Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
J Gen Physiol. 2010 Mar;135(3):217-27. doi: 10.1085/jgp.200910309. Epub 2010 Feb 8.
The state from which channel inactivation occurs is both biologically and mechanistically critical. For example, preferential closed-state inactivation is potentiated in certain Ca(2+) channel splice variants, yielding an enhancement of inactivation during action potential trains, which has important consequences for short-term synaptic plasticity. Mechanistically, the structural substrates of inactivation are now being resolved, yielding a growing library of molecular snapshots, ripe for functional interpretation. For these reasons, there is an increasing need for experimentally direct and systematic means of determining the states from which inactivation proceeds. Although many approaches have been devised, most rely upon numerical models that require detailed knowledge of channel-state topology and gating parameters. Moreover, prior strategies have only addressed voltage-dependent forms of inactivation (VDI), and have not been readily applicable to Ca(2+)-dependent inactivation (CDI), a vital form of regulation in numerous contexts. Here, we devise a simple yet systematic approach, applicable to both VDI and CDI, for semiquantitative mapping of the states from which inactivation occurs, based only on open-channel measurements. The method is relatively insensitive to the specifics of channel gating and does not require detailed knowledge of state topology or gating parameters. Rather than numerical models, we derive analytic equations that permit determination of the states from which inactivation occurs, based on direct manipulation of data. We apply this methodology to both VDI and CDI of Ca(V)1.3 Ca(2+) channels. VDI is found to proceed almost exclusively from the open state. CDI proceeds equally from the open and nearby closed states, but is disfavored from deep closed states distant from the open conformation. In all, these outcomes substantiate and enrich conclusions of our companion paper in this issue (Tadross et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910308) that deduces endpoint mechanisms of VDI and CDI in Ca(V)1.3. More broadly, the methods introduced herein can be readily generalized for the analysis of other channel types.
通道失活状态在生物学和机制上都是至关重要的。例如，在某些钙通道剪接变体中，优先的关闭状态失活增强，导致动作电位列车期间失活增强，这对短期突触可塑性有重要影响。从机制上讲，失活的结构基底正在得到解决，产生越来越多的分子快照，为功能解释做好了准备。出于这些原因，人们越来越需要实验上直接和系统的方法来确定失活所经历的状态。虽然已经设计了许多方法，但大多数方法都依赖于需要详细了解通道状态拓扑和门控参数的数值模型。此外，先前的策略仅解决了电压依赖性失活（VDI），并且不易应用于钙依赖性失活（CDI），这是许多情况下重要的调节形式。在这里，我们设计了一种简单而系统的方法，可应用于 VDI 和 CDI，用于基于开放通道测量对失活所经历的状态进行半定量映射。该方法对通道门控的细节相对不敏感，并且不需要详细了解状态拓扑或门控参数。我们不是使用数值模型，而是推导出允许根据数据直接操作来确定失活所经历的状态的解析方程。我们将这种方法应用于 Ca(V)1.3 钙通道的 VDI 和 CDI。发现 VDI 几乎完全从开放状态进行。CDI 同样从开放和附近的关闭状态进行，但从远离开放构象的深关闭状态不利。总之，这些结果证实并丰富了我们在本期杂志上的另一篇论文（Tadross 等人，2010. J. Gen. Physiol. doi:10.1085/jgp.200910308）中的结论，该结论推断了 Ca(V)1.3 中 VDI 和 CDI 的终点机制。更广泛地说，本文介绍的方法可以很容易地推广到其他通道类型的分析。
