Independent activation of distinct pores in dimeric TMEM16A channels.
Independent activation of distinct pores in dimeric TMEM16A channels.
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DOI：
10.1085/jgp.201611651
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发表时间：
2016-11
期刊：
影响因子：
--
通讯作者：
Chen TY
中科院分区：
文献类型：
--
作者：
Jeng G;Aggarwal M;Yu WP;Chen TY
TMEM 16脂质乱序酶由两个相同的亚基形成，但TMEM 16通道的孔结构仍不清楚。Jeng等人使用具有不同突变的TMEM 16 A亚基的串联二聚体，并显示每个亚基包含独立的孔并含有多于一个的Ca 2+结合位点。TMEM 16家族包括Ca 2+激活的Cl−通道（CaCC）和脂质乱序酶。这些蛋白质由两个相同的亚基形成，如最近解决的TMEM 16脂质乱序酶的晶体结构所证实的。然而，高分辨率结构没有提供关于TMEM 16通道的孔结构的明确信息。在这项研究中，我们表达TMEM 16 A通道构成两个共价连接的亚基与不同的Ca 2+亲和力。异二聚体的剂量-反应曲线似乎是两个剂量-反应曲线的加权和-一个对应于高亲和力亚基，另一个对应于低亲和力亚基。然而，荧光共振能量转移实验表明，共价连接的异二聚体蛋白质折叠和组装为一个分子。总之，这些结果表明，两个TMEM 16 A亚基的激活可能彼此独立地激活。在低Ca 2+浓度范围（[Ca 2 +] < 5 µM）下，异源二聚体的Ca 2+激活曲线与野生型通道相似-两种情况下的希尔系数均显著大于1。这表明Ca 2+结合TMEM 16 A的一个亚基足以激活通道，并且每个亚基含有一个以上的Ca 2+结合位点。我们还利用I-V曲线整流，从孔残基的突变，以解决通道的孔结构的结果。通过引入孔突变和在相同或不同亚基中改变Ca 2+亲和力的突变，我们证明了不同亚基的激活似乎与不同孔的打开相关。这些结果表明，TMEM 16 A CaCC也可以采用“双桶”孔结构，类似于在CLC通道和转运蛋白中发现的。
TMEM16 lipid scramblases are formed from two identical subunits, but the pore architecture of TMEM16 channels remains unclear. Jeng et al. use tandem dimers of TMEM16A subunits with different mutations and show that each subunit comprises an independent pore and contains more than one Ca2+-binding site. The TMEM16 family encompasses Ca2+-activated Cl− channels (CaCCs) and lipid scramblases. These proteins are formed by two identical subunits, as confirmed by the recently solved crystal structure of a TMEM16 lipid scramblase. However, the high-resolution structure did not provide definitive information regarding the pore architecture of the TMEM16 channels. In this study, we express TMEM16A channels constituting two covalently linked subunits with different Ca2+ affinities. The dose–response curve of the heterodimer appears to be a weighted sum of two dose–response curves—one corresponding to the high-affinity subunit and the other to the low-affinity subunit. However, fluorescence resonance energy transfer experiments suggest that the covalently linked heterodimeric proteins fold and assemble as one molecule. Together these results suggest that activation of the two TMEM16A subunits likely activate independently of each other. The Ca2+ activation curve for the heterodimer at a low Ca2+ concentration range ([Ca2+] < 5 µM) is similar to that of the wild-type channel—the Hill coefficients in both cases are significantly greater than one. This suggests that Ca2+ binding to one subunit of TMEM16A is sufficient to activate the channel and that each subunit contains more than one Ca2+-binding site. We also take advantage of the I-V curve rectification that results from mutation of a pore residue to address the pore architecture of the channel. By introducing the pore mutation and the mutation that alters Ca2+ affinity in the same or different subunits, we demonstrate that activation of different subunits appears to be associated with the opening of different pores. These results suggest that the TMEM16A CaCC may also adopt a “double-barrel” pore architecture, similar to that found in CLC channels and transporters.
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影响因子：
3.7
作者：
Ni YL;Kuan AS;Chen TY
通讯作者：
Chen TY
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
影响因子：
64.8
作者：
Dutzler, R;Campbell, EB;MacKinnon, R
通讯作者：
MacKinnon, R
影响因子：
56.9
作者：
Caputo, Antonella;Caci, Emanuela;Galietta, Luis J. V.
通讯作者：
Galietta, Luis J. V.