Desensitization is a prominent feature of nearly all ligand-gated ion channels. Acid-sensing ion channels (ASICs) undergo desensitization within hundreds of milliseconds to seconds upon continual extracellular acidification. The ASIC mechanism of desensitization is primarily due to the isomerization or “flipping” of a short linker joining the 11th and 12th β sheets in the extracellular domain. In the resting and active states this β11-12 linker adopts an “upward” conformation while in the desensitized conformation the linker assumes a “downward” state. It is unclear if a single linker adopting the downward state is sufficient to desensitize the entire channel, or if all three are needed or some more complex scheme. To accommodate this downward state, specific peptide bonds within the linker adopt either trans-like or cis-like conformations. Since proline-containing peptide bonds undergo cis-trans isomerization very slowly, we hypothesized that introducing proline residues in the linker may slow or even abolish ASIC desensitization, potentially providing a valuable research tool. Proline substitutions in the chicken ASIC1 β11-12 linker (L414P and Y416P) slowed desensitization decays approximately 100- to 1000-fold as measured in excised patches. Both L414P and Y416P shifted the steady-state desensitization curves to more acidic pH values while activation curves and ion selectivity were largely unaffected (except for a left-shifted activation pH50 of L414P). To investigate the functional stoichiometry of desensitization in the trimeric ASIC, we created families of L414P and Y416P concatemers with zero, one, two, or three proline substitutions in all possible configurations. Introducing one or two L414P or Y416P substitutions only slightly attenuated desensitization, suggesting that conformational changes in the single remaining faster wild-type subunits were sufficient to desensitize the channel. These data highlight the unusual cis-trans isomerization mechanism of ASIC desensitization and support a model where ASIC desensitization requires only a single subunit.

中文翻译：

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ASIC1 β11-12 接头中的脯氨酸取代会减慢脱敏速度


脱敏是几乎所有配体门控离子通道的突出特征。酸感应离子通道 （ASIC） 在持续的细胞外酸化后，在数百毫秒到几秒钟内发生脱敏。ASIC 脱敏机制主要是由于短接头的异构化或“翻转”，该接头连接细胞外结构域中的第 11 个和第 12 个β折叠。在静止和活跃状态下，该 β11-12 接头采用“向上”构象，而在脱敏构象中，接头呈“向下”状态。目前尚不清楚采用 DOWNWARD 状态的单个 linker 是否足以使整个 channel 脱敏，或者是否需要所有这三个或更复杂的方案。为了适应这种向下状态，接头内的特异性肽键采用反式或顺式样构象。由于含脯氨酸的肽键经历顺反异构化非常缓慢，我们假设在接头中引入脯氨酸残基可能会减慢甚至消除 ASIC 脱敏，从而可能提供有价值的研究工具。鸡 ASIC1 β11-12 接头（L414P 和 Y416P）中的脯氨酸取代减慢了脱敏衰减，以切除的贴剂测量，衰减约为 100 至 1000 倍。L414P 和 Y416P 都将稳态脱敏曲线转变为更酸性的 pH 值，而活化曲线和离子选择性基本不受影响（除了 L414P 的左移活化 pH50）。为了研究三聚体 ASIC 中脱敏的功能性化学计量，我们创建了 L414P 和 Y416P 连接体家族，在所有可能的配置中具有零、一个、两个或三个脯氨酸取代。 引入一个或两个 L414P 或 Y416P 替换仅略微减弱了脱敏作用，表明单个剩余的更快野生型亚基的构象变化足以使通道脱敏。这些数据突出了 ASIC 脱敏的不寻常的顺反异构化机制，并支持 ASIC 脱敏只需要一个亚基的模型。