Regulation and Function of Lysine-Substituted Na,K Pumps in Salt Adaptation of Artemia franciscana

Abstract

The brine shrimp Artemia thrives at extreme conditions of up to 300 g/l salt in hypersaline lakes, but the molecular aspects of this salt adaptation are not clarified. To examine the influence of salt on the expression of two isoforms of Na,K-ATPase, adult Artemia franciscana were cultured for 39 days with the microalga Dunaliella salina as fodder at increasing salt from 30 to 280 g/l. Quantitative reverse-transcriptase polymerase chain reaction showed that the abundance of mRNA of the lysine-substituted α₂(KK)-subunit was very low at 30 g/l salt but rose steeply in the range of 70–200 g/l to a level at 200–280 g/l salt, similar to the abundance of the mRNA of the α₁(NN)-subunit, which was insignificantly affected by increasing salt. Site-directed mutagenesis showed that Asn324Lys and Asn776Lys in the α₁-subunit of pig kidney Na,K-ATPase reduced the stoichiometry of ²⁰⁴Tl binding from 2 to about 1 Tl⁺(K⁺) per α-subunit and Na⁺-dependent phosphorylation from ATP to 25–30%. In structure models, the ε-amino group of Lys776 is located at cation site 1 in the E₁P form and near cation site 2 in the E₂ conformation, while the side chain of Lys324 points away from the cation sites. Salt-induced expression of the α₂(KK)-subunit Na,K-ATPase in A. franciscana may reduce the Na⁺/ATP ratio and enable the Na,K pump to extrude Na⁺ against steeper gradients and, thus, contribute to salt adaptation.