Chimeras of X+,K+-ATPases - The M1-M6 region of Na+,K+-atpase is required for Na+-activated ATPase activity, whereas the M7-M10 region of H+,K+-atpase is involved in K+ de-occlusion
Jb. Koenderink et al., Chimeras of X+,K+-ATPases - The M1-M6 region of Na+,K+-atpase is required for Na+-activated ATPase activity, whereas the M7-M10 region of H+,K+-atpase is involved in K+ de-occlusion, J BIOL CHEM, 276(15), 2001, pp. 11705-11711
In this study we reveal regions of Na+,K+-ATPase and H+,K+-ATPase that are
involved in cation selectivity. A chimeric enzyme in which transmembrane ha
irpin M5-M6 of H+,K+-ATPase was replaced by that of Na+,K+- ATPase was phos
phorylated in the absence of Naf and showed no K+-dependent reactions. Next
, the part originating from Na+,K+-ATPase was gradually increased in the N-
terminal direction. We demonstrate that chimera HN16, containing the transm
embrane segments one to six and intermediate loops of Na+,K+-ATPase, harbor
s the amino acids responsible for Na+ specificity. Compared with Na+,K+-ATP
ase, this chimera displayed a similar apparent Na+ affinity, a lower appare
nt K+ affinity, a higher apparent ATP affinity, and a lower apparent vanada
te affinity in the ATPase reaction. This indicates that the E2K form of thi
s chimera is less stable than that of Na+,K+-ATPase, suggesting that it, li
ke H+,K+-ATPase, de-occludes K+ ions very rapidly. Comparison of the struct
ures of these chimeras with those of the parent enzymes suggests that the C
-terminal 187 amino acids and the beta -subunit are involved in K+ occlusio
n, Accordingly, chimera HN16 is not only a chimeric enzyme in structure, bu
t also in function. On one hand it possesses the Na+-stimulated ATPase reac
tion of Na+,K+-ATPase, while on the other hand it has the K+ occlusion prop
erties of H+,K+-ATPase.