Protein semisynthesis underscores the role of a conserved lysine in activation and desensitization of acid-sensing ion channels

• Published in: Cell chemical biology Vol. 31; no. 5; p. 1000
• Main Authors: Sarkar, Debayan, Galleano, Iacopo, Heusser, Stephanie Andrea, Ou, Sofie Yuewei, Uzun, Gül Refika, Khoo, Keith K, van der Heden van Noort, Gerbrand Jan, Harrison, Joseph Scott, Pless, Stephan Alexander
• Format: Journal Article
• Language: English
• Published: United States 16-05-2024
• Subjects: Acid Sensing Ion Channels - chemistry; Acid Sensing Ion Channels - genetics; Acid Sensing Ion Channels - metabolism; Animals; Humans; Lysine - chemistry; Lysine - metabolism; Models, Molecular; Protein Splicing; ornithine; homolysine; protein trans-splicing; ligand-gated ion channels; acid-sensing ion channels; native chemical ligation; non-canonical amino acids; desensitization; protein semisynthesis; split inteins
• ISSN: 2451-9448

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.