Interactions between Na,K-ATPase α-Subunit ATP-binding Domains

Abstract

The reaction mechanism of the Na,K-ATPase is thought to involve a number of ligand-induced conformational changes. The specific amino acid residues responsible for binding many of the important ligands have been identified; however, details of the specific conformational changes produced by ligand binding are largely undescribed. The experiments described in this paper begin to identify interactions between domains of the Na,K-ATPase ␣-subunit that depend on the presence of particular ligands. The major cytoplasmic loop (between TM4 and TM5), which we have previously shown contains the ATP-binding domain, was overexpressed in bacteria either with a His 6 tag or as a fusion protein with glutathione S-transferase. We have observed that these polypeptides associate in the presence of MgATP. Incubation with [␥-32 P]ATP under conditions that result in phosphorylation of the full-length Na,K-ATPase did not result in 32 P incorporation into either the His 6 tag or glutathione S-transferase fusion proteins. The MgATPinduced association was strongly inhibited by prior modification of the fusion proteins with fluorescein isothiocyanate or by simultaneous incubation with 10 M eosin, indicating that the effect of MgATP is due to interactions within the nucleotide-binding domain. These data are consistent with Na,K-ATPase associating within cells via interactions in the nucleotide-binding domains. Although any functional significance of these associations for ion transport remains unresolved, they may play a role in cell function and in modulating interactions between the Na,K-ATPase and other proteins.

Oligomerization of the Na,K-ATPase in Cell Membranes

“…Work utilizing Na,K-ATPase and H,K-ATPase chimeras suggests that the cytoplasmic loop of the Na,K-ATPase is important for oligomerization (9). These data are in agreement with the observation that the bacterially expressed major cytoplasmic loop between membrane segments M4 and M5 in isolation can dimerize in an ATP-dependent way (21). However, if the M4M5 loop is exclusively responsible for oligomerization, it is surprising that, in the work presented here, the ␣-4,5LL mutant, from which ϳ65% of the M4M5 cytoplasmic loop had been removed, is able to associate with the wild-type protein.…”

“…Recently published work has shown that the large cytoplasmic loop between the M4 and M5 transmembrane segments, when expressed in isolation, form oligomers in an ATP-dependent fashion (21). This loop contains the phosphorylation and nucleotide binding domains (28) for which significant structural information is provided by crystal structures of sarco(endo)plasmic reticulum calcium ATPase and EM structures of the Na,K-ATPase (29 -31).…”

“…Evidence for the existence of such multimers or for associations among monomers has derived from cross-linking experiments (10, 14 -16), single particle images (17), thermal inactivation studies (18), and association among different isoforms (9,19,20). Other recently published work demonstrates that the major cytoplasmic loop between the M4 and M5 membrane segments associates in an ATP-dependent way when expressed in isolation (21). Conversely, it has been claimed that the monomeric solubilized enzyme is capable of normal enzymatic activity (22).…”

The Amino Acid Sequence 442GDASE446 in Na/K-ATPase Is an Important Motif in Forming the High and Low Affinity ATP Binding Pockets

“…Each mutation in the conserved GDASE sequence induced different affinity changes in the two different ATP effects and the affinity for pNPP providing further support for the presence of a single ATP binding site/␣-chain as proposed (12,37,43). Quite recently, three interesting studies appeared, the association of ␤Ϫ␤ chain (51) and the association of expressed ␣-chains by MgATP (54) and the specific activity of the Na/K-ATPase activity of the tetraprotomeric Na/K-ATPase fraction was approximately half that of the diprotomeric and protomeric fractions, using a solubilized dog kidney enzyme (21), which nicely explains both that protomer is sufficient for Na/K-ATPase activity (46,48) and oligomericity is required for the enzyme (11-13, 19 -20, 37, 43, 51-52, 54). These data are consistent with a hypothesis in which each ␣-subunit contains 1 mol of a high affinity ATP binding site for EP formation and 1 mol of low affinity ATP binding site for EATP formation, in the 2n-mer, not only in Na/K-ATPase but also in H/K-ATPase, possibly as (EP:EATP) 2 (21, 37, 43, 52).…”

“…The apparent activation effect for Mg 2ϩ suggests at least two possibilities. One is the presence of a Mg 2ϩ binding site for the activation, in addition to the MgATP binding to the enzyme preceding phosphorylation (52,54), and the other is that the equilibrium between MgATP and ATP ϩ Mg 2ϩ may have been shifted to the left, which increases MgATP binding to the enzyme, resulting in its activation. Each of the mutations of the conserved sequence affected the high and low affinity ATP effects more strongly than the Mg 2ϩ effect.…”