Cellular and Subcellular Localisation of Kv4-Associated KChIP Proteins in the Rat Cerebellum.

Rocío Alfaro-Ruíz,Carolina Aguado,Rafael Luján,Alejandro Martín-Belmonte,Ana Esther Moreno-Martínez

doi:10.3390/ijms21176403

Rocío Alfaro-Ruíz, Carolina Aguado + Show 3 more

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https://doi.org/10.3390/ijms21176403

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Abstract

The K+ channel interacting proteins (KChIPs) are a family of cytosolic proteins that interact with Kv4 channels, leading to higher current density, modulation of channel inactivation and faster recovery from inactivation. Using immunohistochemical techniques at the light and electron microscopic level combined with quantitative analysis, we investigated the cellular and subcellular localisation of KChIP3 and KChIP4 to compare their distribution patterns with those for Kv4.2 and Kv4.3 in the cerebellar cortex. Immunohistochemistry at the light microscopic level demonstrated that KChIP3, KChIP4, Kv4.2 and Kv4.3 proteins were widely expressed in the cerebellum, with mostly overlapping patterns. Immunoelectron microscopic techniques showed that KChIP3, KChIP4, Kv4.2 and Kv4.3 shared virtually the same somato-dendritic domains of Purkinje cells and granule cells. Application of quantitative approaches showed that KChIP3 and KChIP4 were mainly membrane-associated, but also present at cytoplasmic sites close to the plasma membrane, in dendritic spines and shafts of Purkinje cells (PCs) and dendrites of granule cells (GCs). Similarly, immunoparticles for Kv4.2 and Kv4.3 were observed along the plasma membrane and at intracellular sites in the same neuron populations. In addition to the preferential postsynaptic distribution, KChIPs and Kv4 were also distributed presynaptically in parallel fibres and mossy fibres. Immunoparticles for KChIP3, KChIP4 and Kv4.3 were detected in parallel fibres, and KChIP3, KChIP4, Kv4.2 and Kv4.3 were found in parallel fibres, indicating that composition of KChIP and Kv4 seems to be input-dependent. Together, our findings unravelled previously uncharacterised KChIP and Kv4 subcellular localisation patterns in neurons, revealed that KChIP have additional Kv4-unrelated functions in the cerebellum and support the formation of macromolecular complexes between KChIP3 and KChIP4 with heterotetrameric Kv4.2/Kv4.3 channels.

Highlights

The cerebellum is a foliated structure formed by the vermis and two symmetric hemispheres [1] that plays a major role in the fine motor control, maintenance of balance and posture, and contributes to perception, memory and cognition [2,3]
Immunohistochemistry at the light microscopic level demonstrated that KChIP3, KChIP4, Kv4.2 and Kv4.3 proteins were widely expressed in the cerebellum, with mostly overlapping patterns
Using monoclonal antibodies against auxiliary KChIP3 and KChIP4 subunits, together with the antibodies against Kv4.2 and Kv4.3 subunits, we have investigated the distribution of these molecules in the cerebellum using light microscopy immunohistochemical techniques

Summary

Introduction

The cerebellum is a foliated structure formed by the vermis and two symmetric hemispheres [1] that plays a major role in the fine motor control, maintenance of balance and posture, and contributes to perception, memory and cognition [2,3]. KChIP increases the surface expression and the current amplitude of Kv4 [16,17], slows the turnover rate of Kv4 protein and slows the inactivation kinetics and speeds the rate of recovery from inactivation of Kv4 channels [8,11,16]. Consistent with their function in modulating K+ channels, deletion of Kv4.2 channel eliminates expression of its associated KChIP proteins [18]

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