期刊
JOURNAL OF MEMBRANE BIOLOGY
卷 223, 期 1, 页码 37-47出版社
SPRINGER
DOI: 10.1007/s00232-008-9111-y
关键词
structural/functional Na; K-ATPase; epithelial transport; trout gill; alpha 1a-isoform of Na; K-ATPase; lysine substitution; site-directed mutagenesis; Na+ binding; K+ binding; Tl+ binding; Na+/H+ exchange
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.
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