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Title: 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.Artemiaexpress several NKA catalytic α-subunit subtypes. High-salinity adaptation increases abundance of α2KK, an isoform that contains two lysines (Lys308 and Lys758 in transmembrane segments TM4 and TM5, respectively) at positions where canonical NKAs have asparagines (Xenopusα1’s Asn333 and Asn785). Using de novo transcriptome assembly and qPCR, we found thatArtemiaexpress two salinity-independent canonical α subunits (α1NNand α3NN), as well as two β variants, in addition to the salinity-controlled α2KK. These β subunits permitted heterologous expression of the α2KKpump 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 α2KKpumps and compared it to that ofXenopusα1 (and its α2KK-mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers α2KK-like characteristics toXenopusα1, and mutant cycle analysis reveals energetic coupling between these two residues, illustrating how α2KK’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+-congener86Rb+, 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 allowingArtemiato maintain steeper Na+gradients in hypersaline environments.  more » « less
Award ID(s):
2003251 2309048
PAR ID:
10499698
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ; ;
Publisher / Repository:
National Academy of Science
Date Published:
Journal Name:
Proceedings of the National Academy of Sciences
Volume:
120
Issue:
52
ISSN:
0027-8424
Page Range / eLocation ID:
e2313999120
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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