The Relationship between Functional Inhibition and Binding for KCa2 Channel Blockers
The Relationship between Functional Inhibition and Binding for KCa2 Channel Blockers
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DOI：
10.1371/journal.pone.0073328
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发表时间：
2013-09-10
期刊：
影响因子：
3.7
通讯作者：
Moss, Guy William John
中科院分区：
文献类型：
--
作者：
Benton, David Charles Hammond;Garbarg, Monique;Moss, Guy William John
小电导钙激活钾通道(K(Ca)2.1、2.2、2.3)可被多肽毒素(如阿帕明)和小分子阻滞剂(如UCL 1848)高亲和力阻断。在电生理实验中，阿帕明具有亚型选择性，K(Ca)2.2和K(Ca)2.3的IC(50)S分别接近100 PM和1 nM。然而，在结合研究中，apamin似乎不区分K(Ca)2.2和2.3，据报道具有显著更高的亲和力(类似于20-200倍)(类似于下午5点)。这种结合和阻断之间的差异被认为反映了阿帕明的一种不寻常的作用模式。然而，这些结合和电生理阻断实验并不是在相同的离子条件下进行的，因此也可能仅仅因为实验条件的不同而出现差异。我们现在已经研究了后一种可能性。因此，我们在相同的离子条件(即正常生理条件)下测量了I-125-apamin与表达K(Ca)2通道的完整HEK 293细胞的结合。我们发现，如果使用相同的离子条件，结合实验和嵌段实验很好地吻合。此外，在正常生理溶液中，阿帕明与其他阻滞剂的结合表现出亚型选择性(例如，I-125apamin与K(Ca)2.2与K-L 91+/-40 pm结合，与K(Ca)2.3与K-L 711+/-126 pm结合，但在IC50103+/-2 pm时抑制K(Ca)2.2电流)。我们还研究了无钙和无镁溶液中的K(Ca)2通道阻滞剂，模拟文献报道的结合实验条件。在这些(非生理)条件下，K(Ca)2.2阿帕明阻断的IC50降至20+/-3 pm因此，我们的结果表明，文献中报道的封闭和结合之间的明显差异可以在很大程度上归因于在结合实验中使用非生理离子条件。
Small conductance calcium-activated potassium channels (K(Ca)2.1,2.2,2.3) are blocked with high affinity by both peptide toxins (e. g. apamin) and small molecule blockers (e. g. UCL 1848). In electrophysiological experiments, apamin shows subtype selectivity with IC(50)s of similar to 100 pM and similar to 1 nM for block K(Ca)2.2 and K(Ca)2.3 respectively. In binding studies, however, apamin appears not to discriminate between K(Ca)2.2 and 2.3 and is reported to have a significantly higher (similar to 20-200-fold) affinity (similar to 5 pM). This discrepancy between binding and block has been suggested to reflect an unusual mode of action of apamin. However, these binding and electrophysiological block experiments have not been conducted in the same ionic conditions, so it is also possible that the discrepancy arises simply because of differences in experimental conditions. We have now examined this latter possibility. Thus, we measured I-125-apamin binding to intact HEK 293 cells expressing K(Ca)2 channels under the same ionic conditions (i.e. normal physiological conditions) that we also used for current block measurements. We find that binding and block experiments agree well if the same ionic conditions are used. Further, the binding of apamin and other blockers showed subtype selectivity when measured in normal physiological solutions (e. g. I-125-apamin bound to K(Ca)2.2 with K-L 91 +/- 40 pM and to K(Ca)2.3 with K-L 711 +/- 126 pM, while inhibiting K(Ca)2.2 current at IC50 103 +/- 2 pM). We also examined K(Ca)2 channel block in Ca2+ and Mg2+ free solutions that mimic conditions reported in the literature for binding experiments. Under these (non-physiological) conditions the IC50 for apamin block of K(Ca)2.2 was reduced to 20 +/- 3 pM. Our results therefore suggest that the apparent discrepancy between blocking and binding reported in the literature can be largely accounted for by the use of non-physiological ionic conditions in binding experiments.