FUNCTIONAL EXPRESSION OF THE ALPHA-2-ISOFORMS AND ALPHA-3-ISOFORMS OF THE NA,K-ATPASE IN BACULOVIRUS-INFECTED INSECT CELLS

Multiple isoforms of both the alpha and beta subunits of Na,K-ATPase have been identified. Elucidating their roles has been complicated by the fact that most tissues express multiple isoforms and purification techniques specific for each isoform have not been achieved. The baculovirus expression system, which uses the baculovirus Autographica californica to infect insect cells, is an ideal system for studying the Na,K-ATPase isoforms since high amounts of foreign proteins can be produced and some insect cell lines have low levels of endogenous Na,K-ATPase. Recombinant baculoviruses containing the cDNAs for the alpha2, alpha3, and beta1 isoforms of the rat Na,K-ATPase were prepared and used to infect Sf-9 cells, an insect cell line derived from the ovary of the fall armyworm Spodoptera frugiperda. By using this system, Na,K-ATPase alpha2 and alpha3 subunits that were antigenically and electrophoretically indistinguishable from the native subunits were produced. When each subunit is expressed independently in the Sf-9 cells, it is primarily delivered to the plasma membrane. Although the isolated expression of each Na,K-ATPase subunit did not render active Na,K-ATPase molecules, the coexpression of alpha2 or alpha3 with beta1 resulted in catalytically active molecules. This activity could be measured as a ouabain-sensitive ATPase activity or directly demonstrated using either [gamma-P-32]ATP or P-32i to identify the phosphorylated intermediates of the alpha2 and alpha3 isoforms. [H-3]Ouabain binding studies showed that both isoforms are capable of binding the cardiotonic steroid with high affinity, alpha3 being more sensitive to ouabain. These results demonstrate that the baculovirus system is suitable for the expression of the Na,K-ATPase isoforms and should provide a useful method for the characterization of the enzymatic properties of each isoform.