Direct interaction with 14–3-3γ promotes surface expression of Best1 channel in astrocyte

Molecular Brain

Minhee Cho¹  Eunju Kim¹  Eun Mi Hwang¹  Junsung Woo²  C. Justin Lee²  Soo-Jin Oh²  Ae Nim Pae³  Nam-Chul Cho³  Jae-Yong Park⁴  Young-Sun Lee⁴ 

[1] Center for Functional Connectomics, Korea Institute of Science and Technology;Center for Neuroscience, Korea Institute of Science and Technology;Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and Technology;School of Biosystem and Biomedical Science, College of Health Science, Korea University;

关键词: Astrocyte;    Bestrophin-1;    14–3-3γ;    Surface expression;    Glutamate;   

DOI  :  10.1186/s13041-017-0331-x

来源: DOAJ

【 摘 要 】

Abstract Background Bestrophin-1 (Best1) is a calcium-activated anion channel (CAAC) that is expressed broadly in mammalian tissues including the brain. We have previously reported that Best1 is expressed in hippocampal astrocytes at the distal peri-synaptic regions, called microdomains, right next to synaptic junctions, and that it disappears from the microdomains in Alzheimer’s disease mouse model. Although Best1 appears to be dynamically regulated, the mechanism of its regulation and modulation is poorly understood. It has been reported that a regulatory protein, 14-3-3 affects the surface expression of numerous membrane proteins in mammalian cells. Methods The protein-protein interaction between Best1 and 14-3-3γ was confirmed by yeast-two hybrid assay and BiFC method. The effect of 14-3-3γ on Best1-mediated current was measured by whole-cell patch clamp technique. Results We identified 14-3-3γ as novel binding partner of Best1 in astrocytes: among 7 isoforms of 14-3-3 protein, only 14-3-3γ was found to bind specifically. We determined a binding domain on the C-terminus of Best1 which is critical for an interaction with 14-3-3γ. We also revealed that interaction between Best1 and 14-3-3γ was mediated by phosphorylation of S358 in the C-terminus of Best1. We confirmed that surface expression of Best1 and Best1-mediated whole-cell current were significantly decreased after a gene-silencingof 14-3-3γ without a significant change in total Best1 expression in cultured astrocytes. Furthermore, we discovered that 14-3-3γ-shRNA reduced Best1-mediated glutamate release from hippocampal astrocyte by recording a PAR1 receptor-induced NMDA receptor-mediated current from CA1 pyramidal neurons in hippocampal slices injected with adenovirus carrying 14-3-3γ-shRNA. Finally, through a structural modeling, we found critical amino acid residues containing S358 of Best1 exhibiting binding affinities to 14-3-3γ. Conclusions 14-3-3γ promotes surface expression of Best1 channel in astrocytes through direct interaction.

【 授权许可】

Unknown