β-COP Regulates TWIK1/TREK1 Heterodimeric Channel-Mediated Passive Conductance in Astrocytes

Abstract

Mature astrocytes are characterized by a K+ conductance (passive conductance) that changes with a constant slope with voltage, which is involved in K+ homeostasis in the brain. Recently, we reported that the tandem of pore domains in a weak inward rectifying K+ channel (TWIK1 or KCNK1) and TWIK-related K+ channel 1 (TREK1 or KCNK2) form heterodimeric channels that mediate passive conductance in astrocytes. However, little is known about the binding proteins that regulate the function of the TWIK1/TREK1 heterodimeric channels. Here, we found that β-coat protein (COP) regulated the surface expression and activity of the TWIK1/TREK1 heterodimeric channels in astrocytes. β-COP binds directly to TREK1 but not TWIK1 in a heterologous expression system. However, β-COP also interacts with the TWIK1/TREK1 heterodimeric channel in a TREK1 dependent manner and enhances the surface expression of the heterodimeric channel in astrocytes. Consequently, it regulates TWIK1/TREK1 heterodimeric channel-mediated passive conductance in astrocytes in the mouse brain. Taken together, these results suggest that β-COP is a potential regulator of astrocytic passive conductance in the brain.

β-COP Suppresses the Surface Expression of the TREK2

“…To achieve this, we tagged each TREK subfamily protein, TREK1, TREK2, and TRAAK, with GFP, and β-COP was tagged with mCherry to confirm the specificity of their localization. We observed that TREK1 and β-COP co-localized throughout the cell, particularly near the cell membrane, which is consistent with our previous findings [18,29]. Conversely, the co-localization of TREK2 and β-COP was densely localized in a Golgi-like organelle adjacent to the nucleus.…”

“…When VN-and VC-tagged proteins bind, the separated Venus fluorescent proteins come close to each other, resulting in clear Venus fluorescence. Similar to previous reports [29], a clear BiFC signal was observed throughout the HEK293T cells co-transfected with VN-β-COP and VC-TREK1. In HEK293T cells cotransfected with VN-β-COP and VC-TREK2, the signals were concentrated in a narrow area near the organelles, similar to the Golgi apparatus.…”

“…Previous studies have indicated that β-COP binds to the N-terminus of TREK1 and increases its surface expression [18]. Recent research has also suggested that β-COP binds to the TWIK1/TREK1 heterodimeric channel by binding to TREK1 and consequently regulates the passive conductance of adult mouse hippocampal astrocytes [28,29]. However, although it is unclear whether β-COP is associated with TREK2 or TRAAK, which are members of the TREK subfamily, β-COP may upregulate not only TREK1 but also TREK2, similar to AKAP150 and Mtap2 [16,17].…”

Astrocytic NHERF-1 increases seizure susceptibility by inhibiting surface expression of TREK-1

“…Interestingly, in this study, NHERF-1 was identi ed as the protein that binds to the N-terminus of TREK-1, next to β-COP. β-COP was identi ed as an interacting protein of TREK-1 in astrocytes, binding to the N-terminal domain and increasing both surface expression and channel activity 29,41 . However, NHERF-1 is bound to the N-terminal domain of TREK-1 but reduces both surface expression and channel activity.…”

“…To further determine the binding site of NHERF-1 within TREK-1, the N terminus of TREK-1 was divided into four sections (ΔN1, ΔN1,2, ΔN1,2,3, or ΔN) (Figure S4A) 29 . BiFC signals were detected for TREK-1 ΔN1, TREK-1 ΔN1,2, and TREK-1 ΔN1,2,3 using NHERF-1, but not for TREK-1 ΔN using NHERF-1 (Figure S4B).…”