Tewari Shivendra G, Zhou Yifan, Otto Bradley J, Dash Ranjan K, Kwok Wai-Meng, Beard Daniel A
Department of Molecular and Integrative Physiology, University of Michigan Ann Arbor, MI, USA.
HD Biosciences Corporation Shanghai, China.
Front Physiol. 2015 Jan 13;5:513. doi: 10.3389/fphys.2014.00513. eCollection 2014.
The voltage-dependent anion channel (VDAC) is the main conduit for permeation of solutes (including nucleotides and metabolites) of up to 5 kDa across the mitochondrial outer membrane (MOM). Recent studies suggest that VDAC activity is regulated via post-translational modifications (PTMs). Yet the nature and effect of these modifications is not understood. Herein, single channel currents of wild-type, nitrosated, and phosphorylated VDAC are analyzed using a generalized continuous-time Markov chain Monte Carlo (MCMC) method. This developed method describes three distinct conducting states (open, half-open, and closed) of VDAC activity. Lipid bilayer experiments are also performed to record single VDAC activity under un-phosphorylated and phosphorylated conditions, and are analyzed using the developed stochastic search method. Experimental data show significant alteration in VDAC gating kinetics and conductance as a result of PTMs. The effect of PTMs on VDAC kinetics is captured in the parameters associated with the identified Markov model. Stationary distributions of the Markov model suggest that nitrosation of VDAC not only decreased its conductance but also significantly locked VDAC in a closed state. On the other hand, stationary distributions of the model associated with un-phosphorylated and phosphorylated VDAC suggest a reversal in channel conformation from relatively closed state to an open state. Model analyses of the nitrosated data suggest that faster reaction of nitric oxide with Cys-127 thiol group might be responsible for the biphasic effect of nitric oxide on basal VDAC conductance.
电压依赖性阴离子通道（VDAC）是分子量高达5 kDa的溶质（包括核苷酸和代谢物）穿过线粒体外膜（MOM）的主要通道。最近的研究表明，VDAC活性是通过翻译后修饰（PTM）来调节的。然而，这些修饰的性质和作用尚不清楚。在此，我们使用广义连续时间马尔可夫链蒙特卡罗（MCMC）方法分析了野生型、亚硝化型和磷酸化型VDAC的单通道电流。这种改进的方法描述了VDAC活性的三种不同传导状态（开放、半开放和关闭）。我们还进行了脂质双层实验，以记录未磷酸化和磷酸化条件下的单个VDAC活性，并使用改进的随机搜索方法进行分析。实验数据表明，PTM导致VDAC门控动力学和电导率发生显著变化。PTM对VDAC动力学的影响体现在与所识别的马尔可夫模型相关的参数中。马尔可夫模型的稳态分布表明，VDAC的亚硝化不仅降低了其电导率，还显著将VDAC锁定在关闭状态。另一方面，与未磷酸化和磷酸化VDAC相关的模型稳态分布表明，通道构象从相对关闭状态转变为开放状态。对亚硝化数据的模型分析表明，一氧化氮与Cys-127硫醇基团的更快反应可能是一氧化氮对基础VDAC电导率产生双相效应的原因。
