Guy H R, Durell S R
Laboratory of Mathematical Biology, DCBDC, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Soc Gen Physiol Ser. 1995;50:1-16.
The structure determination of voltage-gated channels by the combination of molecular modeling and mutagenesis experiments is a long term, iterative process. As such, the models should be considered as a work in progress, with changes expected as more data becomes available. The primary role of the models is that they assimilate the known data and provide ideas for further experiments to elucidate the real structures. Although the models presented here have already gone through two or three cycles of development and testing, many aspects remain tentative. Perhaps the most significant result so far is that the P segment was experimentally confirmed to form the ion-selective part of the channel. In a subsequent cycle of testing and modeling, the specific residues responsibility for Na+ and Ca2+ selectivity have been identified and the selectivity filter of K+ channels is now predicted to be formed by the side chains, but rather by the carbonyl oxygens of the conserved Gly-Tyr-Gly sequence backbone. As another example, the 9p residue of the P segment of K+ channels was originally modeled as either being buried in the protein or accessible channels was originally modeled as either being buried in the protein or accessible from inside the cell only. However, once mutation of this residue to histidine was found to affect blockade by extracellular TEA, protons, Zn2+ and histidine reagents (DeBiasi et al., 1993), the models were updated to have this and the hydrophilic residues in the first part of P form a helix that comprises part of the extracellular, outer vestibular of the pore. While this motif was used also for Na+ and Ca2+ pore models (see Fig. 2) where the putative helices are amphipathic, it remains to be verified. Modeling of the size and shape of the outer vestibule of K+ channels was also aided by the data for the binding of CTX in the extracellular entrance to the pore. Similarly, experiments with peptide toxins such as mu and omega conotoxins may prove useful in modeling the outer vestibules of the Na+ and/or Ca2+ channels. While important advances have been made, it is important to realize that these approaches are still very new. In the future we are likely to see improvements on both the theoretical and experimental sides which will greatly advance the process.(ABSTRACT TRUNCATED AT 400 WORDS)
通过分子建模与诱变实验相结合来确定电压门控通道的结构是一个长期的、反复的过程。因此，这些模型应被视为一项正在进行的工作，随着更多数据的获得，预计会有所变化。模型的主要作用是整合已知数据，并为进一步阐明真实结构的实验提供思路。尽管这里展示的模型已经经历了两到三轮的开发和测试，但许多方面仍存在不确定性。目前为止，或许最重要的结果是，P段通过实验被证实形成了通道的离子选择性部分。在后续的测试和建模循环中，已确定了负责Na+和Ca2+选择性的特定残基，现在预测K+通道的选择性过滤器是由保守的Gly-Tyr-Gly序列主链的羰基氧形成，而非侧链。再举个例子，K+通道P段的9p残基最初建模为要么埋在蛋白质中，要么仅从细胞内部可及。然而，一旦发现该残基突变为组氨酸会影响细胞外TEA、质子、Zn2+和组氨酸试剂的阻断作用（德比亚西等人，1993年），模型就被更新，使该残基以及P段第一部分的亲水性残基形成一个螺旋，该螺旋构成孔道细胞外、外前庭的一部分。虽然这个基序也用于Na+和Ca2+孔道模型（见图2），其中假定的螺旋是两亲性的，但仍有待验证。K+通道外前庭大小和形状的建模也得益于CTX在孔道细胞外入口处结合的数据。同样，诸如μ和ω芋螺毒素等肽毒素的实验可能对Na+和/或Ca2+通道外前庭的建模有用。虽然已经取得了重要进展，但必须认识到这些方法仍然非常新颖。未来我们可能会在理论和实验方面看到改进，这将极大地推动这一进程。（摘要截断于400字）
