Pseudechetoxin binds to the pore turret of cyclic nucleotide-gated ion channels.
Pseudechetoxin binds to the pore turret of cyclic nucleotide-gated ion channels.
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DOI：
10.1085/jgp.200308823
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发表时间：
2003-12
期刊：
影响因子：
--
通讯作者：
Arsanjani R
中科院分区：
文献类型：
--
作者：
Brown RL;Lynch LL;Haley TL;Arsanjani R
肽毒素是研究离子通道结构和生理学的宝贵工具。伪蝎毒素（Pseudechetoxin，PsTx）是第一个以环核苷酸门控离子通道（cyclicnucleotide-gatedionchannels，CNG）为靶点的肽类毒素，CNG通道在视觉和嗅觉系统的感觉传导中起重要作用。PsTx抑制通道电流在低nM浓度时，适用于细胞外的膜补丁表达嗅觉CNGA 2亚基。令人惊讶的是，500 nM PsTx不抑制通过由来自视锥光感受器的CNGA 3亚基形成的通道的电流。我们已经利用这种差异来确定CNG通道的细胞外表面上的PsTx结合位点。使用嵌合通道的研究揭示，来自CNGA 2的孔结构域的移植足以赋予CNGA 3背景上的高亲和力PsTx结合。为了进一步定义结合位点，在该区域的10个不相同的氨基酸残基处进行了相互突变。我们发现CNGA 2中的两个残基D316和Y321对于PsTx的高亲和力抑制是必需的。此外，需要替换两个残基以赋予CNGA 3高亲和力PsTx抑制。其他几个残基，包括E325，也与PsTx形成有利的相互作用。在CNGA 2-E325 K突变体中，PsTx亲和力降低了105倍至120 nM。与D316的静电相互作用似乎不是PsTx亲和力的主要决定因素，因为用带负电荷的甲硫基磺酸盐试剂修饰D316 C突变体不能恢复高亲和力抑制。参与PsTx结合的残基在孔转塔和螺旋内发现，在与形成电压门控钾通道中孔阻断毒素的受体的残基相似的位置。此外，PsTx块的生物物理性质，包括与渗透离子的不利相互作用，也表明它充当孔阻断剂。总之，PsTx似乎通过与孔转塔形成高亲和力接触来堵塞孔的入口，所述高亲和力接触可能大于在KcsA结构中发现的接触。
Peptide toxins are invaluable tools for studying the structure and physiology of ion channels. Pseudechetoxin (PsTx) is the first known peptide toxin that targets cyclic nucleotide–gated (CNG) ion channels, which play a critical role in sensory transduction in the visual and olfactory systems. PsTx inhibited channel currents at low nM concentrations when applied to the extracellular face of membrane patches expressing olfactory CNGA2 subunits. Surprisingly, 500 nM PsTx did not inhibit currents through channels formed by the CNGA3 subunit from cone photoreceptors. We have exploited this difference to identify the PsTx-binding site on the extracellular face of CNG channels. Studies using chimeric channels revealed that transplantation of the pore domain from CNGA2 was sufficient to confer high affinity PsTx binding upon a CNGA3 background. To further define the binding site, reciprocal mutations were made at 10 nonidentical amino acid residues in this region. We found that two residues in CNGA2, D316 and Y321, were essential for high-affinity inhibition by PsTx. Furthermore, replacement of both residues was required to confer high-affinity PsTx inhibition upon CNGA3. Several other residues, including E325, also form favorable interactions with PsTx. In the CNGA2-E325K mutant, PsTx affinity was reduced by ∼5-fold to 120 nM. An electrostatic interaction with D316 does not appear to be the primary determinant of PsTx affinity, as modification of the D316C mutant with a negatively charged methanethiosulfonate reagent did not restore high affinity inhibition. The residues involved in PsTx binding are found within the pore turret and helix, in similar positions to residues that form the receptor for pore-blocking toxins in voltage-gated potassium channels. Furthermore, biophysical properties of PsTx block, including an unfavorable interaction with permeant ions, also suggest that it acts as a pore blocker. In summary, PsTx seems to occlude the entrance to the pore by forming high-affinity contacts with the pore turret, which may be larger than that found in the KcsA structure.
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影响因子：
56.9
作者：
Doyle, DA;Cabral, JM;MacKinnon, R
通讯作者：
MacKinnon, R
DOI：
10.1073/pnas.91.19.8890
发表时间：
1994-09-13
影响因子：
11.1
作者：
BRADLEY, J;LI, J;ZINN, K
通讯作者：
ZINN, K
影响因子：
56.9
作者：
BONTEMS, F;ROUMESTAND, C;TOMA, F
通讯作者：
TOMA, F
影响因子：
16.2
作者：
KORSCHEN, HG;ILLING, M;MOLDAY, RS
通讯作者：
MOLDAY, RS
影响因子：
56.9
作者：
HIDALGO, P;MACKINNON, R
通讯作者：
MACKINNON, R