Department of Physiology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 03080, Republic of Korea.
The Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea.
Sci Rep. 2019 Apr 30;9(1):6706. doi: 10.1038/s41598-019-43162-1.
Anoctamin-6 (ANO6) belongs to a family of calcium (Ca)-activated chloride channels (CaCCs), with three splicing variants (V1, V2, and V5) showing plasma membrane expression. Unlike other CaCCs, ANO6 requires a non-physiological intracellular free calcium concentration ([Ca] > 1 μM) and several minutes for full activation under a whole-cell patch clamp. Therefore, its physiological role as an ion channel is uncertain and it is more commonly considered a Ca-dependent phospholipid scramblase. Here, we demonstrate that physiological temperature (37 °C) increases ANO6 Ca sensitivity under a whole-cell patch clamp; V1 was activated by 1 μM [Ca], whereas V2 and V5 were activated by 300 nM [Ca]. Increasing the temperature to 42 °C led to activation of all ANO6 variants by 100 nM [Ca]. The delay tme for activation of the three variants was significantly shortened at 37 °C. Notably, the temperature-dependent Ca-sensitisation of ANO6 became insignificant under inside-out patch clamp, suggesting critical roles of unknown cytosolic factors. Unlike channel activity, 27 °C but not 37 °C (physiological temperature) induced the scramblase activity of ANO6 at submicromolar [Ca] (300 nM), irrespective of variant type. Our results reveal a physiological ion conducting property of ANO6 at 37 °C and suggest that ANO6 channel function acts separately from its scramblase activity.
ANO6（Anoctamin-6）属于钙激活氯离子通道（CaCCs）家族，有三种剪接变体（V1、V2 和 V5）显示质膜表达。与其他 CaCCs 不同，ANO6 需要非生理细胞内游离钙浓度（[Ca]>1 μM），并且在全细胞膜片钳下完全激活需要几分钟。因此，其作为离子通道的生理作用尚不确定，更常被认为是一种 Ca 依赖性磷脂翻转酶。在这里，我们证明生理温度（37°C）在全细胞膜片钳下增加了 ANO6 的 Ca 敏感性；V1 被 1 μM [Ca]激活，而 V2 和 V5 被 300 nM [Ca]激活。将温度升高到 42°C 导致所有 ANO6 变体在 100 nM [Ca]下被激活。三种变体的激活延迟时间在 37°C 时明显缩短。值得注意的是，在细胞内向外膜片钳下，ANO6 的温度依赖性 Ca 敏化变得不重要，这表明了未知细胞溶质因子的关键作用。与通道活性不同，27°C 但不是 37°C（生理温度）在亚微摩尔 [Ca]（300 nM）下诱导 ANO6 的翻转酶活性，而与变体类型无关。我们的结果揭示了 ANO6 在 37°C 时的生理离子传导特性，并表明 ANO6 通道功能与其翻转酶活性分开起作用。
