Movement of the C-helix during the gating of cyclic nucleotide-gated channels
Movement of the C-helix during the gating of cyclic nucleotide-gated channels
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DOI：
10.1016/s0006-3495(02)75329-0
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发表时间：
2002-12-01
影响因子：
3.4
通讯作者：
Torre, V
中科院分区：
文献类型：
--
作者：
Mazzolini, M;Punta, M;Torre, V
环核苷酸门控通道的环核苷酸结合结构域内的运动被认为是通道门控的初始阶段的基础（Tibbs，G. R.，D. T.刘，B。G. Leypold和S. A.西格尔鲍姆1998. J.Biol.Chem.273：4497-4505; Zong，X.，H. Zucker，F. Hofmann和M.比尔1998. EMBO J. 17：353-362; Matulef，K.，G. E. Flynn和W. N.扎戈塔1999. Neuron. 24：443-452; Paoletti，P.，E. C. Young和S. A.西格尔鲍姆1999. J.Gen.Physiol.113：17-33;约翰逊，J. P.，和W. N.扎戈塔2001.自然412：917-921）。为了研究这些运动，在28个残基（Leu-583至Asn-610）中的每一个上进行半胱氨酸突变，所述残基跨越牛杆状环核苷酸门控通道的α亚基的激动剂结合结构域。镉离子，2-trimethylammonioethylmethane thiosulfonate（MTSET）和铜菲咯啉（CuP）对通道活性的影响进行了检查，在切除的内面向外补丁的存在和不存在的饱和浓度的cGMP。应用100 μ M的Cd ~（2+）在饱和浓度的cGMP的存在下，引起突变通道E594 C，1600 C和L 601 C的电流不可逆的和几乎完全的减少。在没有cGMP的情况下，100 μ M Cd ~（2+）的存在导致从Asp-588到Leu-607的所有半胱氨酸突变体的强电流降低，除了突变体通道A589 C、M592 C、M602 C、K603 C和L 606 C。Cd 2+离子的选择性效应与向槽液中加入氧化剂CuP时观察到的非常相似，除了突变体通道G597 C，其中CuP引起的电流降低（67 +/- 7%）比Cd 2+（23 +/- 4%）更强。在cGMP不存在的情况下，MTSET导致突变体通道L 607 C、L 601 C、1600 C、G597 C和E594 C中的电流降低>40%，而在cGMP存在的情况下，仅突变体通道L 601 C受到影响。MTSET的应用保护了许多突变通道免受Cd 2+和CuP的影响。这些结果表明，当CNG通道处于开放状态时，从Asp-588到Leu-607的残基处于α-螺旋结构，与分解代谢物基因激活蛋白的C-螺旋同源（Weber，I. T.，和T. A.史泰茨1987. J. Mol. 198：311-326）。此外，属于两个不同亚基的这些螺旋的残基Glu-594、Gly-597、Ile-600和Leu-601必须非常接近。在闭合状态下，C-螺旋处于不同的构型，并经历显著的波动。
Movements within the cyclic nucleotide-binding domain of cyclic nucleotide-gated channels are thought to underlie the initial phase of channel gating (Tibbs, G. R., D. T. Liu, B. G. Leypold, and S. A. Siegelbaum. 1998. J. Biol. Chem. 273:4497-4505; Zong, X., H. Zucker, F. Hofmann, and M. Biel. 1998. EMBO J. 17:353-362; Matulef, K., G. E. Flynn, and W. N. Zagotta. 1999. Neuron. 24:443-452; Paoletti, P., E. C. Young, and S. A. Siegelbaum. 1999. J. Gen. Physiol. 113:17-33; Johnson, J. P., and W. N. Zagotta. 2001. Nature. 412:917-921). To investigate these movements, cysteine mutation was performed on each of the 28 residues (Leu-583 to Asn-610), which span the agonist-binding domain of the alpha-subunit of the bovine rod cyclic nucleotide-gated channel. The effects of Cd2+ ions, 2-trimethylammonioethylmethane thiosulfonate (MTSET) and copper phenanthroline (CuP) on channel activity were examined, in excised inside-out patches in the presence and in the absence of a saturating concentration of cGMP. The application of 100 muM Cd2+ in the presence of saturating concentration of cGMP caused an irreversible and almost complete reduction of the current in mutant channels E594C, 1600C, and L601C. In the absence of cGMP, the presence of 100 muM Cd2+ caused a strong current reduction in all cysteine mutants from Asp-588 to Leu-607, with the exception of mutant channels A589C, M592C, M602C, K603C, and L606C. The selective effect of Cd2+ ions was very similar to that observed when adding the oxidizing agent CuP to the bath medium, except for mutant channel G597C, where CuP caused a stronger current decrease (67 +/- 7%) than Cd2+ (23 +/- 4%). In the absence of cGMP, MTSET caused a reduction of the current by >40% in mutant channels L607C, L601C, 1600C, G597C, and E594C, whereas in the presence of cGMP only mutant channel L601C was affected. The application of MTSET protected many mutant channels from the effects of Cd2+ and CuP. These results suggest that, when CNG channels are in the open state, residues from Asp-588 to Leu-607 are in an a-helical structure, homologous to the C-helix of the catabolite gene activator protein (Weber, I. T., and T. A. Steitz. 1987. J. Mol. Biol. 198:311-326). Furthermore, residues Glu-594, Gly-597, Ile-600, and Leu-601 of these helices belonging to two different subunits must be in close proximity. In the closed state the C-helices are in a different configuration and undergo significant fluctuations.