Importance for absorption of Na+ stop 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)

In the gills of rainbow trout and Atlantic salmon, the alpha 1a- and alpha 1b-isoforms of Na,K-ATPase are expressed reciprocally during salt acclimation. The alpha 1a-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 alpha 1a-isoform compared to the alpha 1b-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 alpha 1a-isoform, a lysine substitution, Asn783 -> Lys, inserts the epsilon-amino group in cation site 1 in the E-1 form to reduce the Na+/ATP ratio. In the E-2 form the epsilon-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 alpha 1a-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 mu M Na+) to blood (160 mM Na+) and to inhibit binding of K+ to allow Na+/H+ rather than Na+/K+ exchange.