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Cryo-EM structure of Na+,K+-ATPase alpha2 from Artemia salina in cation-free E2P form
Structural highlights
Publication Abstract from PubMedBrine 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 (alphabeta) 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 alpha2(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 alpha1's Asn333 and Asn785). Using de novo transcriptome assembly and qPCR, we found that Artemia express two salinity-independent canonical alpha subunits (alpha1(NN) and alpha3(NN)), as well as two beta variants, in addition to the salinity-controlled alpha2(KK). These beta subunits permitted heterologous expression of the alpha2(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 alpha2(KK) pumps and compared it to that of Xenopus alpha1 (and its alpha2(KK)-mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers alpha2(KK)-like characteristics to Xenopus alpha1, and mutant cycle analysis reveals energetic coupling between these two residues, illustrating how alpha2(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. A Na pump with reduced stoichiometry is up-regulated by brine shrimp in extreme salinities.,Artigas P, Meyer DJ, Young VC, Spontarelli K, Eastman J, Strandquist E, Rui H, Roux B, Birk MA, Nakanishi H, Abe K, Gatto C Proc Natl Acad Sci U S A. 2023 Dec 26;120(52):e2313999120. doi: , 10.1073/pnas.2313999120. Epub 2023 Dec 11. PMID:38079564[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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