Taraska Justin W, Zagotta William N
Department of Physiology and Biophysics, Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195, USA.
Nat Struct Mol Biol. 2007 Sep;14(9):854-60. doi: 10.1038/nsmb1281. Epub 2007 Aug 12.
For ligand-gated ion channels, the binding of a ligand to an intracellular or extracellular domain generates changes in transmembrane pore-forming helices, which alters ion flow. The molecular mechanism for this allostery, however, remains unknown. Here we explore the structure and conformational rearrangements of the C-terminal gating ring of the cyclic nucleotide-gated channel CNGA1 during activation by cyclic nucleotides with patch-clamp fluorometry. By monitoring fluorescent resonance energy transfer (FRET) between membrane-resident quenchers and fluorophores attached to the channel, we detected no movement orthogonal to the membrane during channel activation. By monitoring FRET between fluorophores within the C-terminal region, we determined that the C-terminal end of the C-linker and the end of the C-helix move apart when channels open. We conclude that during channel activation, a portion of the gating ring moves parallel to the plasma membrane, hinging toward the central axis of the channel.
对于配体门控离子通道，配体与细胞内或细胞外结构域的结合会导致跨膜孔形成螺旋发生变化，从而改变离子流动。然而，这种变构的分子机制仍然未知。在这里，我们利用膜片钳荧光法探索了环核苷酸门控通道CNGA1在被环核苷酸激活过程中C端门控环的结构和构象重排。通过监测膜结合淬灭剂与连接在通道上的荧光团之间的荧光共振能量转移（FRET），我们在通道激活过程中未检测到与膜垂直的移动。通过监测C端区域内荧光团之间的FRET，我们确定当通道打开时，C连接子的C端和C螺旋的末端会分开。我们得出结论，在通道激活过程中，门控环的一部分平行于质膜移动，朝着通道的中心轴铰接。
