Chimeras of X+,K+-ATPases

Abstract

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 hairpin M5-M6 of H+,K+-ATPase was replaced by that of Na+,K+-ATPase was phosphorylated in the absence of Na+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 transmembrane segments one to six and intermediate loops of Na+,K+-ATPase, harbors the amino acids responsible for Na+specificity. Compared with Na+,K+-ATPase, this chimera displayed a similar apparent Na+affinity, a lower apparent K+affinity, a higher apparent ATP affinity, and a lower apparent vanadate affinity in the ATPase reaction. This indicates that theE2K form of this chimera is less stable than that of Na+,K+-ATPase, suggesting that it, like H+,K+-ATPase, de-occludes K+ions very rapidly. Comparison of the structures of these chimeras with those of the parent enzymes suggests that the C-terminal 187 amino acids and the β-subunit are involved in K+occlusion. Accordingly, chimera HN16 is not only a chimeric enzyme in structure, but also in function. On one hand it possesses the Na+-stimulated ATPase reaction of Na+,K+-ATPase, while on the other hand it has the K+occlusion properties of H+,K+-ATPase.

Extracellular Domains, Transmembrane Segments, and Intracellular Domains Interact To Determine the Cation Selectivity of Na,K- and Gastric H,K-ATPase

“…This study, together with previous work form our laboratory and from other groups ( , ), demonstrates the critical importance of synergistic interactions of transmembrane segments and extracellular domains, as well as interactions of residues within these domains. Our findings underscore the complex and cooperative nature of cation translocation by P-type ATPases.…”

“…Recent observations suggest that the domain containing TM3, TM4, and the intervening ectodomain of the Na,K-ATPase is in part necessary to confer high ouabain sensitivity to chimeras generated from Na,K-and gastric H,K-ATPase (27). The same domain is also included in the stretch comprising TM1-TM6 of the Na,K-ATPase that is able to confer Na + -dependence upon H,K/Na,K-ATPase chimeras (28). These findings are consistent with our conclusion that cooperative interactions between extracellular domains and transmembrane segments may significantly influence functional properties of P-type ATPases.…”

Cysteine‐scanning mutagenesis study of the sixth transmembrane segment of the Na,K‐ATPase α subunit

“…This threonine is also a very highly conserved residue in all known vertebrate Na,K‐ or H,K‐ATPases. These differences may be related to the different cation selectivity (H + or Na + ) of the two ATPases in question and suggest a specific role of these residues in the selectivity for the cation transported in exchange for potassium as also proposed by Koenderink et al [19]. Another possible explanation is the fact that methionine and cysteine are both sulfur‐containing residues and their main difference is the larger size of methionine.…”

Residues within Transmembrane Domains 4 and 6 of the Na,K-ATPase α Subunit Are Important for Na+ Selectivity

“…28, 2004 Sa ´nchez and Blanco properties of those of the H,K-ATPase (38). Likewise the H,K/Na,K chimeras engineered by Koenderink et al show that introduction of H,K-ATPase sequences to the Na,K-ATPase results in a shift of the enzyme toward the E1 conformation (70).…”