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 (alpha beta) pump that usually exports 3Na(+) in exchange for 2 K+ per hydrolyzed ATP. Artemia express several NKA catalytic alpha-subunit subtypes. High-salinity adaptation increases abundance of alpha 2(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 alpha 1's Asn333 and Asn785). Using de novo transcriptome assembly and qPCR, we found that Artemia express two salinity-independent canonical alpha subunits (alpha 1(NN) and alpha 3(NN)), as well as two beta variants, in addition to the salinity-controlled alpha 2(KK). These beta subunits permitted heterologous expression of the alpha 2(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 alpha 2(KK) pumps and compared it to that of Xenopus alpha 1 (and its alpha 2(KK)-mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers alpha 2(KK)-like characteristics to Xenopus alpha 1, and mutant cycle analysis reveals energetic coupling between these two residues, illustrating how alpha 2(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 Rb-86(+), 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.