Barium (Ba²⁺) is a classic permeant blocker of potassium (K⁺) channels. The “external lock-in effect” in barium block experiments, whereby the binding of external K⁺ impedes the forward translocation of the blocker, provides a powerful avenue to investigate the selectivity of the binding sites along the pore of potassium channels. Barium block experiments show that the external lock-in site is highly selective for K⁺ over Na⁺. Wild-type KcsA was crystallized in low K⁺ conditions, and the crystals were soaked in solutions containing various concentrations of barium. Structural analysis reveals open and closed gate conformations of the KcsA channel. Anomalous diffraction experiments show that Ba²⁺ primarily binds to the innermost site S4 of the selectivity filter of the open-gate conformation and also the site S2, but no binding is detected with the closed-gate conformation. Alchemical free-energy perturbation calculations indicate that the presence of a Ba²⁺ ion in the selectivity filter boosts the specificity of K⁺ binding relative to Na⁺ in the external sites S0–S2.

钡 (Ba ²⁺ ) 是钾 (K ⁺ ) 通道的经典渗透阻滞剂。钡阻滞实验中的“外部锁定效应”，即外部 K ⁺的结合阻碍了阻滞剂的正向易位，为研究沿钾通道孔的结合位点的选择性提供了有力的途径。钡块实验表明，与Na ^{+ 相比}，外部锁定位点对 K ⁺具有高度选择性。野生型 KcsA 在低 K ^{+ 中}结晶条件，并将晶体浸泡在含有不同浓度钡的溶液中。结构分析揭示了 KcsA 通道的开放和封闭门构象。异常衍射实验表明，Ba ²⁺主要与开门构象的选择性过滤器的最内位点S4以及位点S2结合，但未检测到与闭门构象的结合。炼金术自由能扰动计算表明，选择性过滤器中Ba ²⁺离子的存在提高了 K ⁺相对于外部位点 S0–S2 中Na ⁺的特异性。