Importance for Absorption of Na+ from Freshwater of Lysine, Valine and Serine Substitutions in the α1a-Isoform of Na,K-ATPase in the Gills of Rainbow Trout (Oncorhynchus mykiss) and Atlantic Salmon (Salmo salar)

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Abstract
In the gills of rainbow trout and Atlantic salmon, the α1a- and α1b-isoforms of Na,K-ATPase are expressed reciprocally during salt acclimation. The α1a-isoform is important for Na+ uptake in freshwater, but the molecular basis for the functional differences between the two isoforms is not known. Here, three amino acid substitutions are identified in transmembrane segment 5 (TM5), TM8 and TM9 of the α1a-isoform compared to the α1b-isoform, and the functional consequences are examined by mutagenesis and molecular modeling on the crystal structures of Ca-ATPase or porcine kidney Na,K-ATPase. In TM5 of the α1a-isoform, a lysine substitution, Asn783 → Lys, inserts the ε-amino group in cation site 1 in the E1 form to reduce the Na+/ATP ratio. In the E2 form the ε-amino group approaches cation site 2 to force ejection of Na+ to the blood phase and to interfere with binding of K+. In TM8, a Asp933 → Val substitution further reduces K+ binding, while a Glu961 → Ser substitution in TM9 can prevent interaction of FXYD peptides with TM9 and alter Na+ or K+ affinities. Together, the three substitutions in the α1a-isoform of Na,K-ATPase act to promote binding of Na+ over K+ from the cytoplasm, to reduce the Na+/ATP ratio and the work done in one Na,K pump cycle of active Na+ transport against the steep gradient from freshwater (10–100 μm Na+) to blood (160 mm Na+) and to inhibit binding of K+ to allow Na+/H+ rather than Na+/K+ exchange.

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