A Na pump with reduced stoichiometry is up-regulated by brine shrimp in extreme salinities

Brine shrimp ( Artemia ) are the only animals to thrive at sodium concentrations above 4 M. Salt excretion is powered by the Na ⁺ ,K ⁺ -ATPase (NKA), a heterodimeric (αβ) pump that usually exports 3Na ⁺ in exchange for 2 K ⁺ per hydrolyzed ATP. Artemia express several NKA catalytic α-subunit subtypes. High-salinity adaptation increases abundance of α2 _KK , an isoform that contains two lysines (Lys308 and Lys758 in transmembrane segments TM4 and TM5, respectively) at positions where canonical NKAs have asparagines ( Xenopus α1’s Asn333 and Asn785). Using de novo transcriptome assembly and qPCR, we found that Artemia express two salinity-independent canonical α subunits (α1 _NN and α3 _NN ), as well as two β variants, in addition to the salinity-controlled α2 _KK . These β subunits permitted heterologous expression of the α2 _KK pump and determination of its CryoEM structure in a closed, ion-free conformation, showing Lys758 residing within the ion-binding cavity. We used electrophysiology to characterize the function of α2 _KK pumps and compared it to that of Xenopus α1 (and its α2 _KK -mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers α2 _KK -like characteristics to Xenopus α1, and mutant cycle analysis reveals energetic coupling between these two residues, illustrating how α2 _KK ’s Lys308 helps to maintain high affinity for external K ⁺ when Lys758 occupies an ion-binding site. By measuring uptake under voltage clamp of the K ⁺ -congener ⁸⁶ Rb ⁺ , we prove that double-lysine-substituted pumps transport 2Na ⁺ and 1 K ⁺ per catalytic cycle. Our results show how the two lysines contribute to generate a pump with reduced stoichiometry allowing Artemia to maintain steeper Na ⁺ gradients in hypersaline environments.