The bestrophin family of calcium (Ca²⁺)-activated chloride (Cl⁻) channels, which mediate the influx and efflux of monovalent anions in response to the levels of intracellular Ca²⁺, comprises four members in mammals (bestrophin 1–4). Here we report cryo-EM structures of bovine bestrophin-2 (bBest2) bound and unbound by Ca²⁺ at 2.4- and 2.2-Å resolution, respectively. The bBest2 structure highlights four previously underappreciated pore-lining residues specifically conserved in Best2 but not in Best1, illustrating the differences between these paralogs. Structure-inspired electrophysiological analysis reveals that, although the channel is sensitive to Ca²⁺, it has substantial Ca²⁺-independent activity for Cl⁻, reflecting the opening at the cytoplasmic restriction of the ion conducting pathway even when Ca²⁺ is absent. Moreover, the ion selectivity of bBest2 is controlled by multiple residues, including those involved in gating.

钙 (Ca ²⁺ ) 激活氯 (Cl ⁻ ) 通道的 bestropin 家族可根据细胞内 Ca ²⁺水平介导单价阴离子的流入和流出，在哺乳动物中由四个成员组成（bestropin 1–4） 。在这里，我们分别以 2.4-Å 和 2.2-Å 分辨率报道了与 Ca ²⁺结合和未结合的牛 bestropin-2 (bBest2) 的冷冻电镜结构。 bBest2 结构突出显示了 Best2 中特别保守但 Best1 中不保守的四个先前未被充分认识的孔衬残基，说明了这些旁系同源物之间的差异。结构启发的电生理学分析表明，尽管该通道对 Ca ²⁺敏感，但它对 Cl ^-具有显着的与 Ca ²⁺无关的活性，这反映了即使在 Ca ²⁺不存在时，离子传导通路的细胞质限制也是开放的。此外，bBest2 的离子选择性由多个残基控制，包括参与门控的残基。