Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
J Gen Physiol. 2011 Nov;138(5):475-93. doi: 10.1085/jgp.201110679. Epub 2011 Oct 17.
The recently published crystal structure of the Cx26 gap junction channel provides a unique opportunity for elucidation of the structure of the conductive connexin pore and the molecular determinants of its ion permeation properties (conductance, current-voltage [I-V] relations, and charge selectivity). However, the crystal structure was incomplete, most notably lacking the coordinates of the N-terminal methionine residue, which resides within the pore, and also lacking two cytosolic domains. To allow computational studies for comparison with the known channel properties, we completed the structure. Grand canonical Monte Carlo Brownian dynamics (GCMC/BD) simulations of the completed and the published Cx26 hemichannel crystal structure indicate that the pore is too narrow to permit significant ion flux. The GCMC/BD simulations predict marked inward current rectification and almost perfect anion selectivity, both inconsistent with known channel properties. The completed structure was refined by all-atom molecular dynamics (MD) simulations (220 ns total) in an explicit solvent and POPC membrane system. These MD simulations produced an equilibrated structure with a larger minimal pore diameter, which decreased the height of the permeation barrier formed by the N terminus. GCMC/BD simulations of the MD-equilibrated structure yielded more appropriate single-channel conductance and less anion/cation selectivity. However, the simulations much more closely matched experimentally determined I-V relations when the charge effects of specific co- and posttranslational modifications of Cx26 previously identified by mass spectrometry were incorporated. We conclude that the average equilibrated structure obtained after MD simulations more closely represents the open Cx26 hemichannel structure than does the crystal structure, and that co- and posttranslational modifications of Cx26 hemichannels are likely to play an important physiological role by defining the conductance and ion selectivity of Cx26 channels. Furthermore, the simulations and data suggest that experimentally observed heterogeneity in Cx26 I-V relations can be accounted for by variation in co- and posttranslational modifications.
最近发表的 Cx26 缝隙连接通道晶体结构为阐明导电连接子孔的结构及其离子渗透特性（传导率、电流-电压 [I-V] 关系和电荷选择性）的分子决定因素提供了独特的机会。然而，该晶体结构并不完整，特别是缺乏位于孔内的 N 端蛋氨酸残基的坐标，也缺乏两个胞质结构域。为了进行与已知通道特性进行比较的计算研究，我们完成了结构的补充。对完整的和已发表的 Cx26 半通道晶体结构进行巨正则蒙特卡罗布朗动力学（GCMC/BD）模拟表明，该孔太窄，无法允许大量离子通过。GCMC/BD 模拟预测会出现明显的内向电流整流和几乎完美的阴离子选择性，这与已知的通道特性都不一致。通过在明确定义的溶剂和 POPC 膜系统中进行全原子分子动力学（MD）模拟（总计 220 ns），对完整结构进行了细化。这些 MD 模拟产生了一个平衡结构，其最小孔径更大，从而降低了由 N 端形成的渗透屏障的高度。对 MD 平衡结构进行 GCMC/BD 模拟后，得出了更合适的单通道传导率和更小的阴离子/阳离子选择性。然而，当纳入先前通过质谱鉴定的 Cx26 的特定共翻译和后翻译修饰的电荷效应时，模拟更接近实验确定的 I-V 关系。我们得出的结论是，与晶体结构相比，MD 模拟后获得的平均平衡结构更能代表开放的 Cx26 半通道结构，并且 Cx26 半通道的共翻译和后翻译修饰很可能通过定义 Cx26 通道的传导率和离子选择性来发挥重要的生理作用。此外，模拟和数据表明，实验观察到的 Cx26 I-V 关系的异质性可以通过共翻译和后翻译修饰的变化来解释。
