Large diameter of palytoxin-induced Na/K pump channels and modulation of palytoxin interaction by Na/K pump ligands
Large diameter of palytoxin-induced Na/K pump channels and modulation of palytoxin interaction by Na/K pump ligands
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DOI：
10.1085/jgp.200308964
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发表时间：
2004-04-01
影响因子：
3.8
通讯作者：
Gadsby, DC
中科院分区：
文献类型：
--
作者：
Artigas, P;Gadsby, DC
海葵毒素结合Na/K泵以产生非选择性阳离子通道，其孔可能包含泵的离子易位途径的至少一部分。我们系统地分析了在广泛的离子和核苷酸条件下，在HEK 293细胞的外向补丁中，在有或没有强心类固醇的情况下，海葵毒素与天然人Na/K泵的相互作用。用5 mM内部（移液管）[MgATP]，沙冬青毒素激活电导，具有表观亲和力，对含Na+（无K+）的外部和内部溶液最高，对含K+（无K+）的外部和内部溶液最低。（无Na+）外部和内部溶液，以及混合外部Na+/内部K+和外部K+/内部Na+条件的中间体;在Na+溶液和MgATP的情况下，沙冬青毒素在Na/K泵上的平均停留时间约为1天。用Na+溶液，在用无核苷酸移液器溶液平衡贴片后，对沙冬青毒素作用的表观亲和力较低。当平衡[MgATP]在亚微摩尔和亚毫摩尔范围内升高时，表观亲和力在两相中增加，但在亚毫摩尔范围内，通过移液器在单相中增加MgAMPPNP;饱和[MgAMPPNP]的表观亲和力保持类似于饱和[MgATP]的30倍。在沙冬青毒素洗脱后，反映沙冬青毒素解结合的电导衰减被强心类固醇加速。当Na/K泵与强心类固醇预孵育时，即使在强心类固醇洗脱后，随后的激活也大大减缓了泥盆草毒素诱导的电导，但激活仍然可以通过增加泥盆草毒素浓度来加速。这些结果表明，海葵毒素和强心类固醇可以同时占据正常的Na/K泵，使彼此不稳定。几种大的有机阳离子，包括N-甲基-D-葡糖胺（+），都能透过由泥盆纪毒素诱导的通道，这表明孔的最西面部分必须与7.5埃宽相似。增强的理解，泥藻毒素的行动，现在允许其用于检查的结构和机制的门，在正常的Na/K泵循环过程中，闭塞/deocolude运输离子。
Palytoxin binds to Na/K pumps to generate nonselective cation channels whose pore likely comprises at least part of the pump's ion translocation pathway. We systematically analyzed palytoxin's interactions with native human Na/K pumps in outside-out patches from HEK293 cells over a broad range of ionic and nucleotide conditions, and with or without cardiotonic steroids. With 5 mM internal (pipette) [MgATP], palytoxin activated the conductance with an apparent affinity that was highest for Na+-containing (K+-free) external and internal solutions, lowest for K+-containing (Na+-free) external and internal solutions, and intermediate for the mixed external Na+/internal K+, and external K+/internal Na+ conditions; with Na+ solutions and MgATP, the mean dwell time of palytoxin on the Na/K pump was about one day. With Na+ solutions, the apparent affinity for palytoxin action was low after equilibration of patches with nucleotide-free pipette solution. That apparent affinity was increased in two phases as the equilibrating [MgATP] was raised over the submicromolar, and submillimolar, ranges, but was increased by pipette MgAMPPNP in a single phase, over the submillimolar range; the apparent affinity at saturating [MgAMPPNP] remained similar to30-fold lower than at saturating [MgATP]. After palytoxin washout, the conductance decay that reflects palytoxin unbinding was accelerated by cardiotonic steroid. When Na/K pumps were preincubated with cardiotonic steroid, subsequent activation of palytoxin-induced conductance was greatly slowed, even after washout of the cardiotonic steroid, but activation could still be accelerated by increasing palytoxin concentration. These results indicate that palytoxin and a cardiotonic steroid can simultaneously occupy the sane Na/K pump, each destabilizing the other. The palytoxin-induced channels were permeable to several large organic cations, including N-methyl-d-glucamine(+), suggesting that the narrowest section of the pore must be similar to7.5 Angstrom wide. Enhanced understanding of palytoxin action now allows its use for examining the structures and mechanisms of the gates that occlude/deocclude transported ions during the normal Na/K pump cycle.