SUR-dependent modulation of KATP channels by an N-terminal KIR6.2 peptide -: Defining intersubunit gating interactions
SUR-dependent modulation of KATP channels by an N-terminal KIR6.2 peptide -: Defining intersubunit gating interactions
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DOI：
10.1074/jbc.m208085200
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发表时间：
2002-11-15
影响因子：
4.8
通讯作者：
Bryan, J
中科院分区：
文献类型：
--
作者：
Babenko, AP;Bryan, J
Ntp和Ctp分别是基于K(IR)6.0的N端和c端序列合成的肽，用于探测K(IR)6.0/SUR K- atp通道的门控。微摩尔Ntp剂量依赖性地增加了无配体溶液中平均开放通道概率（P-O(max)），减弱了K(IR)6.2/SUR1的ATP抑制，但对同质K(IR)6.2通道没有影响。Ntp（高达10(-4)M）没有显著影响平均打开或“快速”，K+驱动力依赖，突发内关闭时间，验证Ntp有选择性地调节平均突发与突发间时间的比率。随机Ntp的Ctp和Rnp没有作用，说明Ntp的作用是结构特异性的。Ntp打开了K(IR)6.1/SUR1通道，通常在没有刺激Mg-核苷酸的情况下沉默，并减弱了高亲和力磺酰脲结合与KATp孔闭合的偶联。这些效应类似于K(IR)6.0的n端缺失（DeltaN）所见，并且在没有Mg2+的情况下，Ntp应用于DeltaNK（ATP）通道降低了它们的P-O（max）和表观IC50。结果与Ntp和内源性N端在通道细胞质面相互作用位点上的竞争或Ntp对缺失的N端的部分替代相一致。已知SUR1的K-IR N端和TMD0-L0片段控制P-O（max）。据报道，L0连接体是格列本脲结合所必需的，DeltaNK(IR)6.2/SUR1通道显示i -125-叠氮格列本脲对KIR的标记减少，这意味着KIR N端和SUR1的L0很接近。我们假设L0在配体抑制的K-ATP通道中与KIR N端相互作用，并基于BtuCD、MsbA和KcsA通道的结构提出了一个模型，其中TMD0-L0将SUR的mdr样核心与K-IR孔连接起来。
Ntp and Ctp, synthetic peptides based on the N- and C-terminal sequences of K(IR)6.0, respectively, were used to probe gating of K(IR)6.0/SUR K-ATP channels. Micromolar Ntp dose-dependently increased the mean open channel probability in ligand-free solution (P-O(max)) and attenuated the ATP inhibition of K(IR)6.2/SUR1, but had no effect on homomeric K(IR)6.2 channels. Ntp (up to similar to10(-4) M) did not affect significantly the mean open or "fast," K+ driving force-dependent, intraburst closed times, verifying that Ntp selectively modulates the ratio of mean burst to interburst times. Ctp and Rnp, a randomized Ntp, had no effect, indicating that the effects of Ntp are structure specific. Ntp opened K(IR)6.1/SUR1 channels normally silent in the absence of stimulatory Mg- nucleotide(s) and attenuated the coupling of high-affinity sulfonylurea binding with KATp pore closure. These effects resemble those seen with N-terminal deletions (DeltaN) of K(IR)6.0, and application of Ntp to DeltaNK(ATP) channels decreased their P-O(max) and apparent IC50 for ATP in the absence of Mg2+. The results are consistent with a competition between Ntp and the endogenous N terminus for a site of interaction on the cytoplasmic face of the channel or with partial replacement of the deleted N terminus by Ntp, respectively. The K-IR N terminus and the TMD0-L0 segment of SUR1 are known to control the P-O(max). The L0 linker has been reported to be required for glibenclamide binding, and DeltaNK(IR)6.2/SUR1 channels exhibit reduced labeling of KIR with I-125-azidoglibenclamide, implying that the KIR N terminus and L0 of SUR1 are in proximity. We hypothesize that L0 interacts with the KIR N terminus in ligand-inhibited K-ATP channels and put forward a model, based on the architecture of BtuCD, MsbA, and the KcsA channel, in which TMD0-L0 links the MDR-like core of SUR with the K-IR pore.