress has been made in our understanding of AMPA receptor trafficking during synaptic plasticity, less is known about the molecules that recruit AMPA receptors to nascent synapses during synaptogenesis. Here we identify a type II transmembrane protein (SynDIG1) that regulates AMPA receptor content at developing synapses in dissociated rat hippocampal neurons. SynDIG1 colocalizes with AMPA receptors at synapses and at extrasynaptic sites and associates with AMPA receptors in heterologous cells and brain. Altered levels of SynDIG1 in cultured neurons result in striking changes in excitatory synapse number and function. SynDIG1-mediated synapse development is dependent on association with AMPA receptors via its extracellular C terminus. Intriguingly, SynDIG1 content in dendritic spines is regulated by neuronal activity. Altogether, we define SynDIG1 as an activity-regulated transmembrane protein that regulates excitatory synapse development.
Neuron 01/2010; 65(1):80-93. · 14.74 Impact Factor
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ABSTRACT: The intrinsic electrical properties and the synaptic input-output relationships of neurons are governed by the action of voltage-dependent ion channels. The localization of specific populations of ion channels with distinct functional properties at discrete sites in neurons dramatically impacts excitability and synaptic transmission. Molecular cloning studies have revealed a large family of genes encoding voltage-dependent ion channel principal and auxiliary subunits, most of which are expressed in mammalian central neurons. Much recent effort has focused on determining which of these subunits coassemble into native neuronal channel complexes, and the cellular and subcellular distributions of these complexes, as a crucial step in understanding the contribution of these channels to specific aspects of neuronal function. Here we review progress made on recent studies aimed to determine the cellular and subcellular distribution of specific ion channel subunits in mammalian brain neurons using in situ hybridization and immunohistochemistry. We also discuss the repertoire of ion channel subunits in specific neuronal compartments and implications for neuronal physiology. Finally, we discuss the emerging mechanisms for determining the discrete subcellular distributions observed for many neuronal ion channels.
Physiological Reviews 11/2008; 88(4):1407-47. · 26.87 Impact Factor
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ABSTRACT: Voltage-gated potassium (Kv) channels comprise four transmembrane alpha subunits, often associated with cytoplasmic beta subunits that impact channel expression and function. Here, we show that cell surface expression, voltage-dependent activation gating, and phosphorylation-dependent modulation of Kv2.1 are regulated by cytoplasmic N/C interaction within the alpha subunit. Kv2.1 surface expression is greatly reduced by C-terminal truncation. Tailless Kv2.1 channels exhibit altered voltage-dependent gating properties and lack the bulk of the phosphorylation-dependent modulation of channel gating. Remarkably, the soluble C terminus of Kv2.1 associates with tailless channels and rescues their expression, function, and phosphorylation-dependent modulation. Soluble N and C termini of Kv2.1 can also interact directly. We also show that the N/C-terminal interaction in Kv2.1 is governed by a 34 aa motif in the juxtamembrane cytoplasmic C terminus, and a 17 aa motif located in the N terminus at a position equivalent to the beta subunit binding site in other Kv channels. Deletion of either motif disrupts N/C-terminal interaction and surface expression, function, and phosphorylation-dependent modulation of Kv2.1 channels. These findings provide novel insights into intrinsic mechanisms for the regulation of Kv2.1 trafficking, gating, and phosphorylation-dependent modulation through cytoplasmic N/C-terminal interaction, which resembles alpha/beta subunit interaction in other Kv channels.
Journal of Neuroscience 06/2008; 28(19):4982-94. · 7.11 Impact Factor
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ABSTRACT: Among ion channels, voltage-gated calcium channels have been considered unique in their ability to mediate signaling events independent of the flow of ions through their pore. A voltage-gated potassium channel termed Kv2.1 has been identified as playing a role remarkably similar to one ion-independent function of calcium channels, facilitating regulated exocytosis through a direct interaction with a t-SNARE [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor] component of the vesicle release machinery. Kv2.1 overexpression enhances depolarization-induced secretion from the neuroendocrine-like PC12 cell line, and a nonconducting Kv2.1 mutant can accomplish the same feat. Kv2.1 interacts directly with syntaxin 1A, a plasma membrane t-SNARE component of the vesicle docking and fusion apparatus. Deletion of the syntaxin 1A-binding segment from Kv2.1 abolishes its ability to promote vesicle release, supporting a mechanism whereby Kv2.1 presumably transfers voltage-dependent conformational changes induced by membrane depolarization to interacting t-SNAREs to affect exocytosis. Kv2.1, a major mediator of electrical events in central neurons, cardiac and smooth muscle, and pancreatic beta cells, must now also be recognized as a physical mediator of secretion. That Kv2.1 is phosphorylated at numerous sites within the syntaxin 1A binding segment raises the possibility that its role in secretion may be dynamically regulated by diverse signaling events.
Science s STKE 08/2007; 2007(393):pe37.
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ABSTRACT: Modulation of voltage-gated potassium (Kv) channel surface expression can profoundly affect neuronal excitability. Some, but not all, mammalian Shaker or Kv1 alpha subunits contain a dominant endoplasmic reticulum (ER) retention signal in their pore region, preventing surface expression of Kv1.1 homotetrameric channels and of heteromeric Kv1 channels containing more than one Kv1.1 subunit. The critical amino acid residues within this ER pore-region retention signal are also critical for high-affinity binding of snake dendrotoxins (DTX). This suggests that ER retention may be mediated by an ER protein with a domain structurally similar to that of DTX. One facet of such a model is that expression of soluble DTX in the ER lumen should compete for binding to the retention protein and allow for surface expression of retained Kv1.1. Here, we show that luminal DTX expression dramatically increased both the level of cell surface Kv1.1 immunofluorescence staining and the proportion of Kv1.1 with processed N-linked oligosaccharides. Electrophysiological analyses showed that luminal DTX expression led to significant increases in Kv1.1 currents. Together, these data showed that luminal DTX expression increases surface expression of functional Kv1.1 homotetrameric channels and support a model whereby a DTX-like ER protein regulates abundance of cell surface Kv1 channels.
The FASEB Journal 04/2007; 21(3):906-14. · 5.71 Impact Factor
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ABSTRACT: Activity-dependent dephosphorylation of neuronal Kv2.1 channels yields hyperpolarizing shifts in their voltage-dependent activation and homoeostatic suppression of neuronal excitability. We recently identified 16 phosphorylation sites that modulate Kv2.1 function. Here, we show that in mammalian neurons, compared with other regulated sites, such as serine (S)563, phosphorylation at S603 is supersensitive to calcineurin-mediated dephosphorylation in response to kainate-induced seizures in vivo, and brief glutamate stimulation of cultured hippocampal neurons. In vitro calcineurin digestion shows that supersensitivity of S603 dephosphorylation is an inherent property of Kv2.1. Conversely, suppression of neuronal activity by anesthetic in vivo causes hyperphosphorylation at S603 but not S563. Distinct regulation of individual phosphorylation sites allows for graded and bidirectional homeostatic regulation of Kv2.1 function. S603 phosphorylation represents a sensitive bidirectional biosensor of neuronal activity.
Journal of Neuroscience 01/2007; 26(52):13505-14. · 7.11 Impact Factor
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ABSTRACT: Dynamic modulation of ion channels by phosphorylation underlies neuronal plasticity. The Kv2.1 potassium channel is highly phosphorylated in resting mammalian neurons. Activity-dependent Kv2.1 dephosphorylation by calcineurin induces graded hyperpolarizing shifts in voltage-dependent activation, causing suppression of neuronal excitability. Mass spectrometry-SILAC (stable isotope labeling with amino acids in cell culture) identified 16 Kv2.1 phosphorylation sites, of which 7 were dephosphorylated by calcineurin. Mutation of individual calcineurin-regulated sites to alanine produced incremental shifts mimicking dephosphorylation, whereas mutation to aspartate yielded equivalent resistance to calcineurin. Mutations at multiple sites were additive, showing that variable phosphorylation of Kv2.1 at a large number of sites allows graded activity-dependent regulation of channel gating and neuronal firing properties.
Science 09/2006; 313(5789):976-9. · 31.20 Impact Factor
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ABSTRACT: Modulation of K+ channels is widely used to dynamically regulate neuronal membrane excitability. The voltage-gated K+ channel Kv2.1 is an abundant delayed rectifier K+ (IK) channel expressed at high levels in many types of mammalian central neurons where it regulates diverse aspects of membrane excitability. Neuronal Kv2.1 is constitutively phosphorylated, localized in high-density somatodendritic clusters, and has a relatively depolarized voltage dependence of activation. Here, we show that the clustering and voltage-dependent gating of endogenous Kv2.1 in cultured rat hippocampal neurons are modulated by cholinergic stimulation, a common form of neuromodulation. The properties of neuronal Kv2.1 are recapitulated in recombinant Kv2.1 expressed in human embryonic kidney 293 (HEK293) cells, but not COS-1 cells, because of cell background-specific differences in Kv2.1 phosphorylation. As in neurons, Kv2.1 in HEK293 cells is dynamically regulated by cholinergic stimulation, which leads to Ca2+/calcineurin-dependent dephosphorylation of Kv2.1, dispersion of channel clusters, and hyperpolarizing shifts in the voltage-dependent gating properties of the channel. Immunocytochemical, biochemical, and biophysical analyses of chimeric Kv channels show that the Kv2.1 cytoplasmic C-terminal domain can act as an autonomous domain sufficient to transfer Kv2.1-like clustering, voltage-dependent activation, and cholinergic modulation to diverse Kv channels. These findings provide novel mechanistic insights into cholinergic modulation of ion channels and regulation of the localization and voltage-dependent gating properties of the abundant neuronal Kv2.1 channel by cholinergic and other neuromodulatory stimuli.
Journal of Neuroscience 02/2006; 26(2):685-95. · 7.11 Impact Factor
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ABSTRACT: Ischemic stroke is often accompanied by neuronal hyperexcitability (i.e., seizures), which aggravates brain damage. Therefore, suppressing stroke-induced hyperexcitability and associated excitoxicity is a major focus of treatment for ischemic insults. Both ATP-dependent and Ca2+-activated K+ channels have been implicated in protective mechanisms to suppress ischemia-induced hyperexcitability. Here we provide evidence that the localization and function of Kv2.1, the major somatodendritic delayed rectifier voltage-dependent K+ channel in central neurons, is regulated by hypoxia/ischemia-induced changes in metabolic state and intracellular Ca2+ levels. Hypoxia/ischemia in rat brain induced a dramatic dephosphorylation of Kv2.1 and the translocation of surface Kv2.1 from clusters to a uniform localization. In cultured rat hippocampal neurons, chemical ischemia (CI) elicited a similar dephosphorylation and translocation of Kv2.1. These events were reversible and were mediated by Ca2+ release from intracellular stores and calcineurin-mediated Kv2.1 dephosphorylation. CI also induced a hyperpolarizing shift in the voltage-dependent activation of neuronal delayed rectifier currents (IK), leading to enhanced IK and suppressed neuronal excitability. The IK blocker tetraethylammonium reversed the ischemia-induced suppression of excitability and aggravated ischemic neuronal damage. Our results show that Kv2.1 can act as a novel Ca2+- and metabolic state-sensitive K+ channel and suggest that dynamic modulation of IK/Kv2.1 in response to hypoxia/ischemia suppresses neuronal excitability and could confer neuroprotection in response to brief ischemic insults.
Journal of Neuroscience 12/2005; 25(48):11184-93. · 7.11 Impact Factor
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ABSTRACT: Neurons use a variety of mechanisms to dynamically control their own signaling capabilities. Regulation of voltage-dependent K+ channel localization and function has long been recognized as a major mechanism to achieve dynamic regulation of intrinsic neuronal excitability in a number of mammalian and non-mammalian neurons. Our recent evidence, together with compelling data from other laboratories, suggests that in mammalian neurons the Kv2.1 channel may play an especially prominent role in determining intrinsic neuronal excitability. Kv2.1 is widely expressed in brain and composes the majority of delayed rectifier K+ current in pyramidal neurons in cortex and hippocampus, and is also widely expressed in interneurons. Dynamic modulation of Kv2.1 localization and function by a mechanism involving activity-dependent Kv2.1 dephosphorylation dramatically impacts intrinsic excitability of neurons. Here we review previous studies of Kv2.1 localization and function in neurons, and summarize recent work regarding dynamic regulation of these characteristics. We also discuss possible roles of the Kv2.1 channel in neuronal and network excitability.
NeuroToxicology 11/2005; 26(5):743-52. · 3.10 Impact Factor
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ABSTRACT: Voltage-dependent Kv2.1 K(+) channels, which mediate delayed rectifier Kv currents (I(K)), are expressed in large clusters on the somata and dendrites of principal pyramidal neurons, where they regulate neuronal excitability. Here we report activity-dependent changes in the localization and biophysical properties of Kv2.1. In the kainate model of continuous seizures in rat, we find a loss of Kv2.1 clustering in pyramidal neurons in vivo. Biochemical analysis of Kv2.1 in the brains of these rats shows a marked dephosphorylation of Kv2.1. In cultured rat hippocampal pyramidal neurons, glutamate stimulation rapidly causes dephosphorylation of Kv2.1, translocation of Kv2.1 from clusters to a more uniform localization, and a shift in the voltage-dependent activation of I(K). An influx of Ca(2+) leading to calcineurin activation is both necessary and sufficient for these effects. Our finding that neuronal activity modifies the phosphorylation state, localization and function of Kv2.1 suggests an important link between excitatory neurotransmission and the intrinsic excitability of pyramidal neurons.
Nature Neuroscience 08/2004; 7(7):711-8. · 15.53 Impact Factor
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ABSTRACT: The K(v)2.1 potassium channel plays an important role in regulating membrane excitability and is highly phosphorylated in mammalian neurons. Our previous results showed that variable phosphorylation of K(v)2.1 at multiple sites allows graded activity-dependent regulation of channel gating. Our previous studies also found functional differences between recombinant K(v)2.1 channels expressed in HEK293 cells and COS-1 cells that were eliminated upon complete dephosphorylation of K(v)2.1. To better understand how phosphorylation affects K(v)2.1 gating in HEK293 and COS-1 cells we used stable isotope labeling by amino acids in cell culture (SILAC) and mass spectrometry to determine the level of phosphorylation at one newly and thirteen previously identified sites on K(v)2.1 purified from HEK293 and COS-1 cells. We identified seven phosphorylation sites on the K(v)2.1 C-terminus that exhibit different levels of phosphorylation in HEK293 and COS-1 cells. Six sites have enhanced phosphorylation in HEK293 compared to COS-1, while one site exhibits enhanced phosphorylation in COS-1 cells. No sites were found phosphorylated in one cell type and not the other. Interestingly, the sites exhibiting differential phosphorylation in HEK293 and COS-1 cells under basal conditions are similar to the subset targeted by calcineurin-mediated signaling pathways. The data presented here suggests that differential phosphorylation at a specific subset of sites, as opposed to utilization of novel cell-specific phosphorylation sites, can explain differences in the gating properties of K(v)2.1 in different cell types under basal conditions, and in the same cell type under basal versus stimulated conditions.
Channels (Austin, Tex.) 1(2):59-61. · 1.91 Impact Factor
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ABSTRACT: KV2.1 is the prominent somatodendritic sustained or delayed rectifier voltage-gated potassium (KV) channel in mammalian central neurons, and is a target for activity-dependent modulation via calcineurin-dependent dephosphorylation. Using hanatoxin-mediated block of KV2.1 we show that, in cultured rat hippocampal neurons, glutamate stimulation leads to significant hyperpolarizing shifts in the voltage-dependent activation and inactivation gating properties of the KV2.1-component of delayed rectifier K+ (IK) currents. In computer models of hippocampal neurons, these glutamate- stimulated shifts in the gating of the KV2.1-component of IK lead to a dramatic suppression of action potential firing frequency. Current-clamp experiments in cultured rat hippocampal neurons showed glutamate stimulation induced a similar suppression of neuronal firing frequency. Membrane depolarization also resulted in similar hyperpolarizing shifts in the voltage-dependent gating properties of neuronal IK currents, and suppression of neuronal firing. The glutamate-induced effects on neuronal firing were eliminated by hanatoxin, but not by dendrotoxin-K, a blocker of KV1.1-containing channels. These studies together demonstrate a specific contribution of modulation of KV2.1 channels in the activity-dependent regulation of intrinsic neuronal excitability.
Channels (Austin, Tex.) 3(1):46-56. · 1.91 Impact Factor
