PT  - JOURNAL ARTICLE
AU  - Debayan Sarkar
AU  - Iacopo Galleano
AU  - Sofie Yuewei Ou
AU  - Gül Refika Uzun
AU  - Keith K. Khoo
AU  - Gerbrand Jan van der Heden van Noort
AU  - Stephan Alexander Pless
TI  - Protein semisynthesis underscores role of a conserved lysine in activation and desensitization of acid-sensing ion channels
AID  - 10.1101/2023.01.23.525296
DP  - 2023 Jan 01
TA  - bioRxiv
PG  - 2023.01.23.525296
4099  - http://biorxiv.org/content/early/2023/01/24/2023.01.23.525296.short
4100  - http://biorxiv.org/content/early/2023/01/24/2023.01.23.525296.full
AB  - Acid-sensing ion channels (ASICs) are trimeric ligand-gated ion channels that open a cation-conducting pore in response to proton-binding to their extracellular domain (ECD). This activation is typically short-lived, as channel opening is followed by a rapid and spontaneous pore closure due to a process termed fast desensitization. This intrinsic channel closing mechanism is important, because excessive activation of ASICs during periods of prolonged acidosis in conditions such as inflammation and ischemia, has been linked to several pathologies, including pain and stroke. Structural and functional data suggest that a conserved lysine in the ECD (Lys211 in mASIC1a) plays a role in receptor function. However, the precise physico-chemical contributions to activation and fast desensitization are difficult to dissect with conventional mutagenesis, as replacement of Lys211 with naturally occurring side chains invariably changes multiple parameters, such as size, charge, and H-bonding pattern simultaneously. Here, we study the contribution of Lys211 to activation and fast desensitization of mASIC1a using tandem protein-trans splicing (tPTS) to incorporate non-canonical lysine analogs. We conduct extensive optimization efforts to improve apparent splicing yields and subsequently interrogate the function of semisynthetic mASIC1a using two-electrode voltage clamp. Our data show that both charge and side chain length of Lys211 are crucial to activation and fast desensitization of mASIC1a, with the latter being sensitive to even the most subtle mutational disruptions. Our work emphasizes that tPTS can enable atomic-scale interrogations of how side chains contribute to the function of complex membrane proteins.Competing Interest StatementThe authors have declared no competing interest.