Activity of TREK-2-like Channels in the Pyramidal Neurons of Rat Medial Prefrontal Cortex Depends on Cytoplasmic Calcium.

Beata Dworakowska,Ewa Nurowska,Maciej Gawlak

doi:10.3390/biology10111119

Abstract

Simple SummaryThe pyramidal neurons of rat prefrontal cortex express potassium channels identified as a non-canonical splice variant of the TREK-2 channel. The main function of TREK channels is to regulate the resting membrane potential. We showed that cytoplasmic Ca2+ upregulates the activity of TREK-2-like channels. Previous studies have indicated that the activation of TREK-2 channels is mediated by PI(4,5)P2, a polyanionic lipid in the inner leaflet of the plasma membrane. While TREK channels are believed to not be regulated by calcium, our work shows otherwise. We propose a model in which calcium ions enable the formation of PI(4,5)P2 nanoclusters, which stabilize active conformation of the channel.TREK-2-like channels in the pyramidal neurons of rat prefrontal cortex are characterized by a wide range of spontaneous activity—from very low to very high—independent of the membrane potential and the stimuli that are known to activate TREK-2 channels, such as temperature or membrane stretching. The aim of this study was to discover what factors are involved in high levels of TREK-2-like channel activity in these cells. Our research focused on the PI(4,5)P2-dependent mechanism of channel activity. Single-channel patch clamp recordings were performed on freshly dissociated pyramidal neurons of rat prefrontal cortexes in both the cell-attached and inside-out configurations. To evaluate the role of endogenous stimulants, the activity of the channels was recorded in the presence of a PI(4,5)P2 analogue (PI(4,5)P2DiC8) and Ca2+. Our research revealed that calcium ions are an important factor affecting TREK-2-like channel activity and kinetics. The observation that calcium participates in the activation of TREK-2-like channels is a new finding. We showed that PI(4,5)P2-dependent TREK-2 activity occurs when the conditions for PI(4,5)P2/Ca2+ nanocluster formation are met. We present a possible model explaining the mechanism of calcium action.

Highlights

The human prefrontal cortex is involved in a variety of cognitive functions [1]
The cryoprotectant was washed with phosphate-buffered saline (PBS) and the brain slices were blocked in 5% normal goat serum in PBS with 0.01% Triton X-100 (NGST) and immersed overnight (4 ◦C) in rabbit anti-TREK2 primary antibody (Invitrogen, PA5-77608) solution in NGST
We already showed that pyramidal neurons in the medial prefrontal cortex of rats express high-conductance TREK-2-like channels [36,46]

Summary

Introduction

The human prefrontal cortex is involved in a variety of cognitive functions [1]. The prefrontal cortex can be divided into two regions: the medial prefrontal cortex (mPFC) and the lateral prefrontal cortex [2]. Medial prefrontal cortex lesions leading to cognitive impairment have been associated with various human brain disorders, such as depression, anxiety disorders, schizophrenia, autism spectrum disorders, Alzheimer’s disease, Parkinson’s disease, and addiction [3] Some of these pathological processes may involve two-pore domain potassium channels (K2P), which are thought to play a significant role in neuronal excitability and in the resting membrane potential [4]. The protonation of glutamic acid in the C-terminus at low pH enhances the cluster of positive charges in the C-terminus and tightens the electrostatic interaction with PI(4,5)P2 Polycationic molecules such as poly-L-lysine disturb that interaction, and in their presence, the TREK channel remains in an inactive state insensitive to low pH and less sensitive to membrane stretch [26]. Phosphorylation adds negative charges to the C-terminus domain, which may reduce binding to the plasma membrane and, the channel activity [18,37]

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