Protein semisynthesis underscores the role of a conserved lysine in activation and desensitization of acid-sensing ion channels
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Acid-sensing ion channels (ASICs) are trimeric ion channels that open a cation-conducting pore in response to proton binding. Excessive ASIC activation during prolonged acidosis in conditions such as inflammation and ischemia is linked to pain and stroke. A conserved lysine in the extracellular domain (Lys211 in mASIC1a) is suggested to play a key role in ASIC function. However, the precise contributions are difficult to dissect with conventional mutagenesis, as replacement of Lys211 with naturally occurring amino acids invariably changes multiple physico-chemical parameters. Here, we study the contribution of Lys211 to mASIC1a function using tandem protein trans-splicing (tPTS) to incorporate non-canonical lysine analogs. We conduct optimization efforts to improve splicing and functionally interrogate semisynthetic mASIC1a. In combination with molecular modeling, we show that Lys211 charge and side-chain length are crucial to activation and desensitization, thus emphasizing that tPTS can enable atomic-scale interrogations of membrane proteins in live cells.
Original language | English |
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Journal | Cell Chemical Biology |
ISSN | 2451-9456 |
DOIs | |
Publication status | Accepted/In press - 2024 |
Bibliographical note
Publisher Copyright:
© 2023 Elsevier Ltd
- acid-sensing ion channels, desensitization, homolysine, ligand-gated ion channels, native chemical ligation, non-canonical amino acids, ornithine, protein semisynthesis, protein trans-splicing, split inteins
Research areas
ID: 384876528