Developmental Expression of Kv Potassium Channels at the Axon Initial Segment of Cultured Hippocampal Neurons

AbstractThe palmitoyl acyltransferase (PAT) ZDHHC14 is highly expressed in the hippocampus and is the only PAT predicted to bind Type I PDZ domain-containing proteins. However, ZDHHC14’s neuronal roles are unknown. Here, we identify the PDZ domain-containing Membrane-associated Guanylate Kinase (MaGUK) PSD93 as a direct ZDHHC14 interactor and substrate. PSD93, but not other MaGUKs, localizes to the Axon Initial Segment (AIS). Using lentiviral-mediated shRNA knockdown in rat hippocampal neurons, we find that ZDHHC14 controls palmitoylation and AIS clustering of PSD93 and also of Kv1 potassium channels, which directly bind PSD93. Neurodevelopmental expression of ZDHHC14 mirrors that of PSD93 and Kv1 channels and, consistent with ZDHHC14’s importance for Kv1 channel clustering, loss of ZDHHC14 decreases outward currents and increases action potential firing in hippocampal neurons. To our knowledge, these findings identify the first neuronal roles and substrates for ZDHHC14 and reveal a previously unappreciated role for palmitoylation in control of neuronal excitability.Impact StatementZDHHC14 controls palmitoylation and axon initial segment targeting of PSD93 and Kv1-family potassium channels, events that are essential for normal neuronal excitability.

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Ankyrin G membrane partners drive the establishment and maintenance of the axon initial segment

10.1101/073163 ◽

2016 ◽

Cited By ~ 2

Author(s):

Christophe Leterrier ◽

Nadine Clerc ◽

Fanny Rueda-Boroni ◽

Audrey Montersino ◽

Bénédicte Dargent ◽

...

Keyword(s):

Cell Adhesion ◽

Cell Adhesion Molecules ◽

Hippocampal Neurons ◽

Initial Segment ◽

Neuronal Development ◽

Axon Initial Segment ◽

Membrane Anchor ◽

Ankyrin G ◽

Membrane Components ◽

Cultured Hippocampal Neurons

The axon initial segment (AIS) is a specialized neuronal compartment that plays a key role in neuronal development and excitability. It concentrates multiple ion channels and cell adhesion molecules. The anchoring of these AIS membrane components is known to be highly dependent of the scaffold protein ankyrin G (ankG) but whether ankG membrane partners play a reciprocal role in ankG targeting and stabilization has not been studied yet. In cultured hippocampal neurons and cortical organotypic slices, we found that shRNA-mediated knockdown of ankG membrane partners led to a decrease of ankG concentration and perturbed the AIS formation and maintenance. These perturbations were rescued by expressing an AIS-targeted sodium channel, or a minimal construct containing the ankyrin-binding domain of Nav1.2 and a membrane anchor. We thus demonstrate that a tight and precocious association of ankG to its membrane partners is crucial for the establishment and maintenance of the AIS.

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Brain-derived neurotrophic factor/neurotrophin 3 regulate axon initial segment location and affect neuronal excitability in cultured hippocampal neurons

Journal of Neurochemistry ◽

10.1111/jnc.14050 ◽

2017 ◽

Vol 142 (2) ◽

pp. 260-271 ◽

Cited By ~ 10

Author(s):

Yu Guo ◽

Zi-jun Su ◽

Yi-kun Chen ◽

Zhen Chai

Keyword(s):

Neurotrophic Factor ◽

Hippocampal Neurons ◽

Initial Segment ◽

Neuronal Excitability ◽

Brain Derived Neurotrophic Factor ◽

Axon Initial Segment ◽

Neurotrophin 3 ◽

Cultured Hippocampal Neurons

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Formin Activity and mDia1 Contribute to Maintain Axon Initial Segment Composition and Structure

Molecular Neurobiology ◽

10.1007/s12035-021-02531-6 ◽

2021 ◽

Author(s):

Wei Zhang ◽

María Ciorraga ◽

Pablo Mendez ◽

Diana Retana ◽

Norah Boumedine-Guignon ◽

...

Keyword(s):

Hippocampal Neurons ◽

Initial Segment ◽

Protein Transport ◽

Neuronal Excitability ◽

Brain Slices ◽

Axon Initial Segment ◽

Composition And Structure ◽

The Stability ◽

Protein Density ◽

Voltage Gated Ion Channels

AbstractThe axon initial segment (AIS) is essential for maintaining neuronal polarity, modulating protein transport into the axon, and action potential generation. These functions are supported by a distinctive actin and microtubule cytoskeleton that controls axonal trafficking and maintains a high density of voltage-gated ion channels linked by scaffold proteins to the AIS cytoskeleton. However, our knowledge of the mechanisms and proteins involved in AIS cytoskeleton regulation to maintain or modulate AIS structure is limited. In this context, formins play a significant role in the modulation of actin and microtubules. We show that pharmacological inhibition of formins modifies AIS actin and microtubule characteristics in cultured hippocampal neurons, reducing F-actin density and decreasing microtubule acetylation. Moreover, formin inhibition diminishes sodium channels, ankyrinG and βIV-spectrin AIS density, and AIS length, in cultured neurons and brain slices, accompanied by decreased neuronal excitability. We show that genetic downregulation of the mDia1 formin by interference RNAs also decreases AIS protein density and shortens AIS length. The ankyrinG decrease and AIS shortening observed in pharmacologically inhibited neurons and neuron-expressing mDia1 shRNAs were impaired by HDAC6 downregulation or EB1-GFP expression, known to increase microtubule acetylation or stability. However, actin stabilization only partially prevented AIS shortening without affecting AIS protein density loss. These results suggest that mDia1 maintain AIS composition and length contributing to the stability of AIS microtubules.

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Tropomyosin Tpm3.1 is required to maintain the structure and function of the axon initial segment

10.1101/711614 ◽

2019 ◽

Cited By ~ 1

Author(s):

Amr Abouelezz ◽

Holly Stefen ◽

Mikael Segerstråle ◽

David Micinski ◽

Rimante Minkeviciene ◽

...

Keyword(s):

Actin Filaments ◽

Hippocampal Neurons ◽

Initial Segment ◽

Actin Polymerization ◽

Firing Frequency ◽

Axon Initial Segment ◽

Action Potential Initiation ◽

Sodium Ion Channels ◽

And Function ◽

Filament Network

ABSTRACTThe axon initial segment (AIS) is the site of action potential initiation and serves as a vesicular filter and diffusion barrier that help maintain neuronal polarity. Recent studies have revealed details about a specialized structural complex in the AIS. While an intact actin cytoskeleton is required for AIS formation, pharmacological disruption of actin polymerization compromises the AIS vesicle filter but does not affect overall AIS structure. In this study, we found that the tropomyosin isoform Tpm3.1 decorates a population of relatively stable actin filaments in the AIS. Inhibiting Tpm3.1 in cultured hippocampal neurons led to the loss of AIS structure, the AIS vesicle filter, the clustering of sodium ion channels, and reduced firing frequency. We propose that Tpm3.1-decorated actin filaments form a stable actin filament network under the AIS membrane which provides a scaffold for membrane organization and AIS proteins.

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Potassium channels activated by GABAB agonists and serotonin in cultured hippocampal neurons

Journal of Neurophysiology ◽

10.1152/jn.1994.71.6.2570 ◽

1994 ◽

Vol 71 (6) ◽

pp. 2570-2575 ◽

Cited By ~ 23

Author(s):

L. S. Premkumar ◽

P. W. Gage

Keyword(s):

Potassium Channels ◽

Hippocampal Neurons ◽

Single Channel ◽

Gamma Aminobutyric Acid ◽

Kinetic Behavior ◽

Open Time ◽

Voltage Dependent ◽

Single Channel Currents ◽

Channel Currents ◽

Cultured Hippocampal Neurons

1. Single-channel currents were recorded in cell-attached patches on cultured hippocampal neurons in response to gamma-aminobutyric acid-B (GABAB) agonists or serotonin applied to the cell surface outside the patch area. 2. The channels activated by GABAB agonists and serotonin were potassium selective but had a different conductance and kinetic behavior. Channels activated by GABAB agonists had a higher conductance, longer open-time, and longer burst-length than channels activated by serotonin. 3. The kinetic behavior of channels activated by GABAB agonists varied with potential whereas channels activated by serotonin did not show voltage-dependent changes in kinetics. 4. In a few cell-attached patches, both types of channel were activated when the cell was exposed to GABA together with serotonin. 5. It was concluded that GABAB agonists and serotonin activate different potassium channels in the soma of cultured hippocampal neurons.

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The Cell Adhesion Molecule Neurofascin Stabilizes Axo-axonic GABAergic Terminals at the Axon Initial Segment

Journal of Biological Chemistry ◽

10.1074/jbc.m110.212191 ◽

2011 ◽

Vol 286 (27) ◽

pp. 24385-24393 ◽

Cited By ~ 38

Author(s):

Martin Kriebel ◽

Jennifer Metzger ◽

Sabine Trinks ◽

Deepti Chugh ◽

Robert J. Harvey ◽

...

Keyword(s):

Cell Adhesion ◽

Adhesion Molecule ◽

Hippocampal Neurons ◽

Initial Segment ◽

Cell Adhesion Molecule ◽

Synapse Formation ◽

Growth Factor Receptor ◽

Axon Initial Segment ◽

Adult Rat ◽

Receptor Clusters

Cell adhesion molecules regulate synapse formation and maintenance via transsynaptic contact stabilization involving both extracellular interactions and intracellular postsynaptic scaffold assembly. The cell adhesion molecule neurofascin is localized at the axon initial segment of granular cells in rat dentate gyrus, which is mainly targeted by chandelier cells. Lentiviral shRNA-mediated knockdown of neurofascin in adult rat brain indicates that neurofascin regulates the number and size of postsynaptic gephyrin scaffolds, the number of GABAA receptor clusters as well as presynaptic glutamate decarboxylase-positive terminals at the axon initial segment. By contrast, overexpression of neurofascin in hippocampal neurons increases gephyrin cluster size presumably via stimulation of fibroblast growth factor receptor 1 signaling pathways.

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The palmitoyl acyltransferase ZDHHC14 controls Kv1-family potassium channel clustering at the axon initial segment

eLife ◽

10.7554/elife.56058 ◽

2020 ◽

Vol 9 ◽

Author(s):

Shaun S Sanders ◽

Luiselys M Hernandez ◽

Heun Soh ◽

Santi Karnam ◽

Randall S Walikonis ◽

...

Keyword(s):

Hippocampal Neurons ◽

Initial Segment ◽

Neuronal Excitability ◽

Pdz Domain ◽

Axon Initial Segment ◽

Type I ◽

Action Potential Firing ◽

Outward Currents ◽

Potential Firing ◽

Palmitoyl Acyltransferase

The palmitoyl acyltransferase (PAT) ZDHHC14 is highly expressed in the hippocampus and is the only PAT predicted to bind Type-I PDZ domain-containing proteins. However, ZDHHC14’s neuronal roles are unknown. Here, we identify the PDZ domain-containing Membrane-associated Guanylate Kinase (MaGUK) PSD93 as a direct ZDHHC14 interactor and substrate. PSD93, but not other MaGUKs, localizes to the axon initial segment (AIS). Using lentiviral-mediated shRNA knockdown in rat hippocampal neurons, we find that ZDHHC14 controls palmitoylation and AIS clustering of PSD93 and also of Kv1 potassium channels, which directly bind PSD93. Neurodevelopmental expression of ZDHHC14 mirrors that of PSD93 and Kv1 channels and, consistent with ZDHHC14’s importance for Kv1 channel clustering, loss of ZDHHC14 decreases outward currents and increases action potential firing in hippocampal neurons. To our knowledge, these findings identify the first neuronal roles and substrates for ZDHHC14 and reveal a previously unappreciated role for palmitoylation in control of neuronal excitability.

Download Full-text