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

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.

Structure-guided unlocking of NaX reveals a non-selective tetrodotoxin-sensitive cation channel.

Unlike classical voltage-gated sodium (NaV) channels, NaX has been characterized as a voltage-insensitive, tetrodotoxin-resistant, sodium (Na+)-activated channel involved in regulating Na+ homeostasis. However, NaX remains refractory to functional characterization in traditional heterologous systems. Here, to gain insight into its atypical physiology, we determine structures of the human NaX channel in complex with the auxiliary ß3-subunit. NaX reveals structural alterations within the selectivity filter, voltage sensor-like domains, and pore module. We do not identify an extracellular Na+-sensor or any evidence for a Na+-based activation mechanism in NaX. Instead, the S6-gate remains closed, membrane lipids fill the central cavity, and the domain III-IV linker restricts S6-dilation. We use protein engineering to identify three pore-wetting mutations targeting the hydrophobic S6-gate that unlock a robust voltage-insensitive leak conductance. This constitutively active NaX-QTT channel construct is non-selective among monovalent cations, inhibited by extracellular calcium, and sensitive to classical NaV channel blockers, including tetrodotoxin. Our findings highlight a functional diversity across the NaV channel scaffold, reshape our understanding of NaX physiology, and provide a template to demystify recalcitrant ion channels.