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bioRxiv · 10.1101/2023.01.23.525296

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

Sarkar, D.·Galleano, I.·Ou, S. Y.·Uzun, G. R.·Khoo, K. K.·van Noort, G. J. v. d. H.·Pless, S. A.
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Abstract

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.

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Sarkar, D., Galleano, I., Ou, S. Y., Uzun, G. R., Khoo, K. K., van Noort, G. J. v. d. H., Pless, S. A.. 2023-01-24. Protein semisynthesis underscores role of a conserved lysine in activation and desensitization of acid-sensing ion channels. https://doi.org/10.1101/2023.01.23.525296

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