Regulation of intrinsic excitability in hippocampal neurons by activity-dependent modulation of the KV2.1 potassium channel

K_V2.1 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 K_V2.1 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 K_V2.1-component of delayed rectifier K⁺ (IK) currents. In computer models of hippocampal neurons, these glutamate-stimulated shifts in the gating of the K_V2.1-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 K_V1.1-containing channels. These studies together demonstrate a specific contribution of modulation of K_V2.1 channels in the activity-dependent regulation of intrinsic neuronal excitability.     

Comparison of the endogenous IK currents in rat hippocampal neurons and cloned Kv2.1 channels in CHO cells

The Kv2.1 potassium channel is a principal component of the delayed rectifier I(K) current in the pyramidal neurons of cortex and hippocampus. We used whole-cell patch-clamp recording techniques to systemically compare the electrophysiological properties between the native neuronal I(K) current of cultured rat hippocampal neurons and the cloned Kv2.1 channel currents in the CHO cells. The slope factors for the activation curves of both currents obtained at different prepulse holding potentials and holding times were similar, suggesting similar voltage-dependent gating. However, the half-maximal activation voltage for I(K) was approximately 20 mV more negative than the Kv2.1 channel in CHO cells at a given prepulse condition, indicating that the neuronal I(K) current had a lower threshold for activation than that of the Kv2.1 channel. In addition, the neuronal I(K) showed a stronger holding membrane potential and holding time-dependence than Kv2.1. The Kv2.1 channel gave a U-shaped inactivation, while the I(K) current did not. The I(K) current also had much stronger voltage-dependent inactivation than Kv2.1. These results imply that the neuronal factors could make Kv2.1 channels easier to activate. The information obtained from these comparative studies help elucidate the mechanism of molecular regulation of the native neuronal I(K) current in neurons.     

Expression of Kv1.3 potassium channels regulates density of cortical interneurons

The Kv1.3 protein is a member of the large family of voltage‐dependent K⁺ subunits (Kv channels), which assemble to form tetrameric membrane‐spanning channels that provide a selective pore for the conductance of K⁺ across the cell membrane. Kv1.3 differs from most other Kv channels in that deletion of Kv1.3 gene produces very striking changes in development and structure of the olfactory bulb, where Kv1.3 is expressed at high levels, resulting in a lower threshold for detection of odors, an increased number of synaptic glomeruli and alterations in the levels of a variety of neuronal signaling molecules. Because Kv1.3 is also expressed in the cerebral cortex, we have now examined the effects of deletion of the Kv1.3 gene on the expression of interneuron populations of the cerebral cortex. Using unbiased stereology we found an increase in the number of parvalbumin (PV) cells in whole cerebral cortex of Kv1.3^?/? mice relative to that in wild‐type mice, and a decrease in the number of calbindin (CB), calretinin (CR), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), and somatostatin (SOM) interneurons. These changes are accompanied by a decrease in the cortical volume such that the cell density of PV interneurons is significantly increased and that of SOM neurons is decreased in Kv1.3^?/? animals. Our studies suggest that, as in the olfactory bulb, Kv1.3 plays a unique role in neuronal differentiation and/or survival of interneuron populations and that expression of Kv1.3 is required for normal cortical function. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73:841–855, 2013     

Age-dependent axonal expression of potassium channel proteins during development in mouse hippocampus

The development of the hippocampal network requires neuronal activity, which is shaped by the differential expression and sorting of a variety of potassium channels. Parallel to their maturation, hippocampal neurons undergo a distinct development of their ion channel profile. The age-dependent dimension of ion channel occurrence is of utmost importance as it is interdependently linked to network formation. However, data regarding the exact temporal expression of potassium channels during postnatal hippocampal development are scarce. We therefore studied the expression of several voltage-gated potassium channel proteins during hippocampal development in vivo and in primary cultures, focusing on channels that were sorted to the axonal compartment. The Kv1.1, Kv1.2, Kv1.4, and Kv3.4 proteins showed a considerable temporal variation of axonal localization among neuronal subpopulations. It is possible, therefore, that hippocampal neurons possess cell type-specific mechanisms for channel compartmentalization. Thus, age-dependent axonal sorting of the potassium channel proteins offers a new approach to functionally distinguish classes of hippocampal neurons and may extend our understanding of hippocampal circuitry and memory processing.     

Defective trafficking of Kv2.1 channels in MPTP‐induced nigrostriatal degeneration

Intracellular protein trafficking is tightly regulated, and improper trafficking might be the fundamental provocateur for human diseases including neurodegeneration. In neurons, protein trafficking to and from the plasma membrane affects synaptic plasticity. Voltage‐gated potassium channel 2.1 (Kv2.1) is a predominant delayed rectifier potassium (K⁺) current, and electrical activity patterns of dopamine (DA) neurons within the substantia nigra are generated and modulated by the orchestrated function of different ion channels. The pathological hallmark of Parkinson's disease (PD) is the progressive loss of these DA neurons, resulting in the degeneration of striatal dopaminergic terminals. However, whether trafficking of Kv2.1 channels contributes to PD remains unclear. In this study, we demonstrated that MPTP/MPP⁺ increases the surface expression of the Kv2.1 channel and causes nigrostriatal degeneration by using a subchronic MPTP mouse model. The inhibition of the Kv2.1 channel by using a specific blocker, guangxitoxin‐1E, protected nigrostriatal projections against MPTP/MPP⁺ insult and thus facilitated the recovery of motor coordination. These findings highlight the importance of trafficking of Kv2.1 channels in the pathogenesis of PD.

     

Heteromeric channels formed by TRPC1, TRPC4 and TRPC5 define hippocampal synaptic transmission and working memory

Canonical transient receptor potential (TRPC) channels influence various neuronal functions. Using quantitative high‐resolution mass spectrometry, we demonstrate that TRPC1, TRPC4, and TRPC5 assemble into heteromultimers with each other, but not with other TRP family members in the mouse brain and hippocampus. In hippocampal neurons from Trpc1/Trpc4/Trpc5‐triple‐knockout (Trpc1/4/5^?/?) mice, lacking any TRPC1‐, TRPC4‐, or TRPC5‐containing channels, action potential‐triggered excitatory postsynaptic currents (EPSCs) were significantly reduced, whereas frequency, amplitude, and kinetics of quantal miniature EPSC signaling remained unchanged. Likewise, evoked postsynaptic responses in hippocampal slice recordings and transient potentiation after tetanic stimulation were decreased. In vivo, Trpc1/4/5^?/? mice displayed impaired cross‐frequency coupling in hippocampal networks and deficits in spatial working memory, while spatial reference memory was unaltered. Trpc1/4/5^?/? animals also exhibited deficiencies in adapting to a new challenge in a relearning task. Our results indicate the contribution of heteromultimeric channels from TRPC1, TRPC4, and TRPC5 subunits to the regulation of mechanisms underlying spatial working memory and flexible relearning by facilitating proper synaptic transmission in hippocampal neurons.     

Involvement of α2‐antiplasmin in dendritic growth of hippocampal neurons

The α2‐Antiplasmin (α2AP) protein is known as a principal physiological inhibitor of plasmin, but we previously demonstrated that it acts as a regulatory factor for cellular functions independent of plasmin. α2AP is highly expressed in the hippocampus, suggesting a potential role for α2AP in hippocampal neuronal functions. However, the role for α2AP was unclear. This study is the first to investigate the involvement of α2AP in the dendritic growth of hippocampal neurons. The expression of microtubule‐associated protein 2, which contributes to neurite initiation and neuronal growth, was lower in the neurons from α2AP^?/? mice than in the neurons from α2AP^+/+ mice. Exogenous treatment with α2AP enhanced the microtubule‐associated protein 2 expression, dendritic growth and filopodia formation in the neurons. This study also elucidated the mechanism underlying the α2AP‐induced dendritic growth. Aprotinin, another plasmin inhibitor, had little effect on the dendritic growth of neurons, and α2AP induced its expression in the neurons from plaminogen^?/? mice. The activation of p38 MAPK was involved in the α2AP‐induced dendritic growth. Therefore, our findings suggest that α2AP induces dendritic growth in hippocampal neurons through p38 MAPK activation, independent of plasmin, providing new insights into the role of α2AP in the CNS.     

Physiological and behavioral effects of amphetamine in BACE1−/− mice

β‐Site APP‐cleaving Enzyme 1 (BACE1) is a protease that has been linked to schizophrenia, a severe mental illness that is potentially characterized by enhanced dopamine (DA) release in the striatum. Here, we used acute amphetamine administration to stimulate neuronal activity and investigated the neurophysiological and locomotor‐activity response in BACE1‐deficient (BACE1^?/?) mice. We measured locomotor activity at baseline and after treatment with amphetamine (3.2 and 10 mg/kg). While baseline locomotor activity did not vary between groups, BACE1^?/? mice exhibited reduced sensitivity to the locomotor‐enhancing effects of amphetamine. Using high‐performance liquid chromatography (HPLC) to measure DA and DA metabolites in the striatum, we found no significant differences in BACE1^?/? compared with wild‐type mice. To determine if DA neuron excitability is altered in BACE1^?/? mice, we performed patch‐clamp electrophysiology in putative DA neurons from brain slices that contained the substantia nigra. Pacemaker firing rate was slightly increased in slices from BACE1^?/? mice. We next measured G protein‐coupled potassium currents produced by activation of D2 autoreceptors, which strongly inhibit firing of these neurons. The maximal amplitude and decay times of D2 autoreceptor currents were not altered in BACE1^?/? mice, indicating no change in D2 autoreceptor‐sensitivity and DA transporter‐mediated reuptake. However, amphetamine (30 µm )‐induced potassium currents produced by efflux of DA were enhanced in BACE1^?/? mice, perhaps indicating increased vesicular DA content in the midbrain. This suggests a plausible mechanism to explain the decreased sensitivity to amphetamine‐induced locomotion, and provides evidence that decreased availability of BACE1 can produce persistent adaptations in the dopaminergic system.     

Kv 1.1 is associated with neuronal apoptosis and modulated by protein kinase C in the rat cerebellar granule cell

Previously, we reported that apoptosis of cerebellar granular neurons induced by low‐K⁺ and serum‐free (LK‐S) was associated with an increase in the A‐type K⁺ channel current (I_A), and an elevated expression of main α‐subunit of the I_A channel, which is known as Kv4.2 and Kv4.3. Here, we show, as assessed by quantitative RT‐PCR and whole‐cell recording, that besides Kv4.2 and Kv4.3, Kv1.1 is very important for I_A channel. The expression of Kv1.1 was elevated in the apoptotic neurons, while silencing Kv1.1 expression by siRNA reduced the I_A amplitude of the apoptotic neuron, and increased neuron viability. Inhibiting Kv1.1 current by dendrotoxin‐K evoked a similar effect of reduction of I_A amplitude and protection of neurons. Applying a protein kinase C (PKC) activator, phorbol ester acetate A (PMA) mimicked the LK‐S‐induced neuronal apoptotic effect, enhanced the I_A amplitude and reduced the granule cell viability. The PKC inhibitor, bisindolylmaleimide I and Gö6976 protected the cell against apoptosis induced by LK‐S. After silencing the Kv1.1 gene, the effect of PMA on the residual K⁺ current was reduced significantly. Quantitative RT‐PCR and Western immunoblot techniques revealed that LK‐S treatment and PMA increased the level of the expression of Kv1.1, in contrast, bisindolylmaleimide I inhibited Kv1.1 expression. In addition, the activation of the PKC isoform was identified in apoptotic neurons. We thus conclude that in the rat cerebellar granule cell, the I_A channel associated with apoptotic neurons is encoded mainly by the Kv1.1 gene, and that the PKC pathway promotes neuronal apoptosis by a brief modulation of the I_A amplitude and a permanent increase in the levels of expression of the Kv1.1 α‐subunit.     

HCN1 subunits contribute to the kinetics of Ih in neonatal cortical plate neurons

The distribution of ion channels in neurons regulates neuronal activity and proper formation of neuronal networks during neuronal development. One of the channels is the hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channel constituting the molecular substrate of hyperpolarization‐activated current (I_h). Our previous study implied a role for the fastest activating subunit HCN1 in the generation of I_h in rat neonatal cortical plate neurons. To better understand the impact of HCN1 in early neocortical development, we here performed biochemical analysis and whole‐cell recordings in neonatal cortical plate and juvenile layer 5 somatosensory neurons of HCN1^?/? and control HCN1^+/+ mice. Western Blot analysis revealed that HCN1 protein expression in neonatal cortical plate tissue of HCN^+/+ mice amounted to only 3% of the HCN1 in young adult cortex and suggested that in HCN1^?/? mice other isoforms (particularly HCN4) might be compensatory up‐regulated. At the first day after birth, functional ablation of the HCN1 subunit did not affect the proportion of I_h expressing pyramidal cortical plate neurons. Although the contribution of individual subunit proteins remains open, the lack of HCN1 markedly slowed the current activation and deactivation in individual I_h expressing neurons. However, it did not impair maximal amplitude/density, voltage dependence of activation, and cAMP sensitivity. In conclusion, our data imply that, although expression is relatively low, HCN1 contributes substantially to I_h properties in individual cortical plate neurons. These properties are significantly changed in HCN1^?/?, either due to the lack of HCN1 itself or due to compensatory mechanisms. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 785–797, 2013     

The cell adhesion molecule EphA4 is involved in circadian clock functions

Circadian (～24 h) rhythms of cellular network plasticity in the central circadian clock, the suprachiasmatic nucleus (SCN), have been described. The neuronal network in the SCN regulates photic resetting of the circadian clock as well as stability of the circadian system during both entrained and constant conditions. EphA4, a cell adhesion molecule regulating synaptic plasticity by controlling connections of neurons and astrocytes, is expressed in the SCN. To address whether EphA4 plays a role in circadian photoreception and influences the neuronal network of the SCN, we have analyzed circadian wheel‐running behavior of EphA4 knockout (EphA4^?/?) mice under different light conditions and upon photic resetting, as well as their light‐induced protein response in the SCN. EphA4^?/? mice exhibited reduced wheel‐running activity, longer endogenous periods under constant darkness and shorter periods under constant light conditions, suggesting an effect of EphA4 on SCN function. Moreover, EphA4^?/? mice exhibited suppressed phase delays of their wheel‐running activity following a light pulse during the beginning of the subjective night (CT15). Accordingly, light‐induced c‐FOS (FBJ murine osteosarcoma viral oncogene homolog) expression was diminished. Our results suggest a circadian role for EphA4 in the SCN neuronal network, affecting the circadian system and contributing to the circadian response to light.     

HPC‐1/syntaxin 1A and syntaxin 1B play distinct roles in neuronal survival

Two types of syntaxin 1 isoforms, HPC‐1/syntaxin 1A (STX1A) and syntaxin 1B (STX1B), are thought to have similar functions in exocytosis of synaptic vesicles. STX1A^?/? mice which we generated previously develop normally, possibly because of compensation by STX1B. We produced STX1B^?/? mice using targeted gene disruption and investigated their phenotypes. STX1B^?/? mice were born alive, but died before postnatal day 14, unlike STX1A^?/? mice. Morphologically, brain development in STX1B^?/? mice was impaired. In hippocampal neuronal culture, the cell viability of STX1B^?/? neurons was lower than that of WT or STX1A^?/? neurons after 9 days. Interestingly, STX1B^?/? neurons survived on WT or STX1A^?/? glial feeder layers as well as WT neurons. However, STX1B^?/? glial feeder layers were less effective at promoting survival of STX1B^?/? neurons. Conditioned medium from WT or STX1A^?/? glial cells had a similar effect on survival, but that from STX1B^?/? did not promote survival. Furthermore, brain‐derived neurotrophic factor (BDNF) or neurotrophin‐3 supported survival of STX1B^?/? neurons. BDNF localization in STX1B^?/? glial cells was disrupted, and BDNF secretion from STX1B^?/? glial cells was impaired. These results suggest that STX1A and STX1B may play distinct roles in supporting neuronal survival by glia.

     

Concerted Trafficking Regulation of Kv2.1 and KATP Channels by Leptin in Pancreatic β-Cells

In pancreatic β-cells, voltage-gated potassium 2.1 (Kv2.1) channels are the dominant delayed rectifier potassium channels responsible for action potential repolarization. Here, we report that leptin, a hormone secreted by adipocytes known to inhibit insulin secretion, causes a transient increase in surface expression of Kv2.1 channels in rodent and human β-cells. The effect of leptin on Kv2.1 surface expression is mediated by the AMP-activated protein kinase (AMPK). Activation of AMPK mimics whereas inhibition of AMPK occludes the effect of leptin. Inhibition of Ca²⁺/calmodulin-dependent protein kinase kinase β, a known upstream kinase of AMPK, also blocks the effect of leptin. In addition, the cAMP-dependent protein kinase (PKA) is involved in Kv2.1 channel trafficking regulation. Inhibition of PKA prevents leptin or AMPK activators from increasing Kv2.1 channel density, whereas stimulation of PKA is sufficient to promote Kv2.1 channel surface expression. The increased Kv2.1 surface expression by leptin is dependent on actin depolymerization, and pharmacologically induced actin depolymerization is sufficient to enhance Kv2.1 surface expression. The signaling and cellular mechanisms underlying Kv2.1 channel trafficking regulation by leptin mirror those reported recently for ATP-sensitive potassium (K_ATP) channels, which are critical for coupling glucose stimulation with membrane depolarization. We show that the leptin-induced increase in surface K_ATP channels results in more hyperpolarized membrane potentials than control cells at stimulating glucose concentrations, and the increase in Kv2.1 channels leads to a more rapid repolarization of membrane potential in cells firing action potentials. This study supports a model in which leptin exerts concerted trafficking regulation of K_ATP and Kv2.1 channels to coordinately inhibit insulin secretion.     

Formation of Kv2.1-FAK complex as a mechanism of FAK activation, cell polarization and enhanced motility

Focal adhesion kinase (FAK) plays key roles in cell adhesion and migration. We now report that the delayed rectifier Kv2.1 potassium channel, through its LD-like motif in N-terminus, may interact with FAK and enhance phosphorylation of FAK(397) and FAK(576/577). Overlapping distribution of Kv2.1 and FAK was observed on soma and proximal dendrites of cortical neurons. FAK expression promotes a polarized membrane distribution of the Kv2.1 channel. In Kv2.1-transfected CHO cells, formation of the Kv2.1-FAK complex was stimulated by fibronectin/integrin and inhibited by the K(+) channel blocker tetraethylammonium (TEA). FAK phosphorylation was minimized by shRNA knockdown of the Kv2.1 channel, point mutations of the N-terminus, and TEA, respectively. Cell migration morphology was altered by Kv2.1 knockdown or TEA, hindering cell migration activity. In wound healing tests in vitro and a traumatic injury animal model, Kv2.1 expression and co-localization of Kv2.1 and FAK significantly enhanced directional cell migration and wound closure. It is suggested that the Kv2.1 channel may function as a promoting signal for FAK activation and cell motility.     

Hypomyelination and increased activity of voltage-gated K(+) channels in mice lacking protein tyrosine phosphatase epsilon

Protein tyrosine phosphatase epsilon (PTP epsilon) is strongly expressed in the nervous system; however, little is known about its physiological role. We report that mice lacking PTP epsilon exhibit hypomyelination of sciatic nerve axons at an early post-natal age. This occurs together with increased activity of delayed- rectifier, voltage-gated potassium (Kv) channels and with hyperphosphorylation of Kv1.5 and Kv2.1 Kv channel alpha-subunits in sciatic nerve tissue and in primary Schwann cells. PTP epsilon markedly reduces Kv1.5 or Kv2.1 current amplitudes in XENOPUS: oocytes. Kv2.1 associates with a substrate-trapping mutant of PTP epsilon, and PTP epsilon profoundly reduces Src- or Fyn-stimulated Kv2.1 currents and tyrosine phosphorylation in transfected HEK 293 cells. In all, PTP epsilon antagonizes activation of Kv channels by tyrosine kinases in vivo, and affects Schwann cell function during a critical period of Schwann cell growth and myelination.     

Novel function of Tau in regulating the effects of external stimuli on adult hippocampal neurogenesis

Tau is a microtubule‐associated neuronal protein found mainly in axons. However, its presence in dendrites and dendritic spines is particularly relevant due to its involvement in synaptic plasticity and neurodegeneration. Here, we show that Tau plays a novel in vivo role in the morphological and synaptic maturation of newborn hippocampal granule neurons under basal conditions. Furthermore, we reveal that Tau is involved in the selective cell death of immature granule neurons caused by acute stress. Also, Tau deficiency protects newborn neurons from the stress‐induced dendritic atrophy and loss of postsynaptic densities (PSDs). Strikingly, we also demonstrate that Tau regulates the increase in newborn neuron survival triggered by environmental enrichment (EE). Moreover, newborn granule neurons from Tau^?/? mice did not show any stimulatory effect of EE on dendritic development or on PSD generation. Thus, our data demonstrate that Tau^?/? mice show impairments in the maturation of newborn granule neurons under basal conditions and that they are insensitive to the modulation of adult hippocampal neurogenesis exerted by both stimulatory and detrimental stimuli.     

Activity-dependent phosphorylation of neuronal Kv2.1 potassium channels by CDK5

Dynamic modulation of ion channel expression, localization, and/or function drives plasticity in intrinsic neuronal excitability. Voltage-gated Kv2.1 potassium channels are constitutively maintained in a highly phosphorylated state in neurons. Increased neuronal activity triggers rapid calcineurin-dependent dephosphorylation, loss of channel clustering, and hyperpolarizing shifts in voltage-dependent activation that homeostatically suppress neuronal excitability. These changes are reversible, such that rephosphorylation occurs after removal of excitatory stimuli. Here, we show that cyclin-dependent kinase 5 (CDK5), a Pro-directed Ser/Thr protein kinase, directly phosphorylates Kv2.1, and determines the constitutive level of Kv2.1 phosphorylation, the rapid increase in Kv2.1 phosphorylation upon acute blockade of neuronal activity, and the recovery of Kv2.1 phosphorylation after stimulus-induced dephosphorylation. We also demonstrate that although the phosphorylation state of Kv2.1 is also shaped by the activity of the PP1 protein phosphatase, the regulation of Kv2.1 phosphorylation by CDK5 is not mediated through the previously described regulation of PP1 activity by CDK5. Together, these studies support a novel role for CDK5 in regulating Kv2.1 channels through direct phosphorylation.     

Altered synaptic plasticity and behavioral abnormalities in CNGA3‐deficient mice

The role of the cyclic nucleotide‐gated (CNG) channel CNGA3 is well established in cone photoreceptors and guanylyl cyclase‐D‐expressing olfactory neurons. To assess a potential function of CNGA3 in the mouse amygdala and hippocampus, we examined synaptic plasticity and performed a comparative analysis of spatial learning, fear conditioning and step‐down avoidance in wild‐type mice and CNGA3 null mutants (CNGA3^?/?). CNGA3^?/? mice showed normal basal synaptic transmission in the amygdala and the hippocampus. However, cornu Ammonis (CA1) hippocampal long‐term potentiation (LTP) induced by a strong tetanus was significantly enhanced in CNGA3^?/? mice as compared with their wild‐type littermates. Unlike in the hippocampus, LTP was not significantly altered in the amygdala of CNGA3^?/? mice. Enhanced hippocampal LTP did not coincide with changes in hippocampus‐dependent learning, as both wild‐type and mutant mice showed a similar performance in water maze tasks and contextual fear conditioning, except for a trend toward higher step‐down latencies in a passive avoidance task. In contrast, CNGA3^?/? mice showed markedly reduced freezing to the conditioned tone in the amygdala‐dependent cued fear conditioning task. In conclusion, our study adds a new entry on the list of physiological functions of the CNGA3 channel. Despite the dissociation between physiological and behavioral parameters, our data describe a so far unrecognized role of CNGA3 in modulation of hippocampal plasticity and amydgala‐dependent fear memory.     

Membrane electrical excitability is necessary for the free‐running larval Drosophila circadian clock

Drosophila larvae and adult pacemaker neurons both express free‐running oscillations of period (PER) and timeless (TIM) proteins that constitute the core of the cell‐autonomous circadian molecular clock. Despite similarities between the adult and larval molecular oscillators, adults and larvae differ substantially in the complexity and organization of their pacemaker neural circuits, as well as in behavioral manifestations of circadian rhythmicity. We have shown previously that electrical silencing of adult Drosophila circadian pacemaker neurons through targeted expression of either an open rectifier or inward rectifier K⁺ channel stops the free‐running oscillations of the circadian molecular clock. This indicates that neuronal electrical activity in the pacemaker neurons is essential to the normal function of the adult intracellular clock. In the current study, we show that in constant darkness the free‐running larval pacemaker clock—like that of the adult pacemaker neurons they give rise to—requires membrane electrical activity to oscillate. In contrast to the free‐running clock, the molecular clock of electrically silenced larval pacemaker neurons continues to oscillate in diurnal (light–dark) conditions. This specific disruption of the free‐running clock caused by targeted K⁺ channel expression likely reflects a specific cell‐autonomous clock‐membrane feedback loop that is common to both larval and adult neurons, and is not due to blocking pacemaker synaptic outputs or disruption of pacemaker neuronal morphology. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

AMIGO is an auxiliary subunit of the Kv2.1 potassium channel

Kv2.1 is a potassium channel α-subunit abundantly expressed throughout the brain. It is a main component of delayed rectifier current (I(K)) in several neuronal types and a regulator of excitability during high-frequency firing. Here we identify AMIGO (amphoterin-induced gene and ORF), a neuronal adhesion protein with leucine-rich repeat and immunoglobin domains, as an integral part of the Kv2.1 channel complex. AMIGO shows extensive spatial and temporal colocalization and association with Kv2.1 in the mouse brain. The colocalization of AMIGO and Kv2.1 is retained even during stimulus-induced changes in Kv2.1 localization. AMIGO increases Kv2.1 conductance in a voltage-dependent manner in HEK cells. Accordingly, inhibition of endogenous AMIGO suppresses neuronal I(K) at negative membrane voltages. In conclusion, our data indicate AMIGO as a function-modulating auxiliary subunit for Kv2.1 and thus provide new insights into regulation of neuronal excitability.