Kim Kwon-Woo, Lee Euna, Ko Ara, Hwang Junmo, Park Kunwoong, Lee Byoung-Cheol, Kim Ki Woo, Oh Won-Jong, Kim Kyuhyung, Lim Hyun-Ho
Neurovascular Unit Research Group, Korea Brain Research Institute (KBRI), Daegu 41062, Republic of Korea.
Neurovascular Unit Research Group, Korea Brain Research Institute (KBRI), Daegu 41062, Republic of Korea; Department of Brain Sciences, DGIST, Daegu 42988, Republic of Korea.
Mol Cells. 2025 May;48(5):100208. doi: 10.1016/j.mocell.2025.100208. Epub 2025 Mar 3.
Bestrophin 1 (BEST1) channels are calcium-activated Cl channels involved in diverse physiological processes, including gliotransmitter release in astrocytes. Although human and chicken BEST1 orthologs have been extensively studied, the structural and functional properties of mouse BEST1 (mBEST1) remain poorly understood. In this study, we characterized the structure-function of mBEST1-BF, a C-terminally tagged variant, using whole-cell patch-clamp recordings, surface biotinylation assays, and single-particle cryo-electron microscopy. Cryo-electron microscopy structural analysis of mBEST1-BF revealed closed and partially open conformations. Comparative analysis with human and chicken BEST1 orthologs highlighted conserved calcium-binding and gating mechanisms, with distinct features in mBEST1, including a wider aperture sufficient to accommodate dehydrated Cl ions and potential anion-binding sites near Val205 and Gln208 residues. The disordered C-terminal region of mBEST1 remains unresolved, suggesting it may require stabilizing factors for structural determination. Additionally, the autoinhibitory domain, which includes Ser354, likely plays a key role in regulating gating, with Ser354 potentially serving as a phosphorylation site that modulates channel activity. Our findings provide structural and functional insights into mBEST1 and suggest mechanisms underlying its unique gating and ion permeation properties.
最佳rophin 1（BEST1）通道是钙激活的氯离子通道，参与多种生理过程，包括星形胶质细胞中神经递质的释放。尽管人类和鸡的BEST1直系同源物已得到广泛研究，但小鼠BEST1（mBEST1）的结构和功能特性仍知之甚少。在本研究中，我们使用全细胞膜片钳记录、表面生物素化分析和单颗粒冷冻电子显微镜对C末端标记变体mBEST1-BF的结构功能进行了表征。mBEST1-BF的冷冻电子显微镜结构分析揭示了关闭和部分开放的构象。与人类和鸡的BEST1直系同源物的比较分析突出了保守的钙结合和门控机制，mBEST1具有独特的特征，包括一个足以容纳脱水氯离子的更宽孔径以及靠近Val205和Gln208残基的潜在阴离子结合位点。mBEST1的无序C末端区域仍未解析，这表明其结构测定可能需要稳定因子。此外，包括Ser354的自抑制结构域可能在调节门控中起关键作用，Ser354可能作为调节通道活性的磷酸化位点。我们的研究结果提供了对mBEST1的结构和功能的见解，并提出了其独特的门控和离子渗透特性的潜在机制。
