Molecular Distance Measurements Reveal an (αβ)2Dimeric Structure of Na+/K+-ATPase

Abstract

ATP hydrolysis by Na؉ /K ؉ -ATPase proceeds via the interaction of simultaneously existing and cooperating high (E 1 ATP) and low (E 2 ATP) substrate binding sites. It is unclear whether both ATP sites reside on the same or on different catalytic ␣-subunits. To answer this question, we looked for a fluorescent label for the E 2 ATP site that would be suitable for distance measurements by Fö rster energy transfer after affinity labeling of the E 1 ATP site by fluorescein 5-isothiocyanate (FITC). Naϩ /K ϩ -ATPase is an integral membrane protein that transports sodium and potassium ions against an electrochemical gradient. The transport of Na ϩ and K ϩ ions is presumably connected to an oscillation of the enzyme between two major conformational states, namely the E 1 Na ϩ and the E 2 K ϩ conformations. The E 1 and E 2 states have different affinities for ATP. The pumping mechanism may be described by conformational changes of a single ATP site of the catalytic ␣-subunit between a high affinity E 1 ATP 1 site (from where Na ϩ export starts by phosphorylation) and a low affinity E 2 ATP site (which is involved in K ϩ import) (1-3). Yet a model assuming consecutive changes of a single ATP site during the catalytic process, the so-called Albers-Post model, is inconsistent with the recent kinetic demonstration of simultaneously existing and cooperating ATP binding sites (4, 5) and the finding that specific labeling of the E 1 ATP or the E 2 ATP sites does not block labeling and partial activities of the other empty site (6 -13). The recent demonstration of a "superphosphorylation," i.e. that at least 2 mol of phosphate can be incorporated into the catalytic ␣-subunit per mol of ouabain binding sites (14), is consistent with the coexistence of phosphorylated intermediates (E 1 P, E*P, and E 2 P (15)) at different places in an oligomeric enzyme. Also, the observations of phosphorylation from P i during Na ϩ -ATPase activity (16) and in an FITC-treated enzyme (10) are consistent with the possibility that Na ϩ /K ϩ -ATPase is phosphorylated from both ATP sites (12, 17).Since two ATP binding sites of Na ϩ /K ϩ -ATPase cooperate during ATP hydrolysis (4, 5), it is of great interest to obtain more detailed information on the mutual interaction of both ATP sites in the absence (8,10,18,19) and presence of the transported cations (20) and on their distance. There are a great number of experiments favoring the idea that both ATP sites reside on different ␣-subunits (21, 22). But studies with detergent-solubilized Na ϩ /K ϩ -ATPase seem to contradict this assumption (13,23,24). Ward and Cavieres (13, 24) demonstrated that the detergent-solubilized putative (␣␤) promoter of Na ϩ /K ϩ -ATPase shows negative cooperativity of ATP hydrolysis. This finding is in conflict with the assumption of cooperating catalytic ␣-subunits during ATP hydrolysis but supports the possibility of two interacting ATP sites residing on the same catalytic ␣-subunit. The amino acid sequence forming the high affinity E 1 ATP site has been defined by affinity...

Ligands Presumed to Label High Affinity and Low Affinity ATP Binding Sites Do Not Interact in an (αβ)2 Diprotomer in Duck Nasal Gland Na+,K+-ATPase, nor Do the Sites Coexist in Native Enzyme

“…Fluorescence resonance energy transfer measurements between bound FITC and bound ErITC indicated that the two probes were too far apart to be bound to the same protomer (11). ATP at high concentration prevented the binding of ErITC to the FITC-labeled enzyme (11). Fig.…”

“…The putative low affinity ATP binding site resides on the same ␣ chain as the putative high affinity ATP binding site. -ATPase with about half-maximal theoretical phosphorylation capacity binds about 0.5 FITC molecules per ␣␤ protomer and then binds an additional 0.5 molecules of ErITC per ␣␤ protomer to the FITC-labeled enzyme with inhibition of the residual K ϩ -dependent pNPPase activity (11). In this calculation, the entire protein mass of the Na ϩ ,K ϩ -ATPase preparation was taken to consist only of ␣␤ protomers although only half could be phosphorylated.…”

“…In the second study, erythrosin 5-isothiocyanate (ErITC) was used to label and inactivate the low affinity site. Fluorescence resonance energy transfer measurements were used to show that the probes resided on separate ␣␤ protomers (11). Our results show that, in the nasal gland enzyme, FITC and TNP-ADP or ErITC react with sites on a single ␣␤ protomer.…”

“…ErITC inhibits K ϩ -dependent pNPPase activity of FITCmodified renal enzyme, and ATP protects against inhibition (11). Fig.…”

Oligomerization of the Na,K-ATPase in Cell Membranes

“…7 and 8. Briefly, physical interactions between ␣␤, ␣␣, and ␤␤ have been implicated from cross-linking studies (14,16,24,(37)(38)(39), co-immunoprecipitations have demonstrated the interaction of different ␣-subunit isoforms (19), thermal inactivation studies suggest ␣␣ contact (18), fluorescence resonance energy transfer provides distance measurements consistent with (␣␤) 2 (40), and low angle laser light scattering photometry coupled with high performance gel chromatography (41) as well as single molecule detection (17) indicate the presence of ␣␤, (␣␤) 2 , and higher order oligomers. Numerous studies in which half-site phosphorylation and halfor quarter-site ATP-analog binding stoichiometries have been interpreted in a model that proposes that the Na,K-ATPase co-exits simultaneously in the E1ATP, E2ATP, or EPATP forms (42).…”