Kv4.2 trafficking in CA1 pyramidal neuron dendrites. We previously reported that neuronal stimulation results in a redistribution of Kv4.2 channels away from dendritic spines to the dendritic shaft. This activity-dependent redistribution of Kv4.2 required activation of NMDA-type glutamate receptors and calcium influx, two requirements shared with synaptic plasticity, which is thought to underlie learning and memory. Given the nonuniform distribution of Kv4.2 channels in CA1 dendrites, Mike Nestor performed experiments to test the hypothesis that Kv4.2 channels are differentially trafficked at different regions along the apical dendrite during basal activity and upon stimulation in CA1 neurons. Proximal (50-150 μm from the soma, primary and oblique) and distal (>200 μm) apical dendrites were selected. The fluorescence recovery after photobleaching (FRAP) technique was used to measure basal cycling rates of EGFP-tagged Kv4.2 (Kv4.2g). We found that the cycling rate of Kv4.2 channels was one order of magnitude slower at both primary and oblique dendrites between 50-150 μm from the soma. Kv4.2 channel cycling increased significantly at 200-250 μm from the soma. Expression of a Kv4.2 mutant lacking a phosphorylation site for protein kinase-A (Kv4.2gS552A) abolished this distance-dependent change in channel cycling;demonstrating that phosphorylation by PKA underlies the increased mobility in distal dendrites. Neuronal stimulation increased cycling of Kv4.2 channels significantly at distal sites only. This activity-dependent increase in Kv4.2 cycling at distal dendrites was blocked by expression of Kv4.2gS552A. These results indicate that distance-dependent Kv4.2 mobility is regulated by activity-dependent phosphorylation of Kv4.2 by PKA. Functional role of the Kv4.2 auxiliary subunits: AKAPs A potential source of PKA modulation of Kv4.2 was uncovered this year by research fellow Lin Lin and Wei Sun when they identified A-kinase anchoring proteins (AKAPs) as novel accessory subunits for Kv4.2. AKAPs target PKA to glutamate receptor and ion channel complexes to allow for discrete, local signaling. We determined that the C-terminal domain of Kv4.2 interacts with an internal region of AKAP79/150 that overlaps with its MAGUK binding domain. AKAP79/150-anchored PKA activity was shown to control Kv4.2 surface expression in heterologous cells and hippocampal neurons. Consistent with these findings, disrupting PKA anchoring leads to a decrease in neuronal excitability while preventing dephosphorylation by the phosphatase calcineurin results in increased excitability. These results demonstrate that AKAP79/150 provides a platform for dynamic PKA regulation of Kv4.2 expression, fundamentally impacting neuronal excitability. Functional role of the Kv4.2 auxiliary subunits: KChIP4a KChIPs (KChIP1-4), associate with the N-terminal of Kv4.2 and modulate the channels biophysical properties, turnover rate and surface expression. We investigated the role of Kv4.2 C-terminal PKA phosphorylation site S552 in the KChIP4a-mediated effects on Kv4.2 channel trafficking. We found that while interaction between Kv4.2 and KChIP4a does not require PKA phosphorylation of Kv4.2S552, phosphorylation of this site is necessary for both enhanced stabilization and membrane expression of Kv4.2 channel complexes produced by KChIP4a. Enhanced surface expression and protein stability conferred by co-expression of Kv4.2 with other KChIP isoforms did not require PKA phosphorylation of Kv4.2 S552. These data demonstrate that PKA phosphorylation of Kv4.2 plays an important role in the trafficking of Kv4.2 through its specific interaction with KChIP4a. Functional role of the Kv4.2 auxiliary subunits: DPP6 Studies in heterologous expression systems have shown that Kv4 α-subunits interact with transmembrane DPP6 proteins to regulate channel trafficking and properties. The DPP6 auxiliary subunit protein, which is expressed in CA1 neurons, has recently been identified in large copy-number variants screens from some populations as an Autism Spectrum Disorder and ALS target gene. DPP6 enhances the opening probability of Kv4 channels and increases channel surface expression in heterologous systems. In dendritic recordings from DPP6 knock out mice, graduate student Wei Sun discovered that DPP6 is critical for generating the A-type K+ current gradient observed in CA1 dendrites. The loss this gradient led to hyper-excitable dendrites, with implications for information storage and coding. Additional, preliminary results show a critical role for DPP6 in synapse formation during development. We are currently investigating the possibility, suggested from these results, that dendritic excitability might be a common factor altered in neurological disorders recently associated with the DPP6 gene. Role of Kv4.2 channels in synaptic plasticity and development We have found that altering functional Kv4.2 expression level leads to a rapid, bidirectional remodeling of CA1 synapses. Neurons exhibiting enhanced A-type K+ current (IA) showed a decrease in relative synaptic NR2B/NR2A subunit composition and do not exhibit a form of synaptic plasticity called long-term potentiation or 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. Our data suggest that A-type K+ channels are an integral part of a synaptic complex that regulates Ca2+ signaling through spontaneous NMDA receptor activation to control synaptic NMDA receptor expression and plasticity. Additional advances included an investigation into the role of Kv4.2 in controlling the expression of synaptic NMDA receptors in vivo and during development. Synaptic NR2B fraction is developmentally regulated with implications for synaptic plasticity and learning and memory as well as diseases associated with learning impairments. Eunyoung Kim has found that in vivo injection of virus to alter Kv4.2 expression levels bidirectionally regulates NR2B subunit expression throughout development. Dendritic intrinsic plasticity in memory and disease We have shown previously, a role of A-type K+ channels in regulating intrinsic excitability of CA1 pyramidal neurons of the hippocampus after the induction of synaptic plasticity. This non-synaptic plasticity, called intrinsic plasticity, might represent an information storage mechanism available to neurons in addition to synaptic plasticity. Emilie Campanac is attempting to dissociate the signals involved in the induction of synaptic and intrinsic plasticity by the use of GluA1- lacking mice. In CA1 pyramidal neurons, the GluA1 subunit is differentially recruited by different patterns of activity known to induce long-term potentiation. Intrinsic plasticity has also been observed upon drug addiction. Cocaine is an addictive drug with psychostimulant effects that are attributed to inhibition of the dopamine transporter, which increases dopaminergic transmission. Chronic exposure to cocaine leads to neurodaptations in several voltage membrane conductances of neurons localized in the medial prefrontal cortex (mPCF) and nucleus accumbens. To date, all of these modifications have been characterized in the soma. Our goal is to identify more precisely which conductances are regulated in dendrites. Adult male mice were injected for 5 consecutive days with cocaine or saline. No significant difference was observed in intrinsic excitability in pyramidal neurons of the mPCF after cocaine injection. We did, however, find a left shift in the EPSP-Spike coupling curve after cocaine injection. This shift is abolished in the presence of an inhibitor of GABAA receptors suggesting a decrease of inhibition after cocaine.

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Piard, Juliette; Hu, Jia-Hua; Campeau, Philippe M et al. (2018) FRMPD4 mutations cause X-linked intellectual disability and disrupt dendritic spine morphogenesis. Hum Mol Genet 27:589-600
Lin, Lin; Murphy, Jonathan G; Karlsson, Rose-Marie et al. (2018) DPP6 Loss Impacts Hippocampal Synaptic Development and Induces Behavioral Impairments in Recognition, Learning and Memory. Front Cell Neurosci 12:84
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