Activity-dependent Internalization Research Articles

A receptor-inactivation model for single-celled habituation in Stentor coeruleus.

The single-celled ciliate Stentor coeruleus demonstrates habituation to mechanical stimuli, showing that even single cells can manifest a basic form of learning. Although the ability of Stentor to habituate has been extensively documented, the mechanism of learning is currently not known. Here we take a bottom-up approach and investigate a simple biochemistry-based model based on prior electrophysiological measurements in Stentor along with general properties of receptor molecules. In this model, a mechanoreceptor senses the stimulus and leads to channel opening to change membrane potential, with a sufficient change in polarization triggering an action potential that drives contraction. Receptors that are activated can become internalized, after which they can either be degraded or recycled back to the cell surface. This activity-dependent internalization provides a potential means for the cell to learn. Stochastic simulations of this model confirm that it is capable of showing habituation similar to what is seen in actual Stentor cells, including the lack of dishabituation by strong stimuli and the apparently step-like response of individual cells during habituation. The model also can account for several habituation hallmarks that a previous two-state Markov model could not, namely, the dependence of habituation rate on stimulus magnitude, which had to be added onto the two state model but arises naturally in the receptor inactivation model; the rate of response recovery after cessation of stimulation; the ability of high frequency stimulus sequences to drive faster habituation that results in a lower response probability, and the potentiation of habituation by repeated rounds of training and recovery. The model makes the prediction that application of high force stimuli that do not normally habituate should drive habituation to weaker stimuli due to decrease in the receptor number, which serves as an internal hidden variable. We confirmed this prediction using two new sets of experiments involving alternation of weak and strong stimuli. Furthermore, the model predicts that training with high force stimuli delays response recovery to low force stimuli, which aligns with our new experimental data. The model also predicts subliminal accumulation, wherein continuation of training even after habituation has reached asymptotic levels should lead to delayed response recovery, which was also confirmed by new experiments. The model is unable to account for the phenomenon of rate sensitivity, in which habituation caused by higher frequency stimuli is more easily reversed leading to a frequency dependence of response recovery. Such rate sensitivity has not been reported in Stentor . Here we carried out a new set of experiments which are consistent with the model's prediction of the lack of rate sensitivity. This work demonstrates how a simple model can suggest new ways to probe single-cell learning at an experimental level. Finally, we interpret the model in terms of a kernel estimator that the cell may use to guide its decisions about how to response to new stimuli as they arise based on information, or the lack thereof, from past stimuli.

Activity-Dependent Internalization of Glun2B-Containing NMDARS Is Required For Synaptic Incorporation of Glun2A And Synaptic Plasticity.

NMDA-type glutamate receptors are heterotetrameric complexes composed of two GluN1 and two GluN2 subunits. The precise composition of the GluN2 subunits determines the channel's biophysical properties and influences its interaction with postsynaptic scaffolding proteins and signaling molecules involved in synaptic physiology and plasticity. The precise regulation of NMDAR subunit composition at synapses is crucial for proper synaptogenesis, neuronal circuit development, and synaptic plasticity, a cellular model of memory formation.In the forebrain during early development, NMDARs contain solely the GluN2B subunit, which is necessary for proper synaptogenesis and synaptic plasticity. In rodents, GluN2A subunit expression begins in the second postnatal week, replacing GluN2B-containing NMDARs at synapses in an activity- or sensory experience-dependent process. This switch in NMDAR subunit composition at synapses alters channel properties and reduces synaptic plasticity. The molecular mechanism regulating the switch remains unclear.We have investigated the role of activity-dependent internalization of GluN2B-containing receptors in shaping synaptic NMDAR subunit composition. Using molecular, pharmacological, and electrophysiological approaches in cultured organotypic hippocampal slices from rats of both sexes, we show that the process of incorporating GluN2A-containing NMDARs receptors requires activity-dependent internalization of GluN2B-containing NMDARs. Interestingly, blockade of GluN2A synaptic incorporation was associated with impaired potentiation of AMPA-mediated synaptic transmission, suggesting a potential coupling between the trafficking of AMPARs into synapses and that of GluN2A-containing NMDARs.These insights contribute to our understanding of the molecular mechanisms underlying synaptic trafficking of glutamate receptors and synaptic plasticity. They may also have implications for therapeutic strategies targeting NMDAR function in neurological disorders.Significance statement NMDARs play a critical role in synaptogenesis, synaptic stability, and activity-dependent regulation of synaptic strength. The developmental switch in their GluN2 subunits composition is part of normal synapse development and crucial for proper synaptic physiology, plasticity, and the formation of functional neuronal circuits, though the mechanisms governing it remain unclear. We show that internalization of GluN2B-containing NMDARs is required for synaptic incorporation of GluN2A-containing receptors. This process can be induced by long-term potentiation and requires Ca+2 Notably, GluN2A trafficking to synapses is linked to the incorporation of AMPA-type glutamate receptors, suggesting a shared pathway for synaptic incorporation. These findings provide greater insight into the molecular mechanisms behind glutamate receptor trafficking and synaptic plasticity, potentially informing therapeutic strategies for neurological disorders.

The Alzheimer’s disease risk gene BIN1 regulates activity-dependent gene expression in human-induced glutamatergic neurons

Bridging Integrator 1 (BIN1) is the second most important Alzheimer’s disease (AD) risk gene, but its physiological roles in neurons and its contribution to brain pathology remain largely elusive. In this work, we show that BIN1 plays a critical role in the regulation of calcium homeostasis, electrical activity, and gene expression of glutamatergic neurons. Using single-cell RNA-sequencing on cerebral organoids generated from isogenic BIN1 wild type (WT), heterozygous (HET) and homozygous knockout (KO) human-induced pluripotent stem cells (hiPSCs), we show that BIN1 is mainly expressed by oligodendrocytes and glutamatergic neurons, like in the human brain. Both BIN1 HET and KO cerebral organoids show specific transcriptional alterations, mainly associated with ion transport and synapses in glutamatergic neurons. We then demonstrate that BIN1 cell-autonomously regulates gene expression in glutamatergic neurons by using a novel protocol to generate pure culture of hiPSC-derived induced neurons (hiNs). Using this system, we also show that BIN1 plays a key role in the regulation of neuronal calcium transients and electrical activity via its interaction with the L-type voltage-gated calcium channel Cav1.2. BIN1 KO hiNs show reduced activity-dependent internalization and higher Cav1.2 expression compared to WT hiNs. Pharmacological blocking of this channel with clinically relevant doses of nifedipine, a calcium channel blocker, partly rescues electrical and gene expression alterations in BIN1 KO glutamatergic neurons. Further, we show that transcriptional alterations in BIN1 KO hiNs that affect biological processes related to calcium homeostasis are also present in glutamatergic neurons of the human brain at late stages of AD pathology. Together, these findings suggest that BIN1-dependent alterations in neuronal properties could contribute to AD pathophysiology and that treatment with low doses of clinically approved calcium blockers should be considered as an option to slow disease-onset and progression.

Visualizing endocytic recycling and trafficking in live neurons by subdiffractional tracking of internalized molecules.

Our understanding of endocytic pathway dynamics is restricted by the diffraction limit of light microscopy. Although super-resolution techniques can overcome this issue, highly crowded cellular environments, such as nerve terminals, can also dramatically limit the tracking of multiple endocytic vesicles such as synaptic vesicles (SVs), which in turn restricts the analytical dissection of their discrete diffusional and transport states. We recently introduced a pulse-chase technique for subdiffractional tracking of internalized molecules (sdTIM) that allows the visualization of fluorescently tagged molecules trapped in individual signaling endosomes and SVs in presynapses or axons with 30- to 50-nm localization precision. We originally developed this approach for tracking single molecules of botulinum neurotoxin type A, which undergoes activity-dependent internalization and retrograde transport in autophagosomes. This method was then adapted to localize the signaling endosomes containing cholera toxin subunit-B that undergo retrograde transport in axons and to track SVs in the crowded environment of hippocampal presynapses. We describe (i) the construction of a custom-made microfluidic device that enables control over neuronal orientation; (ii) the 3D printing of a perfusion system for sdTIM experiments performed on glass-bottom dishes; (iii) the dissection, culturing and transfection of hippocampal neurons in microfluidic devices; and (iv) guidance on how to perform the pulse-chase experiments and data analysis. In addition, we describe the use of single-molecule-tracking analytical tools to reveal the average and the heterogeneous single-molecule mobility behaviors. We also discuss alternative reagents and equipment that can, in principle, be used for sdTIM experiments and describe how to adapt sdTIM to image nanocluster formation and/or tubulation in early endosomes during sorting events. The procedures described in this protocol take ∼1 week.

Activated G Protein Gαs Samples Multiple Endomembrane Compartments

Heterotrimeric G proteins are localized to the plasma membrane where they transduce extracellular signals to intracellular effectors. G proteins also act at intracellular locations, and can translocate between cellular compartments. For example, Gαs can leave the plasma membrane and move to the cell interior after activation. However, the mechanism of Gαs translocation and its intracellular destination are not known. Here we use bioluminescence resonance energy transfer (BRET) to show that after activation, Gαs rapidly associates with the endoplasmic reticulum, mitochondria, and endosomes, consistent with indiscriminate sampling of intracellular membranes from the cytosol rather than transport via a specific vesicular pathway. The primary source of Gαs for endosomal compartments is constitutive endocytosis rather than activity-dependent internalization. Recycling of Gαs to the plasma membrane is complete 25 min after stimulation is discontinued. We also show that an acylation-deacylation cycle is important for the steady-state localization of Gαs at the plasma membrane, but our results do not support a role for deacylation in activity-dependent Gαs internalization.

PIKfyve mediates the motility of late endosomes and lysosomes in neuronal dendrites

The endosome/lysosome system in the nervous system is critically important for a variety of neuronal functions such as neurite outgrowth, retrograde transport, and synaptic plasticity. In neurons, the endosome/lysosome system is crucial for the activity-dependent internalization of membrane proteins and contributes to the regulation of lipid level on the plasma membrane. Although homeostasis of membrane dynamics plays important roles in the properties of central nervous systems, it has not been elucidated how endosome/lysosome system is regulated. Here, we report that phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) mediates the motility of late endosomes and lysosomes in neuronal dendrites. Endosomes and lysosomes are highly motile in resting neurons, however knockdown of PIKfyve led to a significant reduction in late endosomes and lysosomes motility. We also found that vesicle acidification is crucial for their motility and PIKfyve is associated with this process indirectly. These data suggest that PIKfyve mediates vesicle motility through the regulation of vesicle integrity in neurons.

Stiripentol in refractory status epilepticus

Benzodiazepines (BZDs), which enhance γ-aminobutyric acid (GABAA ) receptor-mediated inhibition, are the first-line therapy for treatment of status epilepticus (SE). However, pharmacoresistance to BZDs develops rapidly after SE initiation. This is due to an activity-dependent internalization of BZD-sensitive GABAA receptors during SE. Stiripentol (STP) is a positive allosteric modulator of GABAA receptors with a unique subunit selectivity profile. We report that in a rodent model of SE, STP terminates behavioral seizures and remains effective in established SE when seizures have become BZD resistant. The anticonvulsant effects of STP are age dependent, with greater potency in juvenile animals. Whole cell recordings from dentate granule cells in hippocampal slices reveal that STP potentiates GABAergic inhibitory postsynaptic currents (IPSCs) and tonic GABAergic currents by acting at a site on the GABAA receptor that is separate from the benzodiazepine binding site. This potentiation persists in established SE, whereas potentiation of GABAergic inhibition by BZDs is lost. STP potentiates IPSCs in juvenile animals with greater potency than in adult animals. We suggest that STP, either alone or as add-on therapy, may prove useful in treating established and BZD-resistant status epilepticus. Furthermore, STP may be particularly effective in terminating SE in children when SE is most prevalent.

PICK1 interacts with PACSIN to regulate AMPA receptor internalization and cerebellar long-term depression

The dynamic trafficking of AMPA receptors (AMPARs) into and out of synapses is crucial for synaptic transmission, plasticity, learning, and memory. The protein interacting with C-kinase 1 (PICK1) directly interacts with GluA2/3 subunits of the AMPARs. Although the role of PICK1 in regulating AMPAR trafficking and multiple forms of synaptic plasticity is known, the exact molecular mechanisms underlying this process remain unclear. Here, we report a unique interaction between PICK1 and all three members of the protein kinase C and casein kinase II substrate in neurons (PACSIN) family and show that they form a complex with AMPARs. Our results reveal that knockdown of the neuronal-specific protein, PACSIN1, leads to a significant reduction in AMPAR internalization following the activation of NMDA receptors in hippocampal neurons. The interaction between PICK1 and PACSIN1 is regulated by PACSIN1 phosphorylation within the variable region and is required for AMPAR endocytosis. Similarly, the binding of PICK1 to the ubiquitously expressed PACSIN2 is also regulated by the homologous phosphorylation sites within the PACSIN2-variable region. Genetic deletion of PACSIN2, which is highly expressed in Purkinje cells, eliminates cerebellar long-term depression. This deficit can be fully rescued by overexpressing wild-type PACSIN2, but not by a PACSIN2 phosphomimetic mutant, which does not bind PICK1 efficiently. Taken together, our data demonstrate that the interaction of PICK1 and PACSIN is required for the activity-dependent internalization of AMPARs and for the expression of long-term depression in the cerebellum.

Calcyon upregulation in adolescence impairs response inhibition and working memory in adulthood

Calcyon regulates activity-dependent internalization of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) glutamate receptors and long-term depression of excitatory synapses. Elevated levels of calcyon are consistently observed in brains from schizophrenic patients, and the calcyon gene is associated with attention-deficit hyperactivity disorder. Executive function deficits are common to both disorders, and at least for schizophrenia, the etiology appears to involve both heritable and neurodevelopmental factors. Here, we show with calcyon-overexpressing Cal(OE) transgenic mice that lifelong calcyon upregulation impairs executive functions including response inhibition and working memory, without producing learning and memory deficits in general. As response inhibition and working memory, as well as the underlying neural circuitry, continue to mature into early adulthood, we functionally silenced the transgene during postnatal days 28-49, a period corresponding to adolescence. Remarkably, the response inhibition and working memory deficits including perseverative behavior were absent in adult Cal(OE) mice with the transgene silenced in adolescence. Suppressing the calcyon transgene in adulthood only partially rescued the deficits, suggesting calcyon upregulation in adolescence irreversibly alters development of neural circuits supporting mature response inhibition and working memory. Brain regional immunoblots revealed a prominent downregulation of AMPA GluR1 subunits in hippocampus and GluR2/3 subunits in hippocampus and prefrontal cortex of the Cal(OE) mice. Silencing the transgene in adolescence prevented the decrease in hippocampal GluR1, further implicating altered fronto-hippocampal connectivity in the executive function deficits observed in the Cal(OE) mice. Treatments that mitigate the effects of high levels of calcyon during adolescence could preempt adult deficits in executive functions in individuals at risk for serious mental illness.

PICK1 Regulates Incorporation of Calcium-Permeable AMPA Receptors during Cortical Synaptic Strengthening

While AMPA-type glutamate receptors (AMPARs) found at principal neuron excitatory synapses typically contain the GluR2 subunit, several forms of behavioral experience have been linked to the de novo synaptic insertion of calcium-permeable (CP) AMPARs, defined by their lack of GluR2. In particular, whisker experience drives synaptic potentiation as well as the incorporation of CP-AMPARs in the neocortex. Previous studies implicate PICK1 (protein interacting with C kinase-1) in activity-dependent internalization of GluR2, suggesting one potential mechanism leading to the subsequent accumulation of synaptic CP-AMPARs and increased synaptic strength. Here we test this hypothesis by using a whisker stimulation paradigm in PICK1 knock-out mice. We demonstrate that PICK1 facilitates the surface expression of CP-AMPARs and is indispensable for their experience-dependent synaptic insertion. However, the failure to incorporate CP-AMPARs in PICK1 knock-outs does not preclude sensory-induced enhancement of synaptic currents. Our results indicate that synaptic strengthening in the early postnatal cortex does not require PICK1 or the addition of GluR2-lacking AMPARs. Instead, PICK1 permits changes in AMPAR subunit composition to occur in conjunction with synaptic potentiation.

Hedgehog Signaling: Is Smo a G Protein-Coupled Receptor?

The Hedgehog signal transducer Smoothened is structurally similar to G protein-coupled receptors. Now there is direct evidence that Smoothened relies on heterotrimeric G proteins in order to transduce the Hedgehog signal.

Rapid, Bidirectional Remodeling of Synaptic NMDA Receptor Subunit Composition by A-type K+ Channel Activity in Hippocampal CA1 Pyramidal Neurons

The transient, A-type K+ current (IA) controls the excitability of CA1 pyramidal neuron dendrites by regulating the back-propagation of action potentials and by shaping synaptic input. Dendritic A-type K+ channels are targeted for modulation during long-term potentiation (LTP) and we have recently shown that activity-dependent internalization of the A-type channel subunit Kv4.2 enhances synaptic currents. However, the effect of changes in IA on the ability to induce subsequent synaptic plasticity (metaplasticity) has not been investigated. Here, we show that altering functional Kv4.2 expression level leads to a rapid, bidirectional remodeling of CA1 synapses. Neurons exhibiting enhanced IA showed a decrease in relative synaptic NR2B/NR2A subunit composition and did not exhibit LTP. Conversely, reducing IA by expression of a Kv4.2 dominant-negative or through genomic knockout of Kv4.2 led to an increased fraction of synaptic NR2B/NR2A and enhanced LTP. Bidirectional synaptic remodeling was mimicked in experiments manipulating intracellular Ca2+ and dependent on spontaneous activation of NMDA receptors and CaMKII activity. Our data suggest that A-type K+ channels are an integral part of a synaptic complex that regulates Ca2+ signaling through spontaneous NMDAR activation to control synaptic NMDAR expression and plasticity.

Protein Kinase A Mediates Activity-Dependent Kv4.2 Channel Trafficking

The A-type potassium channel subunit Kv4.2 influences hippocampal function through regulation of dendritic excitability, and changes in Kv4.2 surface expression alter synaptic plasticity. Recent data from our laboratory demonstrate that EGFP (enhanced green fluorescent protein)-tagged Kv4.2 channels located in dendritic spines are internalized in an activity-dependent manner after synaptic stimulation and during chemically induced long-term potentiation. However, the molecular trigger for Kv4.2 internalization remains unknown. Here we examined the role of protein kinase A (PKA) in Kv4.2 activity-dependent trafficking. In hippocampal neurons, PKA activation with forskolin or 8-Br-cAMP induced Kv4.2 internalization from dendritic spines, whereas PKA inhibition with H89 prevented AMPA-induced internalization. Furthermore, introduction of a point mutation at the C-terminal PKA phosphorylation site of Kv4.2 (S552A) prevented the AMPA-induced internalization of Kv4.2. Together, these data demonstrate that Kv4.2 activity-dependent internalization requires PKA phosphorylation of Kv4.2 at serine 522.

Mechanisms regulating ApTrkl, a Trk‐like receptor in Aplysia sensory neurons

An Aplysia Trk-like receptor (ApTrkl) was previously shown to be involved in cell wide long-term facilitation (LTF) and activation of ERK when serotonin (5-HT) is applied to the cell soma. The current study investigated the regulation of ApTrkl by overexpressing the receptor and several variants in Aplysia sensory neuron cultures. Kinase activity-dependent constitutive activation of ApTrkl was observed mainly on the plasma membrane. These studies revealed two modes of receptor internalization: (1) kinase activity-dependent internalization and (2) 5-HT-dependent, kinase activity-independent internalization. Both modes of internalization were ligand independent, and the action of 5-HT was mediated through G-protein-coupled receptors (GPCRs). On the other hand, methiothepin, an inverse agonist of 5-HT GPCRs activated endogenous ApTrkl to the same extent as 5-HT, suggesting a transactivation mechanism due to a novel coupling of GPCRs to receptor tyrosine kinase (RTK) activation that is also activated through inverse agonist binding. The neuropeptide sensorin could transiently activate ApTrkl but was not required for 5-HT-induced ApTrkl activation.

The role of kinases in the Hedgehog signalling pathway

The Hedgehog (Hh) signalling pathway has a crucial role in several developmental processes and is aberrantly activated in a variety of cancers. In Drosophila, many of the canonical Hh pathway components are phosphorylated, yet the precise role of these phosphorylation events in the regulation of Hh signal transduction is unclear. Furthermore, the Hh pathway receives input from several kinases that have well-described roles in other cellular functions, some of which have both positive and negative effects on Hh signalling. Several recent studies have characterized the role of specific phosphorylation events in the Hh pathway, and have begun to shed light on how phosphorylation of Hh signalling components affects their subcellular location, stability and activity to mediate the transcriptional response to the Hh gradient.

The Tumor Suppressor eIF3e Mediates Calcium-Dependent Internalization of the L-Type Calcium Channel CaV1.2

Voltage-gated calcium channels (VGCCs) convert electrical activity into calcium (Ca2+) signals that regulate cellular excitability, differentiation, and connectivity. The magnitude and kinetics of Ca2+ signals depend on the number of VGCCs at the plasma membrane, but little is known about the regulation of VGCC surface expression. We report that electrical activity causes internalization of the L-type Ca2+ channel (LTC) CaV1.2 and that this is mediated by binding to the tumor suppressor eIF3e/Int6 (eukaryotic initiation factor 3 subunit e). Using total internal reflection microscopy, we identify a population of CaV1.2 containing endosomes whose rapid trafficking is strongly regulated by Ca2+. We define a domain in the II-III loop of CaV1.2 that binds eIF3e and is essential for the activity dependence of both channel internalization and endosomal trafficking. These findings provide a mechanism for activity-dependent internalization and trafficking of CaV1.2 and provide a tantalizing link between Ca2+ homeostasis and a mammalian oncogene.

Hedgehog, Smoothened, and β-Arrestin

Hedgehog (Hh) proteins carry signals that are essential for pattern formation during vertebrate embryogenesis. Extracellular Hh molecules bind to a receptor on the cell surface and activate Smoothened, a membrane-spanning protein, which transmits signals to the cell interior. β-Arrestin proteins are inhibitors of heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPCR) signaling and also promote internalization and signaling by GPCRs. Chen et al. find that in mammalian cells, activated Smoothened molecules preferentially associate with β-arrestin 2. Reducing levels of β-arrestin 2 inhibited internalization of Smoothened. Furthermore, Wilbanks et al. find that loss of β-arrestin 2 in zebrafish causes developmental abnormalities similar to those of mutants in the Hh signaling pathway. Overexpression of β-arrestin 2 could partially rescue some defects in embryos with deficient Hh signaling, and loss of β-arrestin 2 decreased expression of Hh-responsive genes. Together, the findings provide insight into the roles of β-arrestin 2 during development and the mechanisms by which Hh signaling influences developmental processes from embryogenesis to cancer. W. Chen, X.-R. Ren, C. D. Nelson, L. S. Barak, J. K. Chen, P. A. Beachy, F. de Sauvage, R. J. Lefkowitz, Activity-dependent internalization of Smoothened mediated by β-arrestin 2 and GRK2. Science 306 , 2257-2260 (2004). [Abstract] [Full Text] A. M. Wilbanks, G. B. Fralish, M. L. Kirby, L. S. Barak, Y.-X. Li, M. G. Caron, β-Arrestin 2 regulates zebrafish development through the Hedgehog signaling pathway. Science 306 , 2264-2267 (2004). [Abstract] [Full Text]

Activity-Dependent Internalization of Smoothened Mediated by ß-Arrestin 2 and GRK2

Binding of Sonic Hedgehog (Shh) to Patched (Ptc) relieves the latter's tonic inhibition of Smoothened (Smo), a receptor that spans the cell membrane seven times. This initiates signaling which, by unknown mechanisms, regulates vertebrate developmental processes. We find that two molecules interact with mammalian Smo in an activation-dependent manner: G protein-coupled receptor kinase 2 (GRK2) leads to phosphorylation of Smo, and beta-arrestin 2 fused to green fluorescent protein interacts with Smo. These two processes promote endocytosis of Smo in clathrin-coated pits. Ptc inhibits association of beta-arrestin 2 with Smo, and this inhibition is relieved in cells treated with Shh. A Smo agonist stimulated and a Smo antagonist (cyclopamine) inhibited both phosphorylation of Smo by GRK2 and interaction of beta-arrestin 2 with Smo. beta-Arrestin 2 and GRK2 are thus potential mediators of signaling by activated Smo.

Activity-dependent endocytic sorting of kainate receptors to recycling or degradation pathways.

Kainate receptors (KARs) play important roles in the modulation of neurotransmission and plasticity, but the mechanisms that regulate their surface expression and endocytic sorting remain largely unknown. Here, we show that in cultured hippocampal neurons the surface expression of GluR6-containing KARs is dynamically regulated. Furthermore, internalized KARs are sorted into recycling or degradative pathways depending on the endocytotic stimulus. Kainate activation causes a Ca2+- and PKA-independent but PKC-dependent internalization of KARs that are targeted to lysosomes for degradation. In contrast, NMDAR activation evokes a Ca2+-, PKA- and PKC-dependent endocytosis of KARs to early endosomes with subsequent reinsertion back into the plasma membrane. These results demonstrate that GluR6-containing KARs are subject to activity-dependent endocytic sorting, a process that provides a mechanism for both rapid and chronic changes in the number of functional receptors.

Unraveling Internalization Signals

Du et al . investigated the possibility that neuronal activity could influence internalization of neurotrophin-receptor complexes, a process required for some aspects of neurotrophin signaling, and discovered an unexpected link between calcium, receptor tyrosine kinase activity, and brain-derived neurotrophic factor (BDNF)-TrkB receptor complex internalization. Neuronal activity can promote the efficacy with which BDNF affects neuronal survival, synaptic efficacy, and synaptic plasticity, which provides a possible mechanism for selective activity-dependent enhancement of particular neuronal pathways. Du et al . used fluorescent imaging of biotinylated BDNF, quantification of radiolabeled BDNF on cell surface and the amount internalized, and Western analysis of TrkB in combination with electrical field stimulation to show that stimulus protocols associated with production of long-term potentiation promoted internalization of BDNF and TrkB in cultured hippocampal neurons. The authors used electrical stimulation of cells undergoing pharmacological blockade of voltage-dependent calcium channels or N -methyl-D-aspartate receptors, or in medium made without calcium, to show that activity-dependent internalization of BDNF and TrkB depended on calcium influx. BDNF stimulated tyrosine phosphorylation of TrkB, and electrical stimulation enhanced BDNF-dependent TrkB phosphorylation, an enhancement that depended on calcium influx. Pharmacological blockade of BDNF-dependent TrkB internalization did not affect activity-dependent enhancement of TrkB phosphorylation, whereas inhibition of TrkB kinase activity, either pharmacologically or with a dominant-negative TrkB mutant, blocked receptor internalization. Thus, neuronal activity appears to enhance BDNF-TrkB internalization through calcium-dependent stimulation of TrkB tyrosine phosphorylation. J. Du, L. Feng, E. Zaitsev, H.-S. Je, X.-W. Liu, B. Lu, Regulation of TrkB receptor tyrosine kinase and its internalization by neuronal activity and Ca 2+ influx. J. Cell Biol. 163 , 385-395 (2003). [Abstract] [Full Text]