PDF(Pubmed)
Abstract：
Desensitization is a prominent feature of nearly all ligand gated ion channels. Acid-sensing ion channels (ASIC) 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 beta 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. 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 tools. 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 pHs while activation curves and ion selectivity of these slow-desensitizing currents were largely unaffected. 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 mutations only slightly attenuated desensitization, suggesting that conformational changes in the 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 a single subunit is sufficient to desensitize the entire channel.
摘要：
脱敏是几乎所有配体门控离子通道的突出特征。酸敏感离子通道(ASIC)在数百毫秒到数秒内经历持续的细胞外酸化。脱敏的ASIC机制主要是由于连接胞外域中第11和第12个β折叠的短接头的异构化或“翻转”。在静息和活跃状态下，该β11-12接头采用“向上”构象，而在脱敏构象下，接头呈“向下”状态。为了适应这种“向下”状态，接头内的特异性肽键采用反式样或顺式样构象。由于含脯氨酸的肽键进行顺反异构非常缓慢，我们假设在接头中引入脯氨酸残基可能会减缓甚至取消ASIC脱敏，可能提供有价值的研究工具。鸡ASIC1β11-12接头（L414P和Y416P）中的脯氨酸取代减缓了脱敏衰减约100至1000倍，如在切下的贴片中测量。L414P和Y416P均将稳态脱敏曲线移至更酸性的pH，而这些缓慢脱敏电流的激活曲线和离子选择性在很大程度上不受影响。为了研究三聚体ASIC中脱敏的功能化学计量学，我们用零创建了L414P和Y416P串联家族，一,在所有可能的构型中有两个或三个脯氨酸取代。引入一个或两个L414P或Y416P突变仅轻微减弱脱敏，表明其余较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机制，并支持了单个亚基足以使整个通道脱敏的模型。
