@article{TEXTUAL,
      recid = {10431},
      author = {Artigas, Pablo and Meyer, Dylan J. and Young, Victoria C.  and Spontarelli, Kerri and Eastman, Jessica and  Strandquist, Evan and Rui, Huan and Roux, Benoît and Birk,  Matthew A. and Nakanishi, Hanayo and Abe, Kazuhiro and  Gatto, Craig},
      title = {A Na pump with reduced stoichiometry is up-regulated by  brine shrimp in extreme salinities},
      journal = {PNAS},
      address = {2023-12-11},
      number = {TEXTUAL},
      abstract = {Brine shrimp (Artemia) are the only animals to thrive at  sodium concentrations above 4 M. Salt excretion is powered  by the Na<sup>+</sup>,K<sup>+</sup>-ATPase (NKA), a  heterodimeric (αβ) pump that usually exports  3Na<sup>+</sup> in exchange for 2 K<sup>+</sup> per  hydrolyzed ATP. Artemia express several NKA catalytic  α-subunit subtypes. High-salinity adaptation increases  abundance of α2<sub>KK</sub>, 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<sub>NN</sub> and α3<sub>NN</sub>), as well as two β  variants, in addition to the salinity-controlled α2KK.  These β subunits permitted heterologous expression of the  α2<sub>KK</sub> 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<sub>KK</sub> pumps and compared it to that of Xenopus α1  (and its α2<sub>KK</sub>-mimicking single- and  double-lysine substitutions). The double substitution  N333K/N785K confers α2<sub>KK</sub>-like characteristics to  Xenopus α1, and mutant cycle analysis reveals energetic  coupling between these two residues, illustrating how  α2<sub>KK</sub>’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<sup>+</sup>-congener <sup>86</sup>Rb+, we prove that  double-lysine-substituted pumps transport 2Na<sup>+</sup>  and 1 K<sup>+</sup> per catalytic cycle. Our results show  how the two lysines contribute to generate a pump with  reduced stoichiometry allowing Artemia to maintain steeper  Na<sup>+</sup> gradients in hypersaline environments.},
      url = {http://knowledge.uchicago.edu/record/10431},
}