THE NEGATIVE CHARGE OF GLUTAMIC ACID-820 IN THE GASTRIC H-ATPASE ALPHA-SUBUNIT IS ESSENTIAL FOR K+ ACTIVATION OF THE ENZYME-ACTIVITY(,K+)

To investigate the role of Glu(820), located in transmembrane domain M 6 of the alpha-subunit of gastric H+,K+-ATPase, a number of mutants wa s prepared and expressed in Sf9 cells using a baculovirus encoding for both H+,K+-ATPase subunits. The wild-type enzyme and the E820D (Glu(8 20) --> Asp) mutant showed a similar biphasic activation by K+ on the ATPase activity (maximum at 1 mM). The mutant E820A had a markedly dec reased K+ affinity (maximum at 40-100 mM). The other mutants, E820Q, E 820N, E820L and E820K, showed no K+-activated ATPase activity at all, whereas all mutants formed a phosphorylated intermediate. After preinc ubation with K+ before phosphorylation mutant E820D showed a similar K +-sensitivity as the wild-type enzyme. The mutants E820N and E820Q had a 10-20 times lower sensitivity, whereas the other three mutants were hardly sensitive towards K+. Upon preincubation with nomethyl)-2-meth yl-8-(phenylmethoxy)imidazo[1,2a]- pyridine (SCH 28080), all mutants s howed similar sensitivity for this drug as the wild-type enzyme, excep t mutant E820Q, which could only partly be inhibited, and mutant E820K , which was completely insensitive towards SCH 28080. These experiment s suggest that, with a relatively large residue at position 820, the b inding of SCH 28080 is obstructed. The various mutants showed a behavi our in K+-stimulated-dephosphorylation experiments similar to that for K+-activated-ATPase-activity measurements. These results indicate tha t K+ binding, and indirectly the transition to the E-2, form, is only fully possible when a negatively charged residue is present at positio n 820 in the alpha-subunit.