Feng Z P, Hamid J, Doering C, Bosey G M, Snutch T P, Zamponi G W
Departments of Physiology & Biophysics and Pharmacology & Therapeutics, Neuroscience Research Group, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
J Biol Chem. 2001 May 11;276(19):15728-35. doi: 10.1074/jbc.M100406200. Epub 2001 Feb 2.
We recently reported that amino acid residues contained within a putative EF hand motif in the domain III S5-H5 region of the alpha(1B) subunit affected the relative barium:calcium permeability of N-type calcium channels (Feng, Z. P., Hamid, J., Doering, C., Jarvis, S. E., Bosey, G. M., Bourinet, E., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 5726-5730). Since this region partially overlaps with residues previously implicated in block of the channel by omega-conotoxin GVIA, we assessed the effects of mutations in the putative EF hand domain on channel block by omega-conotoxin GVIA and the structurally related omega-conotoxin MVIIA. Both of the toxins irreversibly block the activity of wild type alpha(1B) N-type channels. We find that in addition to previously identified amino acid residues, residues in positions 1326 and 1332 are important determinants of omega-conotoxin GVIA blockade. Substitution of residue Glu(1332) to arginine slows the time course of development of block. Point mutations in position Gly(1326) to either arginine, glutamic acid, or proline dramatically decrease the time constant for development of the block. Additionally, in the G1326P mutant channel activity was almost completely recovered following washout. A qualitatively similar result was obtained with omega-conotoxin MVIIA, suggesting that common molecular determinants underlie block by these two toxins. Taken together the data suggest that residue Gly(1326) may form a barrier, which controls the access of peptide toxins to their blocking site within the outer vestibule of the channel pore and also stabilizes the toxin-channel interaction.
我们最近报道，α(1B)亚基结构域III的S5-H5区域中一个假定的EF手基序内所含的氨基酸残基，影响了N型钙通道的相对钡：钙通透性（冯，Z.P.，哈米德，J.，多林，C.，贾维斯，S.E.，博西，G.M.，布里内，E.，斯纳奇，T.P.，和赞波尼，G.W.（2001年）《生物化学杂志》276，5726 - 5730）。由于该区域与先前认为参与ω-芋螺毒素GVIA对通道阻断的残基部分重叠，我们评估了假定的EF手结构域中的突变对ω-芋螺毒素GVIA以及结构相关的ω-芋螺毒素MVIIA阻断通道的影响。这两种毒素都不可逆地阻断野生型α(1B) N型通道的活性。我们发现，除了先前鉴定出的氨基酸残基外，1326位和1332位的残基是ω-芋螺毒素GVIA阻断的重要决定因素。将残基Glu(1332)替换为精氨酸会减慢阻断发展的时间进程。1326位的甘氨酸突变为精氨酸、谷氨酸或脯氨酸会显著降低阻断发展的时间常数。此外，在G1326P突变体中，洗脱后通道活性几乎完全恢复。用ω-芋螺毒素MVIIA获得了定性相似的结果，表明这两种毒素的阻断存在共同的分子决定因素。综合这些数据表明，残基Gly(1326)可能形成一个屏障，它控制肽毒素进入通道孔外前庭内的阻断位点，并稳定毒素 - 通道相互作用。
