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

Abstract

Gastric H+,K+-ATPase consists of α-subunit with 10 transmembrane domains and β-subunit with a single transmembrane domain. We constructed cDNAs encoding chimeric β-subunits between the gastric H+,K+ATPase and Na+,K+-ATPase β-subunits and co-transfected them with the H+,K+-ATPase α-subunit cDNA in HEK-293 cells. A chimeric β-subunit that consists of the cytoplasmic plus transmembrane domains of Na+,K+ATPase β-subunit and the ectodomain of H+,K+-ATPase β-subunit assembled with the H+,K+-ATPase α- subunit and expressed the K+-ATPase activity. Therefore, the whole cytoplasmic and transmembrane domains of H+,K+-ATPase β-subunit were replaced by those of Na+,K+-ATPase β-subunit without losing the enzyme activity. However, most parts of the ectodomain of H+,K+ATPase β-subunit were not replaced by the corresponding domains of Na+,K+-ATPase β- subunit. Interestingly, the extracellular segment between Cys152 and Cys178, 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 (67DPYT70) in the ectodomain of H+,K+-ATPase β-subunit were replaced by the corresponding amino acids (63SDFE66) of Na+,K+ATPase β-subunit. The ATPase activity was abolished, however, when 4 amino acids (76QLKS79) in the ectodomain of H+,K+-ATPase β-subunit were replaced by the counterpart (72RVAP75) of Na+,K+-ATPase β- subunit, indicating that this region is the most N-terminal one that discriminates the H+,K+-ATPase β-subunit from that of Na+,K+-ATPase.