Core Conclusion:
This study systematically elucidates the multifunctional transport properties and structural basis of BTR1, a member of the SLC4 family. Using cryo-electron microscopy (cryo-EM) and electrophysiology, it reveals BTR1’s unique mechanism in transporting ammonia, sodium ions, and protons, and identifies key regulatory sites and functional residues.

Key Highlights:

Structure and Conformation: The study resolved BTR1 in 13 distinct conformational states under various buffer and pH conditions, predominantly inward-open and outward-open conformations. BTR1 forms a homodimer with its cytoplasmic domain tightly coupled to the transmembrane region, structurally distinct from other SLC4 members like NDCBE and Band 3.
Multi-Substrate Transport Capacity: BTR1 is identified as an efficient ammonia (NH₃) transporter, and also transports sodium ions (Na⁺) and protons (H⁺). Its functional mode depends on the substrate environment: in the absence of ammonia, it acts as a Na⁺-dependent [H⁺]/[OH⁻] transporter; in the presence of ammonia (especially NH₃), it functions as an NH₃ transporter, with Na⁺ enhancing its transport efficiency.
Regulatory Mechanism: pH regulates BTR1’s conformational transitions, with alkaline conditions favoring the outward-open state. Phospholipid PIP₂ binding within the transmembrane region participates in modulating conformational changes and function. Ammonium salts (NH₄Cl) and sodium ions influence and stabilize its outward-open conformation.
Transport Dynamics and Key Residues: Molecular dynamics simulations reveal differences in the energy pathways and transport efficiencies for different substrates (H⁺ > Na⁺ > NH₃). Key residues (e.g., F399, E572, L579, L575) undergo dynamic conformational changes to facilitate substrate passage. Electrophysiological experiments on point mutants confirm the critical roles of residues T410, Q414, T434, T435, and H719 in substrate binding and transport, with T434A and T435A mutations nearly abolishing NH₃ transport.
Disease Relevance: BTR1 dysfunction is associated with various corneal endothelial dystrophies (e.g., CHED, FECD) and potential neurological and hearing disorders. Clarifying its structure and transport mechanism provides important insights into disease pathogenesis and potential therapeutic targets.

Significance
Integrating structural, functional, and computational analyses, this study not only details BTR1’s multifunctional transport mechanism but also expands the understanding of substrate recognition and transport modes within the SLC4 transporter family, offering new theoretical foundations for intervening in related diseases.
This study reveals BTR1‘s structure and dual transport mechanism, functioning as a Na⁺-dependent H⁺/OH⁻ transporter and a Na⁺-enhanced NH₃ transporter.

DOI：10.1093/procel/pwaf108