Molecular insight into the structural and functional modulation of bestrophin1 channel   

초록

Bestrophin 1 (BEST1) channels are calcium-activated Cl- channels involved in diverse physiological processes, including gliotransmitter release in astrocytes. Interestingly, BEST1 orthologs share high amino acid sequence identity in the N-terminal region (~370 residues), but exhibit only marginal identity in the C-terminal region (~150-200 residues). While human and chicken BEST1 orthologs have been extensively studied, the structural and functional properties of mouse BEST1 (mBEST1) remain poorly understood. To address this gap, we characterized mBEST1 using whole-cell patch-clamp recordings, surface biotinylation assays, and single-particle cryo-electron microscopy (cryo-EM). Additionally, to investigate the molecular basis underlying the functional differences between human and mouse BEST1 channels, we performed immunoprecipitation-mass spectrometry (IP–MS) to identify proteins that specifically interact with hBEST1. This analysis revealed PYCR3, the terminal enzyme in proline biosynthesis, as a specific binding partner of hBEST1. The interaction was further validated through co-immunoprecipitation, size-exclusion chromatography, and split-GFP reconstitution assays. The functional consequences of the hBEST1–PYCR3 interaction were evaluated using whole-cell patch-clamp recordings, and we also determined the structure of the hBEST1-PYCR3 complex at a resolution of ~3 Å. In this symposium, I will present recent advances from my laboratory on the structural and functional characterization of BEST1 channels and discuss their physiological and mechanistic implications.