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

Abstract

Brine shrimp (<i>Artemia</i>) 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. <i>Artemia</i> 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 (<i>Xenopus</i> α1's Asn333 and Asn785). Using de novo transcriptome assembly and qPCR, we found that <i>Artemia</i> 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 <i>Xenopus</i> α1 (and its α2_KK-mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers α2_KK-like characteristics to <i>Xenopus</i> 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 ⁸⁶Rb⁺, 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 <i>Artemia</i> to maintain steeper Na⁺ gradients in hypersaline environments.