You are here: Home > Hoffman Lab

Molecular Nature and Functional Role of Dendritic Voltage-Gated Ion Channels

• Dax Hoffman, PhD, Head, Molecular Neurophysiology and Biophysics Section
• Jiahua Hu, PhD, Staff Scientist
• Lin Lin, PhD, Microbiologist
• Ying Liu, MD, Biologist
• Cole Malloy, PhD, Postdoctoral Fellow
• Jon Murphy, PhD, Postdoctoral Fellow
• Adriano Bellotti, BS, Gates Cambridge Scholar
• Maisie Ahern, BS, Postbaccalaureate Fellow
• Joseph Krzeski, BS, Postbaccalaureate Fellow
• Ashley Pratt, BS, Postbaccalaureate Fellow

The central nervous system (CNS) underlies all our experiences, actions, emotions, knowledge, and memories. With billions of neurons each firing hundreds of times per second, the complexity of the brain is stunning. To pare down the task of understanding something so complex, our research approach calls for studying the workings of a single central neuron: the pyramidal neuron from the CA1 region of the hippocampus. The hippocampus is essential for long-term memory in humans and is among the first brain regions affected by epilepsy and Alzheimer’s disease. To understand how the hippocampus stores and processes information, we focus on one of its principal cell types, the CA1 pyramidal neuron. Each pyramidal neuron in the CA1 region of the hippocampus receives tens of thousands of inputs onto its dendrites, and it is commonly thought that information is stored by altering the strength of individual synapses (synaptic plasticity). Recent evidence suggests that the regulation of synaptic surface expression of glutamate receptors can, in part, determine synaptic strength. However, the dendrites contain an abundance of ion channels that are involved in receiving, transforming, and relaying information in the dendrites, adding an additional layer of complexity to neuronal information processing.

We found that the A-type potassium channel subunit Kv4.2 is highly expressed in the dendritic regions of CA1 neurons in the hippocampus and, as one of the primary regulators of dendritic excitability, plays a pivotal role in information processing. Kv4.2 is targeted for modulation during the types of plasticity thought to underlie learning and memory. Moreover, we found that the functional expression level of Kv4.2 regulates the subtype expression of NMDA–type glutamate receptors, the predominant molecular devices controlling synaptic plasticity and memory. We are currently following up on these findings with more detailed investigations into the mechanisms of activity-dependent Kv4.2 regulation. In addition, we have begun to investigate the role of dendritic voltage-gated potassium and calcium channels in neuronal development and developmental disorders.

Role of voltage-gated ion channels in synaptic development and disease

Regulation of potassium channel trafficking and function

Kv4.2 channels, the major contributors to somatodendritic A-type potassium channels, are key determinants of dendritic excitability and integration, spike timing–dependent plasticity, and long-term potentiation. Downregulation of Kv4.2 channel expression occurs following hippocampal seizures and in epilepsy, suggesting that A-type currents may be targets for novel therapeutics. To identify Kv4.2–binding proteins, Jiahua Hu employed a tandem affinity purification (TAP) approach to isolate the Kv4.2 protein complex from hippocampal neurons. Mass-spectrometry (MS) analysis identified known proteins such as KChIP (see below) family members and DPP6/10. The TAP–MS assay also identified an isomerase as a binding partner of Kv4.2. The binding was confirmed by brain co-immunoprecipitation, co-expression in HEK293T cells, and peptide pull-down in vitro. The isomerase binds to a specific Kv4.2 site, and the association is regulated by neuronal activity and seizure.

We recently identified a novel molecular cascade initiated by the activation of p38 kinase and subsequent isomerization of a C-terminal motif (T607) in Kv4.2, which triggers dissociation from its auxiliary subunit DPP6, a reduction I_A (type A potassium current), and an increase in neuronal excitability. The phosphorylation of the Kv4.2 T607 site is induced by novel environment exposure or seizure and is mediated by p38 MAPK (mitogen-activated protein kinase) but not by ERK MAPK. To investigate the consequences of this cascade on behavior and neuronal physiology, we used CRISP-Cas9 techniques to generate a knockin mouse in which the isomerase binding site is specifically abolished (Kv4.2TA). The mice are viable and appear normal, although activity-dependent dissociation of the Kv4.2-DPP6 complex is impaired.

Cole Malloy used patch-clamp electrophysiology in pyramidal cells of hippocampal slices from Kv4.2TA and wild-type (WT) mice to decipher the role of p38-Pin1 (a peptidyl-prolyl isomerase)–mediated regulation of Kv4.2 on neuronal excitability. He found that Kv4.2TA cells displayed a reduction in action potential (AP) firing compared with WT in response to somatic current injections. The reduced excitability can be traced to increased Kv4.2–mediated current in Kv4.2TA cells in outside-out somatic patches. Pharmacological block of both p38 kinase and Pin1 in WT recapitulated the impact of the mutation on neuronal firing properties and I_A, confirming the specificity of this cascade as underlying these effects.

To detect how such alterations in neuronal physiology are manifested in behavioral changes, Jiahua Hu performed a battery of tests to probe seizure susceptibility and learning and memory capability. In response to intraperitoneal (IP) kainic acid injection, Kv4.2TA mice exhibited reduced seizure intensity over an hour-long period compared with WT mice. The reduced seizure intensity could also be recapitulated in WT with pharmacological block of p38 kinase. We thus identified a novel signaling cascade, which can be a target for therapeutic intervention to mitigate seizure intensity in epilepsy by reducing Kv4.2 downregulation.

Furthermore, Kv4.2TA mice exhibit normal initial learning and memory in the Morris Water maze. However, they exhibited better ‘reversal’ learning in the Morris Water maze (a measure of cognitive flexibility) than did WT mice. In the operant reversal lever press, the Kv4.2TA mice displayed improved reversal learning. The data strongly support the idea that activity-dependent regulation of Kv4.2 plays an important role in cognitive flexibility, the ability to appropriately adjust one’s behavior to a changing environment, which is impaired in various neuro-developmental disorders, such as the autism spectrum disorder. In light of the findings that Kv4.2TA mice exhibit enhanced cognitive flexibility, our ongoing experiments utilize whole-cell recordings from pyramidal neurons in hippocampal slice to investigate potential differences in the synaptic properties of WT and Kv4.2TA mice. Collectively, the experiments will reveal the cellular mechanisms underlying the reversal learning phenotype in Kv4.2TA mice and will provide further insight into mechanisms impacting cognitive flexibility.

Ca²⁺ regulation of potassium channel function

Jonathan Murphy found that Ca²⁺ entry mediated by the voltage-gated Ca²⁺ channel subunit Cav2.3 regulates Kv4.2 function both in a heterologous expression system and, endogenously, in CA1 pyramidal neurons through Ca²⁺ binding to auxiliary subunits known as K⁺ channel–interacting proteins (KChIPs). KChIPs are calcium-sensing molecules that containing four EF-hands (motif with helix-loop-helix topology), and which are dysregulated in several diseases and disorders, including epilepsy, Huntington’s disease, and Alzheimer’s disease. He characterized a KChIP–independent interaction between Cav2.3 and Kv4.2 using immunofluorescence co-localization, co-immunoprecipitation, electron microscopy, FRAP (fluorescence recovery after photobleaching), and FRET (fluorescence resonance energy transfer). We found that Ca²⁺ entry via Cav2.3 increases Kv4.2–mediated whole-cell current, partly as a result of an increase in Kv4.2 surface expression. In hippocampal neurons, pharmacological block of Cav2.3 reduced whole-cell I_A by about 33%. We also found an approximately 20% reduction in whole-cell I_A in Cav2.3 knockout (KO) mouse neurons with a loss of the characteristic dendritic I_A gradient. Furthermore, we found that the Cav2.3–Kv4.2 complex regulates the size of synaptic currents and spine Ca²⁺ transients. The results reveal an intermolecular Cav2.3–Kv4.2 complex that impacts synaptic integration in CA1 hippocampal neurons.

The KChIP protein, but not mRNA expression, has been shown to be reduced in Kv4.2 KO mouse brains, suggesting increased KChIP protein degradation in the absence of Kv4.2. We hypothesized that KChIP protein degradation is dependent on binding to Kv4.2 and that KChIP protein degradation increases in the absence of Kv4.2. We aimed to elucidate the molecular mechanism of KChIP protein degradation and its effect on Kv4.2 protein levels and function. Joe Krzeski identified the pathway through which KChIP is degraded and a novel function for KChIP regulation of Kv4.2 in HEK293 cells. A mechanistic understanding of KChIP protein degradation is important, as it may lead to new therapeutic strategies to treat diseases in which KChIPs are dysregulated.

DPP6 plays a role in brain development, function, and behavior.

We previously showed that the Kv4 auxiliary subunit DPP6 has a novel function in regulating dendritic filopodia formation and stability, affecting synaptic development and function. In 2018, we reported that DPP6–KO mice are impaired in hippocampus-dependent learning and memory, with smaller brain size and weight. Recently, using immunofluorescence and electron microscopy, in a project led by Lin Lin, we discovered a novel structure in hippocampal area CA1 that was significantly more prevalent in DPP6–KO mice than in WT mice of the same age and that the structures were observed earlier in development in DPP6–KO mice, appearing as clusters of large puncta that colocalized the neuronal proteins NeuN, synaptophysin, and chromogranin A. Electron microscopy revealed that the structures are abnormal, enlarged presynaptic swellings filled with mainly fibrous material, with occasional peripheral, presynaptic active zones forming synapses. We found diagnostic biomarkers of Alzheimer’s disease present in abnormal levels in DPP6–KO mice, including accumulation of amyloid β and amyloid precursor protein (APP) in the hippocampal CA1 area and a significant increase in expression of hyper-phosphorylated tau. The amyloid β and phosphorylated tau pathologies were associated with neuro-inflammation characterized by activation of microglia and astrocytes. Multiplex cytokine array detection with WT and DPP6–KO mouse blood serum showed that levels of pro-inflammatory or anti-inflammatory cytokines increased in aged DPP6–KO mice. We also found that the presence of activated astrocytes and microglia was significantly increased in DPP6–KO brain sections and that DPP6-KO mice displayed circadian dysfunction, a common symptom of Alzheimer's disease. Together, the results indicate that DPP6–KO mice show symptoms of enhanced neurodegeneration reminiscent of Alzheimer’s disease and that they are associated with a novel structure resulting from synapse loss and neuronal death.

Publications

1. Hu JH, Malloy C, Tabor GT, Gutzmann JJ, Liu Y, Abebe D, Karlsson RM, Durell S, Cameron HA, Hoffman DA. Activity-dependent isomerization of Kv4.2 by Pin1 regulates cognitive flexibility. Nat Commun 2020;11(1):1567.
2. Hu JH, Malloy C, Hoffman DA. P38 regulates kainic acid-induced seizure and neuronal firing via Kv4.2 phosphorylation. Int J Mol Sci 2020;21(16):5921.
3. Gray EE, Murphy JG, Liu Y, Trang I, Tabor GT, Lin L, Hoffman DA. Disruption of GpI mGluR-dependent Cav2.3 translation in a mouse model of fragile X syndrome. J Neurosci 2019;39(38):7453-7464.
4. Murphy JG, Hoffman DA. A polybasic motif in alternatively spliced KChIP2 isoforms prevents Ca²⁺ regulation of Kv4 channels. J Biol Chem 2019;294(10):3683-3695.
5. Gutzmann JJ, Lin L, Hoffman DA. Functional coupling of Cav2.3 and BK potassium channels regulates action potential repolarization and short-term plasticity in the mouse hippocampus. Front Cell Neurosci 2019;13:27.
6. Tabor GT, Park JM, Murphy JG, Hu JH, Hoffman DA. A novel bungarotoxin binding site-tagged construct reveals MAPK-dependent Kv4.2 trafficking. Mol Cell Neurosci 2019;98:121-130.

Collaborators

• Heather Cameron, Section on Neuroplasticity, NIMH, Bethesda, MD
• Avindra Nath, MD, Translational Neuroscience Center, NINDS, Bethesda, MD
• Constantine A. Stratakis, MD, D(med)Sci, Section on Endocrinology and Genetics, NICHD, Bethesda, MD

Contact

For more information, email hoffmand@mail.nih.gov or visit http://hoffmanlab.nichd.nih.gov.

Top of Page