Chimeric domain analysis of the compatibility between H+,K+-ATPase and Na+,K+-ATPase β-subunits for the functional expression of gastric H+,K+-ATPase

Gastric H+,K+-ATPase consists of alpha-subunit with 10 transmembrane domains and beta-subunit with a single transmembrane domain, We constructed cDNAs encoding chimeric beta-subunits between the gastric H+,K+ ATPase and Na+,K+-ATPase beta-subunits and co-transfected them with the H+,K+-ATPase alpha-subunit cDNA in HEK-293 cells. A chimeric beta-subunit that consists of the cytoplasmic plus transmembrane domains of Na+,K+ ATPase beta-subunit and the ectodomain of H+,K+-ATPase beta-subunit assembled with the H+,K+-ATPase alpha-subunit and expressed the K+-ATPase activity. Therefore, the whole cytoplasmic and transmembrane domains of H+,K+-ATPase beta-subunit were replaced by those of Na+,K+-ATPase beta-subunit without losing the enzyme activity. However, most parts of the ectodomain of H+,K+ ATPase beta-subunit were not replaced by the corresponding domains of Na+,K+-ATPase beta-subunit. interestingly the extracellular segment between Cys(152) and Cys(178), which contains the second disulfide bond, was exchangeable between H+,K+-ATPase and Na+,K+ ATPase, preserving the K+-ATPase activity intact. Furthermore, the K+-ATPase activity was preserved when the N-terminal first 4 amino acids ((DPYT70)-D-67) in the ectodomain of H+,K+-ATPase beta-subunit were replaced by the corresponding amino acids ((SDFE66)-S-63) of Na+,K+ ATPase beta-subunit. The ATPase activity was abolished, however, when 4 amino acids ((76)QLKS(79)) in the ectodomain of H+,K+-ATPase beta-subunit were replaced by the counterpart ((72)RVAP(75)) of Na+,K+-ATPase beta-subunit, indicating that this region is the most N-terminal one that discriminates the H+,K+-ATPase beta-subunit from that of Na+,K+-ATPase.