Determinants of inhibition of transiently expressed voltage-gated calcium channels by ω-conotoxins GVIA and MVIIA
Determinants of inhibition of transiently expressed voltage-gated calcium channels by ω-conotoxins GVIA and MVIIA
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DOI：
10.1074/jbc.m300581200
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发表时间：
2003-05-30
影响因子：
4.8
通讯作者：
Zamponi, GW
中科院分区：
文献类型：
--
作者：
Feng, ZP;Doering, CJ;Zamponi, GW
Conus magus肽毒素- omega- concontoxin MVIIA被认为是一种不可逆的n型钙通道特异性阻滞剂，目前正在临床试验中作为鞘内镇痛药。在这里，我们研究了MVIIA对tsA-201细胞中瞬时表达的突变型和野生型钙通道的作用。虽然我们之前已经表明，在域IIIS5-H5区域假定的外部EF-hand基模的突变会改变omega- concontoxin GVIA和MVIIA的阻断(Feng， Z.P, Hamid， J., Doering， C., Bosey， G.M, Snutch， T.P, and Zamponi， G.W. (2001) J. Biol。化学，276,15728-15735)，与GVIA相比，引入已知影响GVIA阻断的五点突变（位于EF-hand下游）对MVIIA阻断的影响程度较小。这些数据表明，尽管有一些重叠，MVIIA和GVIA块并不共享相同的通道结构决定因素。在较高浓度（类似于3mum）下，MVIIA可逆地阻断L-、P/Q-和r型通道，但不阻断t型通道，这表明MVIIA位点的整体结构在所有类型的高压激活钙通道中都是保守的。MVIIA对n型通道影响的动力学分析表明，MVIIA阻断了静息通道、开放通道和失活通道。虽然MVIIA阻滞的发展似乎不依赖于电压，也不依赖于频率，但阻滞的恢复程度很大程度上取决于冲洗期间施加的电位。有趣的是，冲洗的程度是高度可变的，并且似乎弱依赖于毒素施用期间施加的保持电位。我们提出了一个n型钙通道可以与MVIIA形成可逆和不可逆配合物的模型。
The Conus magus peptide toxin omega-conotoxin MVIIA is considered an irreversible, specific blocker of N-type calcium channels, and is now in clinical trials as an intrathecal analgesic. Here, we have examined the action of MVIIA on mutant and wild type calcium channels transiently expressed in tsA-201 cells. Although we have shown previously that mutations in a putative external EF-hand motif in the domain IIIS5-H5 region alters block by both omega-conotoxin GVIA and MVIIA (Feng, Z.P., Hamid, J., Doering, C., Bosey, G.M., Snutch, T.P., and Zamponi, G.W. (2001) J. Biol. Chem. 276, 15728-15735), the introduction of five point mutations known to affect GVIA blocking ( and located downstream of the EF-hand) affected MVIIA block to a smaller degree compared with GVIA. These data suggest that despite some overlap, MVIIA and GVIA block does not share identical channel structural determinants. At higher concentrations (similar to3 muM), MVIIA reversibly blocked L-, P/Q-, and R-type, but not T-type channels, indicating that the overall architecture of the MVIIA site is conserved in all types of high voltage-activated calcium channels. A kinetic analysis of the MVIIA effects on the N-type channel showed that MVIIA blocked resting, open, and inactivated channels. Although the development of MVIIA block did not appear to be voltage-, nor frequency-dependent, the degree of recovery from block strongly depended on the potential applied during washout. Interestingly, the degree of washout was highly variable and appeared to weakly depend on the holding potential applied during toxin application. We propose a model in which N-type calcium channels can form both reversible and irreversible complexes with MVIIA.