Effects of Mutations in M4 of the Gastric H+,K+-ATPase on Inhibition Kinetics of SCH28080

The effects of site-directed mutagenesis were used to explore the role of residues in M4 on the apparent K i of a selective, K+-competitive inhibitor of the gastric H+,K+ ATPase, SCH28080. A double transfection expression system is described, utilizing HEK293 cells and separate plasmids encoding the α and β subunits of the H+,K+-ATPase. The wild-type enzyme gave specific activity (micromoles of Pi per hour per milligram of expressed H+,K+-ATPase protein), apparent K m for ammonium (a K+ surrogate), and apparent K i for SCH28080 equal to the H+,K+-ATPase purified from hog gastric mucosa. Amino acids in the M4 transmembrane segment of the α subunit were selected from, and substituted with, the nonconserved residues in M4 of the Na+,K+-ATPase, which is insensitive to SCH28080. Most of the mutations produced competent enzyme with similar K m,app values for NH4 + and K i,app for SCH28080. SCH28080 affinity was decreased 2-fold in M330V and 9-fold in both M334I and V337I without significant effect on K m,app. Hence methionine 334 and valine 337 participate in binding but are not part of the NH4 + site. Methionine 330 may be at the periphery of the inhibitor site, which must have minimum dimensions of ∼16 × 8 × 5 Å and be accessible from the lumen in the E2-P conformation. Multiple sequence alignments place the membrane surface near arginine 328, suggesting that the side chains of methionine 334 and valine 337, on one side of the M4 helix, project into a binding cavity within the membrane domain.