Cdk-mediated phosphorylation of the Kvβ2 auxiliary subunit regulates Kv1 channel axonal targeting

Kv1 channels are concentrated at specific sites in the axonal membrane, where they regulate neuronal excitability. Establishing these distributions requires regulated dissociation of Kv1 channels from the neuronal trafficking machinery and their subsequent insertion into the axonal membrane. We find that the auxiliary Kvβ2 subunit of Kv1 channels purified from brain is phosphorylated on serine residues 9 and 31, and that cyclin-dependent kinase (Cdk)–mediated phosphorylation at these sites negatively regulates the interaction of Kvβ2 with the microtubule plus end–tracking protein EB1. Endogenous Cdks, EB1, and Kvβ2 phosphorylated at serine 31 are colocalized in the axons of cultured hippocampal neurons, with enrichment at the axon initial segment (AIS). Acute inhibition of Cdk activity leads to intracellular accumulation of EB1, Kvβ2, and Kv1 channel subunits within the AIS. These studies reveal a new regulatory mechanism for the targeting of Kv1 complexes to the axonal membrane through the reversible Cdk phosphorylation-dependent binding of Kvβ2 to EB1.

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Effect of Lamotrigine on the Ca2+-Sensing Cation Current in Cultured Hippocampal Neurons

Journal of Neurophysiology ◽

10.1152/jn.2001.86.5.2520 ◽

2001 ◽

Vol 86 (5) ◽

pp. 2520-2526 ◽

Cited By ~ 18

Author(s):

Zhi-Gang Xiong ◽

Xiang-Ping Chu ◽

J. F. MacDonald

Keyword(s):

Action Potentials ◽

Hippocampal Neurons ◽

Neuronal Excitability ◽

Membrane Depolarization ◽

Slow Inward Current ◽

Calcium Sensing ◽

Cation Current ◽

Imaging Experiment ◽

Intracellular Ca ◽

Cultured Hippocampal Neurons

Concentrations of extracellular calcium ([Ca2+]e) in the CNS decrease substantially during seizure activity. We have demonstrated previously that decreases in [Ca2+]e activate a novel calcium-sensing nonselective cation (csNSC) channel in hippocampal neurons. Activation of csNSC channels is responsible for a sustained membrane depolarization and increased neuronal excitability. Our study has suggested that the csNSC channel is likely involved in generating and maintaining seizure activities. In the present study, the effects of anti-epileptic agent lamotrigine (LTG) on csNSC channels were studied in cultured mouse hippocampal neurons using patch-clamp techniques. At a holding potential of −60 mV, a slow inward current through csNSC channels was activated by a step reduction of [Ca2+]e from 1.5 to 0.2 mM. LTG decreased the amplitude of csNSC currents dose dependently with an IC50 of 171 ± 25.8 (SE) μM. The effect of LTG was independent of membrane potential. In the presence of 300 μM LTG, the amplitude of csNSC current was decreased by 31 ± 3% at −60 mV and 29 ± 2.9% at +40 mV ( P > 0.05). LTG depressed csNSC current without affecting the potency of Ca2+ block of the current (IC50 for Ca2+block of csNSC currents in the absence of LTG: 145 ± 18 μM; in the presence of 300 μM LTG: 136 ± 10 μM. n = 5, P > 0.05). In current-clamp recordings, activation of csNSC channel by reducing the [Ca2+]e caused a sustained membrane depolarization and an increase in the frequency of spontaneous firing of action potentials. LTG (300 μM) significantly inhibited csNSC channel-mediated membrane depolarization and the excitation of neurons. Fura-2 ratiometric Ca2+imaging experiment showed that LTG also inhibited the increase in intracellular Ca2+ concentration induced by csNSC channel activation. The effect of LTG on csNSC channels may partially contribute to its broad spectrum of anti-epileptic actions.

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Ps in the (Channel) Pod Are Not Alike …

Epilepsy Currents ◽

10.1111/j.1535-7511.2007.00203.x ◽

2007 ◽

Vol 7 (5) ◽

pp. 136-137

Author(s):

Yoav Noam ◽

Tallie Z. Baram

Keyword(s):

Neuronal Activity ◽

Hippocampal Neurons ◽

Neuronal Excitability ◽

Phosphorylation Sites ◽

Voltage Dependent ◽

Activity Dependent ◽

Stimulation Of ◽

Cultured Hippocampal Neurons

Bidirectional Activity-Dependent Regulation of Neuronal Ion Channel Phosphorylation. Misonou H, Menegola M, Mohapatra DP, Guy LK, Park KS, Trimmer JS. J Neurosci 2006;26(52):13505–13514. 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.

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An MBoC Favorite: Axonal membrane proteins are transported in distinct carriers: a two-color video microscopy study in cultured hippocampal neurons

Molecular Biology of the Cell ◽

10.1091/mbc.e12-02-0154 ◽

2012 ◽

Vol 23 (11) ◽

pp. 2015-2015

Author(s):

Pearl V. Ryder ◽

Gary J. Bassell ◽

Victor Faundez

Keyword(s):

Membrane Proteins ◽

Hippocampal Neurons ◽

Microscopy Study ◽

Video Microscopy ◽

Axonal Membrane ◽

Color Video ◽

Cultured Hippocampal Neurons

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Clustering of Extrasynaptic GABAAReceptors Modulates Tonic Inhibition in Cultured Hippocampal Neurons

Journal of Biological Chemistry ◽

10.1074/jbc.m407229200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 45833-45843 ◽

Cited By ~ 35

Author(s):

Enrica Maria Petrini ◽

Ivan Marchionni ◽

Paola Zacchi ◽

Werner Sieghart ◽

Enrico Cherubini

Keyword(s):

Hippocampal Neurons ◽

Neuronal Excitability ◽

Prolonged Exposure ◽

Tonic Inhibition ◽

Aminobutyric Acid ◽

Action Potential Generation ◽

Potential Generation ◽

Acid Type ◽

Ambient Gaba ◽

Cultured Hippocampal Neurons

Tonic inhibition plays a crucial role in regulating neuronal excitability because it sets the threshold for action potential generation and integrates excitatory signals. Tonic currents are known to be largely mediated by extrasynaptic γ-aminobutyric acid type A (GABAA) receptors that are persistently activated by submicromo-lar concentrations of ambient GABA. We recently reported that, in cultured hippocampal neurons, the clustering of synaptic GABAAreceptors significantly affects synaptic transmission (Petrini, E. M., Zacchi, P., Barberis, A., Mozrzymas, J. W., and Cherubini, E. (2003)J. Biol. Chem.278, 16271–16279). In this work, we demonstrated that the clustering of extrasynaptic GABAAreceptors modulated tonic inhibition. Depolymerization of the cytoskeleton with nocodazole promoted the disassembly of extrasynaptic clusters of δ and γ2subunit-containing GABAAreceptors. This effect was associated with a reduction in the amplitude of tonic currents and diminished shunting inhibition. Moreover, diffuse GABAAreceptors were less sensitive to the GAT-1 inhibitor NO-711 and to flurazepam. Quantitative analysis of GABA-evoked currents after prolonged exposure to submicromolar concentrations of GABA and model simulations suggest that clustering affects the gating properties of extrasynaptic GABAAreceptors. In particular, a larger occupancy of the singly and doubly bound desensitized states can account for the modulation of tonic inhibition recorded after nocodazole treatment. Moreover, comparison of tonic currents recorded during spontaneous activity and those elicited by exogenously applied low agonist concentrations allows estimation of the concentration of ambient GABA. In conclusion, receptor clustering appears to be an additional regulating factor for tonic inhibition.

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