Independent activation of ion conduction pores in the double-barreled calcium-activated chloride channel TMEM16A.
Independent activation of ion conduction pores in the double-barreled calcium-activated chloride channel TMEM16A.
复制标题
DOI：
10.1085/jgp.201611650
复制
发表时间：
2016-11
期刊：
影响因子：
--
通讯作者：
Dutzler R
中科院分区：
文献类型：
--
作者：
Lim NK;Lam AK;Dutzler R
TMEM16家族含有由细胞内钙离子激活的二聚体膜蛋白。认识到脂类混乱酶家族成员包含两个独立激活的亚基，Lim等人。使用串联的TMEM16A亚基来显示离子通道成员包含两个独立激活的孔。TMEM16蛋白是一类具有特殊功能的膜蛋白家族，包括脂质扰乱酶和Cl-−通道。这两个分支的成员都被钙激活，作用于细胞内，并可能共享一个共同的结构，这在最近的脂质扰乱酶nhTMEM16的结构中定义了。NhTMEM16亚基的结构特征和钙结合位点的排列表明，该二聚体蛋白含有两个独立于激活和脂质传导的催化位置。在这里，我们问是否在钙激活的氯−通道TMEM16A中观察到类似的独立性。为此，我们生成了包含具有不同激活和渗透属性的亚单位的串联结构。我们的生化研究表明，经过增溶和纯化后的串联体是完整的。在膜片钳电生理学研究中，含有两个野生型(WT)亚基或一个WT亚基与第二个激活受损亚基配对的构建体的功能行为与TMEM16A非常相似。这种相似之处延伸到离子选择性、电导以及钙离子激活通道的浓度和电压依赖性。结合具有不同钙离子潜力的亚基的结构显示了一条两相激活曲线，该曲线可以描述为其组成成分的性质的线性组合。推测的孔衬残基的突变进一步支持了功能独立性，该突变改变了突变亚基的传导性质。我们的结果强烈表明，TMEM16A含有两个离子传导孔，它们独立地被钙离子与嵌入在每个亚单位跨膜部分的位点结合激活。
The TMEM16 family contains dimeric membrane proteins activated by intracellular Ca2+. Realizing that lipid scramblase family members contain two independently activated subunits, Lim et al. use concatenated TMEM16A subunits to show that ion channel members contain two independently activated pores. The TMEM16 proteins constitute a family of membrane proteins with unusual functional breadth, including lipid scramblases and Cl− channels. Members of both these branches are activated by Ca2+, acting from the intracellular side, and probably share a common architecture, which was defined in the recent structure of the lipid scramblase nhTMEM16. The structural features of subunits and the arrangement of Ca2+-binding sites in nhTMEM16 suggest that the dimeric protein harbors two locations for catalysis that are independent with respect to both activation and lipid conduction. Here, we ask whether a similar independence is observed in the Ca2+-activated Cl− channel TMEM16A. For this purpose, we generated concatenated constructs containing subunits with distinct activation and permeation properties. Our biochemical investigations demonstrate the integrity of concatemers after solubilization and purification. During investigation by patch-clamp electrophysiology, the functional behavior of constructs containing either two wild-type (WT) subunits or one WT subunit paired with a second subunit with compromised activation closely resembles TMEM16A. This resemblance extends to ion selectivity, conductance, and the concentration and voltage dependence of channel activation by Ca2+. Constructs combining subunits with different potencies for Ca2+ show a biphasic activation curve that can be described as a linear combination of the properties of its constituents. The functional independence is further supported by mutation of a putative pore-lining residue that changes the conduction properties of the mutated subunit. Our results strongly suggest that TMEM16A contains two ion conduction pores that are independently activated by Ca2+ binding to sites that are embedded within the transmembrane part of each subunit.
登录
查看更多内容
影响因子：
3.7
作者：
Ni YL;Kuan AS;Chen TY
通讯作者：
Chen TY
影响因子：
56.9
作者：
Doyle, DA;Cabral, JM;MacKinnon, R
通讯作者：
MacKinnon, R
影响因子：
6.8
作者：
Brunner, Janine D.;Schenck, Stephan;Dutzler, Raimund
通讯作者：
Dutzler, Raimund
影响因子：
4.8
作者：
Ferrera, Loretta;Caputo, Antonella;Galietta, Luis J. V.
通讯作者：
Galietta, Luis J. V.
影响因子：
56.9
作者：
Dutzler, R;Campbell, EB;MacKinnon, R
通讯作者：
MacKinnon, R