Dart C, Leyland M L, Spencer P J, Stanfield P R, Sutcliffe M J
Ion Channel Group, Department of Cell Physiology and Pharmacology, University of Leicester, PO Box 138, Leicester LE1 9HN, UK.
J Physiol. 1998 Aug 15;511 ( Pt 1)(Pt 1):25-32. doi: 10.1111/j.1469-7793.1998.025bi.x.
We have produced a structural model of the pore-forming H5 (or P) region of the strong inward rectifier K+ channel, Kir2.1, based initially on an existing molecular model of the pore region of the voltage-gated K+ channel, Kv1.3. Cysteine-scanning mutagenesis and subsequent blockage by Ag+ was used to test our model by determining the residues in H5 whose side chains line the ion conduction pathway. Mutations made in eight positions within the highly conserved H5 region resulted in apparently non-functional channels. Constructing covalently linked dimers, which carry a cysteine substitution in only one of the linked subunits, rescued six of these mutants; a covalently linked tetramer, carrying a cysteine substitution on only one of the linked subunits, rescued a further mutant. Our results using the dimers and tetramers suggest that residues Thr141, Thr142, Ile143, Tyr145, Phe147 and Cys149 are accessible to externally applied Ag+ (100-200 nM) and therefore that their side chains line the channel pore. We conclude that the topology of the Kir pore is similar, but not identical, to that of Kv channels. Additionally, the molecular model suggests that selectivity may be conferred both by aromatic residues (Tyr145 and Phe147) via cation-pi interactions and by backbone carbonyl groups (Thr142 and Gly144).
我们最初基于电压门控钾通道Kv1.3孔区的现有分子模型，构建了内向整流钾通道Kir2.1的成孔H5（或P）区的结构模型。通过半胱氨酸扫描诱变及随后的银离子阻断，确定H5中侧链位于离子传导途径的残基，以此来检验我们的模型。在高度保守的H5区域内的八个位置进行突变，得到了明显无功能的通道。构建仅在一个连接亚基中带有半胱氨酸替代的共价连接二聚体，挽救了其中六个突变体；构建仅在一个连接亚基中带有半胱氨酸替代的共价连接四聚体，挽救了另一个突变体。我们使用二聚体和四聚体的结果表明，残基Thr141、Thr142、Ile143、Tyr145、Phe147和Cys149可被外部施加的银离子（100 - 200 nM）接触到，因此它们的侧链位于通道孔内。我们得出结论，Kir孔的拓扑结构与Kv通道的拓扑结构相似，但并不相同。此外，分子模型表明，选择性可能由芳香族残基（Tyr145和Phe147）通过阳离子-π相互作用以及主链羰基（Thr142和Gly144）赋予。
