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

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 14-3-3 gamma was confirmed by yeast-two hybrid assay and BiFC method. The effect of 14-3-3 gamma on Best1-mediated current was measured by whole-cell patch clamp technique. Results: We identified 14-3-3 gamma as novel binding partner of Best1 in astrocytes: among 7 isoforms of 14-3-3 protein, only 14-3-3 gamma was found to bind specifically. We determined a binding domain on the C-terminus of Best1 which is critical for an interaction with 14-3-3 gamma. We also revealed that interaction between Best1 and 14-3-3 gamma 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 14-3-3 gamma without a significant change in total Best1 expression in cultured astrocytes. Furthermore, we discovered that 14-3-3 gamma-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 14-3-3 gamma-shRNA. Finally, through a structural modeling, we found critical amino acid residues containing S358 of Best1 exhibiting binding affinities to 14-3-3 gamma. Conclusions: 14-3-3 gamma promotes surface expression of Best1 channel in astrocytes through direct interaction.