Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, Rochester, New York, USA.
Deparment of Neurology, School of Medicine and Dentistry, University of Rochester, Rochester, New York, USA.
Protein Sci. 2023 Nov;32(11):e4800. doi: 10.1002/pro.4800.
Acid-sensing ion channels (ASICs) are important players in detecting extracellular acidification throughout the brain and body. ASICs have large extracellular domains containing two regions replete with acidic residues: the acidic pocket, and the palm domain. In the resting state, the acidic pocket is in an expanded conformation but collapses in low pH conditions as the acidic side chains are neutralized. Thus, extracellular acidification has been hypothesized to collapse the acidic pocket that, in turn, ultimately drives channel activation. However, several observations run counter to this idea. To explore how collapse or mobility of the acidic pocket is linked to channel gating, we employed two distinct tools. First, we incorporated the photocrosslinkable noncanonical amino acids (ncAAs) 4-azido-L-phenylalanine (AzF) or 4-benzoyl-L-phenylalanine (BzF) into several positions in the acidic pocket. At both E315 and Y318, AzF incorporation followed by UV irradiation led to right shifts in pH response curves and accelerations of desensitization and deactivation, consistent with restrictions of acidic pocket mobility destabilizing the open state. Second, we reasoned that because Cl ions are found in the open and desensitized structures but absent in the resting state structures, Cl substitution would provide insight into how stability of the pocket is linked to gating. Anion substitution resulted in faster deactivation and desensitization, consistent with the acidic pocket regulating the stability of the open state. Taken together, our data support a model where acidic pocket collapse is not essential for channel activation. Rather, collapse of the acidic pocket influences the stability of the open state of the pore.
酸敏离子通道 (ASICs) 在检测脑和全身细胞外酸化方面起着重要作用。ASICs 具有包含两个富含酸性残基区域的大细胞外结构域：酸性口袋和手掌域。在静息状态下，酸性口袋呈扩张构象，但在低 pH 条件下，由于酸性侧链被中和，酸性口袋会塌陷。因此，人们假设细胞外酸化会使酸性口袋塌陷，进而最终导致通道激活。然而，有几个观察结果与这一观点相悖。为了探究酸性口袋的塌陷或流动性与通道门控之间的关系，我们采用了两种不同的工具。首先，我们将光交联非典型氨基酸（ncAA）4-叠氮-L-苯丙氨酸（AzF）或 4-苯甲酰基-L-苯丙氨酸（BzF）引入酸性口袋的几个位置。在 E315 和 Y318 处，AzF 掺入后进行 UV 照射导致 pH 响应曲线右移，脱敏和失活加速，这与酸性口袋流动性受限使开放状态不稳定一致。其次，我们推断，由于 Cl 离子存在于开放和脱敏结构中，但不存在于静息状态结构中，Cl 取代将为了解口袋稳定性与门控之间的关系提供线索。阴离子取代导致失活和脱敏加快，这与酸性口袋调节开放状态的稳定性一致。总之，我们的数据支持这样一种模型，即酸性口袋塌陷对于通道激活不是必需的。相反，酸性口袋的塌陷会影响孔的开放状态的稳定性。
