Experimental Ophthalmology, Department of Ophthalmology, Charité - Universitätsmedizin Berlin, a corporate member of Freie Universität, Humboldt-University, the Berlin Institute of Health, Berlin, Germany.
Department of Gastroenterology, Endocrinology, Rheumatology and Infectious Diseases, University Hospital Regensburg, Regensburg, Germany.
Sci Rep. 2019 Feb 19;9(1):2257. doi: 10.1038/s41598-018-37287-y.
Changes in cell function occur by specific patterns of intracellular Ca, activating Ca-sensitive proteins. The anoctamin (TMEM16) protein family has Ca-dependent ion channel activity, which provides transmembrane ion transport, and/or Ca-dependent phosphatidyl-scramblase activity. Using amino acid sequence analysis combined with measurements of ion channel function, we clarified the so far unknown Ano4 function as Ca-dependent, non-selective monovalent cation channel; heterologous Ano4 expression in HEK293 cells elicits Ca activated conductance with weak selectivity of K > Na > Li. Endogenously expressed Ca-dependent cation channels in the retinal pigment epithelium were identified as Ano4 by KO mouse-derived primary RPE cells and siRNA against Ano4. Exchanging a negatively charged amino acid in the putative pore region (AA702-855) into a positive one (E775K) turns Ano4-elicited currents into Cl currents evidencing its importance for ion selectivity. The molecular identification of Ano4 as a Ca-activated cation channel advances the understanding of its role in Ca signaling.
细胞功能的变化是通过特定的细胞内 Ca 模式来实现的，从而激活 Ca 敏感蛋白。ANOCTAMIN（TMEM16）蛋白家族具有 Ca 依赖性离子通道活性，提供跨膜离子转运和/或 Ca 依赖性磷脂翻转酶活性。通过氨基酸序列分析结合离子通道功能的测量，我们阐明了迄今为止未知的 Ano4 功能是 Ca 依赖性、非选择性单价阳离子通道；在 HEK293 细胞中异源表达 Ano4 可引起 Ca 激活的电导，对 K>Na>Li 具有较弱的选择性。通过 KO 小鼠衍生的原代 RPE 细胞和针对 Ano4 的 siRNA，鉴定出内源性表达的 Ca 依赖性阳离子通道为 Ano4。在假定的孔区（AA702-855）中将带负电荷的氨基酸替换为带正电荷的氨基酸（E775K），将 Ano4 诱导的电流转化为 Cl 电流，证明其对离子选择性很重要。Ano4 作为 Ca 激活的阳离子通道的分子鉴定，推进了对其在 Ca 信号转导中作用的理解。
