Regulation and function of lysine-substituted Na,K pumps in salt adaptation of Artemia franciscana.
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 alpha(2)(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 alpha(1)(NN)-subunit, which was insignificantly affected by increasing salt. Site-directed mutagenesis showed that Asn324Lys and Asn776Lys in the alpha(1)-subunit of pig kidney Na,K-ATPase reduced the stoichiometry of (204)Tl binding from 2 to about 1 Tl(+)(K(+)) per alpha-subunit and Na(+)-dependent phosphorylation from ATP to 25-30%. In structure models, the epsilon-amino group of Lys776 is located at cation site 1 in the E(1)P form and near cation site 2 in the E(2) conformation, while the side chain of Lys324 points away from the cation sites. Salt-induced expression of the alpha(2)(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.
Jorgensen
Peter Leth
PL
Department of Molecular Biology, University of Copenhagen, Universitetsparken 13, Copenhagen OE, Denmark. pljorgensen@aki.ku.dk
Amat
Francisco
F
D000222:Adaptation, Physiological / Q000187:drug effects* / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D001156:Artemia / Q000201:enzymology* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D008239:Lysine / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012965:Sodium Chloride / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship
D021122:Protein Subunits; D012333:RNA, Messenger; D012965:Sodium Chloride; D000254:Sodium-Potassium-Exchanging ATPase; D008239:Lysine