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.
Highlights
Plasma membrane expression is vital for the function of Acid-sensing ion channels (ASICs), which act as extracellular proton sensors
We expanded mutagenesis to adjacent residues, namely Thr104 and Gln149 in rat ASIC3 (rASIC3), which may play a role in stabilizing the conformation of the salt bridge via hydrogen bonding
These analyses suggest that the conserved salt bridge located at the extracellular loop is stringently precise, and it is crucial for the normal surface expression of ASIC3 channels
Summary
Plasma membrane expression is vital for the function of ASICs, which act as extracellular proton sensors. Conclusion: Surface ASIC expression involves an exposed rigid signal patch at the extracellular loop. We examined the role of a highly conserved salt bridge residing at the extracellular loop of rat ASIC3 (Asp107-Arg153) and human ASIC1a (Asp107-Arg160) channels. 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. Using a diversity of approaches, including comprehensive mutagenesis, electrophysiological recording, charge swapping, cysteine cross-linking, Western blotting, immunocytochemistry, molecular dynamics (MD) simulations, normal mode analysis (NMA), and protein flexibility analysis, we demonstrate that this highly conserved extracellular salt bridge, together with its adjacent residues, which form a rigid signal patch, plays an essential role in normal cell surface expression of ASIC channels
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