[No authors listed]
The catalytic alpha-subunits of Na,K- and H,K-ATPase require an accessory beta-subunit for proper folding, maturation, and plasma membrane delivery but also for cation transport. To investigate the functional significance of the beta-N terminus of the gastric H,K-ATPase in vivo, several N-terminally truncated beta-variants were expressed in Xenopus oocytes, together with the S806C alpha-subunit variant. Upon labeling with the reporter fluorophore tetramethylrho da mine-6-maleimide, this construct can be used to determine the voltage-dependent distribution between E(1)P/E(2)P states. Whereas the E(1)P/E(2)P conformational equilibrium was unaffected for the shorter N-terminal deletions betaDelta4 and betaDelta8, we observed significant shifts toward E(1)P for the two larger deletions betaDelta13 and betaDelta29. Moreover, the reduced DeltaF/F ratios of betaDelta13 and betaDelta29 indicated an increased reverse reaction via E(2)P --> E(1)P + ADP --> E(1) + ATP, because cell surface expression was completely unaffected. This interpretation is supported by the reduced sensitivity of the mutants toward the E(2)P-specific inhibitor SCH28080, which becomes especially apparent at high concentrations (100 microm). Despite unaltered apparent Rb(+) affinities, the maximal Rb(+) uptake of these mutants was also significantly lowered. Considering the two putative interaction sites between the beta-N terminus and alpha-subunit revealed by the recent cryo-EM structure, the N-terminal tail of the H,K-ATPase beta-subunit may stabilize the pump in the E(2)P conformation, thereby increasing the efficiency of proton release against the million-fold proton gradient of the stomach lumen. Finally, we demonstrate that a similar truncation of the beta-N terminus of the closely related Na,K-ATPase does not affect the E(1)P/E(2)P distribution or pump activity, indicating that the E(2)P-stabilizing effect by the beta-N terminus is apparently a unique property of the H,K-ATPase.
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