title: Highly conserved salt bridge stabilizes rigid signal patch at extracellular loop critical for surface expression of acid-sensing ion channels.
abstract: Acid-sensing ion channels (ASICs) are non-selective cation channels activated by extracellular acidosis associated with many physiological and pathological conditions. A detailed understanding of the mechanisms that govern cell surface expression of ASICs, therefore, is critical for better understanding of the cell signaling under acidosis conditions. In this study, we examined the role of a highly conserved salt bridge residing at the extracellular loop of rat ASIC3 (Asp(107)-Arg(153)) and human ASIC1a (Asp(107)-Arg(160)) channels. Comprehensive mutagenesis and electrophysiological recordings revealed that the salt bridge is essential for functional expression of ASICs in a pH sensing-independent manner. Surface biotinylation and immunolabeling of an extracellular epitope indicated that mutations, including even minor alterations, at the salt bridge impaired cell surface expression of ASICs. Molecular dynamics simulations, normal mode analysis, and further mutagenesis studies suggested a high stability and structural constrain of the salt bridge, which serves to separate an adjacent structurally rigid signal patch, important for surface expression, from a flexible gating domain. Thus, we provide the first evidence of structural requirement that involves a stabilizing salt bridge and an exposed rigid signal patch at the destined extracellular loop for normal surface expression of ASICs. These findings will allow evaluation of new strategies aimed at preventing excessive excitability and neuronal injury associated with tissue acidosis and ASIC activation.
authors.affiliation: Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.forename: Ye
authors.initials: Y
authors.lastname: Yu
authors.forename: Jin
authors.initials: J
authors.lastname: Cheng
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.forename: Di-Shi
authors.initials: DS
authors.lastname: Liu
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
authors.forename: Michael X
authors.initials: MX
authors.lastname: Zhu
authors.forename: Rui
authors.initials: R
authors.lastname: Wang
authors.forename: Tian-Le
authors.initials: TL
authors.lastname: Xu
mesh_terms: D062565:Acid Sensing Ion Channels; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary; D051381:Rats; D015222:Sodium Channels / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
chemical_list: C568266:ASIC1 protein, human; C112231:ASIC3 protein, human; C568269:ASIC3 protein, rat; D062565:Acid Sensing Ion Channels; D009419:Nerve Tissue Proteins; D015222:Sodium Channels