Spadin Selectively Antagonizes Arachidonic Acid Activation of TREK-1 Channels

Ruolin Ma,Anthony Lewis

doi:10.3389/fphar.2020.00434

Abstract

TREK-1 channel activity is a critical regulator of neuronal, cardiac, and smooth muscle physiology and pathology. The antidepressant peptide, spadin, has been proposed to be a TREK-1-specific blocker. Here we sought to examine the mechanism of action underlying spadin inhibition of TREK-1 channels. Heterologous expression in Xenopus laevis oocytes and electrophysiological analysis using two-electrode voltage clamp in standard bath solutions was used to characterize the pharmacological profile of wild-type and mutant murine TREK-1 and TREK-2 channels using previously established human K2P activators; arachidonic acid (AA), cis-4,7,10,13,16,19-docosahexaenoic acid (DHA), BL-1249, and cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC) and inhibitors; spadin and barium (Ba2+). Mouse TREK-1 and TREK-2 channel currents were both significantly increased by AA, BL-1249, and CDC, similar to their human homologs. Under basal conditions, both TREK-1 and TREK-2 currents were insensitive to application of spadin, but could be blocked by Ba2+. Spadin did not significantly inhibit either TREK-1 or TREK-2 currents either chemically activated by AA, BL-1249, or CDC, or structurally activated via a gating mutation. However, pre-exposure to spadin significantly perturbed the subsequent activation of TREK-1 currents by AA, but not TREK-2. Furthermore, spadin was unable to prevent activation of TREK-1 by BL-1249, CDC, or the related bioactive lipid, DHA. Spadin specifically antagonizes the activation of TREK-1 channels by AA, likely via an allosteric mechanism. Lack of intrinsic activity may explain the absence of clinical side effects during antidepressant therapy.

Highlights

Two-pore domain (K2P) channels generate a background or leak potassium (K+) ion conductance critical to the control of cell membrane potential and excitability (Goldstein et al, 2001; Feliciangeli et al, 2015)
In order to allow a direct comparison to previous work by ourselves and others using mouse model tissue, we conducted a comprehensive biophysical and pharmacological assessment of cloned mouse TREK-1 and TREK-2 channels by two-electrode voltage clamp after heterologous expression in X. laevis oocytes
Robust ionic currents were recorded in standard bath solution containing 4 mM K+ from both Mouse TREK-1 (mTREK-1) (Figure 1A) and mouse TREK-2 (mTREK-2) (Figure 1D) expressing oocytes

Summary

Introduction

Two-pore domain (K2P) channels generate a background or leak potassium (K+) ion conductance critical to the control of cell membrane potential and excitability (Goldstein et al, 2001; Feliciangeli et al, 2015). TREK-1 and TREK-2 channels can be strongly and reversibly activated by polyunsaturated free fatty acids (PUFAs) such as AA and DHA through a mechanism involving the C-terminal domain (Fink et al, 1998; Patel et al, 1998; Maingret et al, 1999; Ferroni et al, 2003). Other activators including caffeic acid esters [such as cinnamyl-3,4-dihydroxya-cyanocinnamate (CDC)] and cyclooxygenase inhibitors including BL-1249 and FFA (Danthi et al, 2004; Veale et al, 2014) work independently of the C-terminal domain and are thought to bind below the selectivity filter to open the filter gate (Schewe et al, 2019). TREK-1 channels are sensitive to selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine (Kennard et al, 2005) which are thought to bind within intramembrane fenestrations in a state-dependent manner (Dong et al, 2015; Mcclenaghan et al, 2016)

Methods

Results

Conclusion