Is the Sodium Pump a Functional Dimer?

To investigate the relationship between the high and the low affinity ATP-binding site, which appears during the Na ؉ /K ؉ -ATPase reaction, four amino acids were mutated, the side chains of which are exposed to inside of the ATP-binding pocket. Six mutants, F475Y, K480A, K480E, K501A, K501E, and R544A, where the numbers correspond to the pig Na ؉ /K ؉ -ATPase ␣-chain, were expressed in HeLa cells. The apparent affinities were determined by high affinity ATP-dependent phosphorylation and by the low affinity activation of Na ؉ /K ؉ -ATPase or low affinity ATP inhibition of K ؉ -para-nitrophenylphosphatase (pNPPase). For the mutants K480A and K501A, little affinity change was detected for either the high affinity or the low affinity effect. In contrast, the other four mutants reduced both apparent affinities. Strikingly, R544A had a 30-fold greater effect on the high affinity ATP site than the low affinity site. For the F475Y mutant, it is likely that there was a greater effect on the low affinity site than the high affinity site, but for both F475Y and K480E the affinity for the low affinity ATP effect was reduced so much that it was not possible to estimate a K 0.5 . However, both the affinities for the K480E were reduced to ϳ1/20. The turnover number of the Na ؉ /K ؉ -ATPase and the apparent affinity for Na ؉ and pNPP was reduced slightly or not at all for these mutants, but the turnover number of K ؉ -pNPPase and the apparent affinity for K ؉ were increased. These and other data suggest the presence of only one ATP-binding site, which can change its conformation to accept ATP with a high and low affinity. The requirement of Arg-544 and possibly Lys-501 is more important in forming a high affinity ATP binding conformation, and Phe-475 and possibly Lys-480 are more important in forming the low affinity ATP binding conformation.

Section: Nasupporting

confidence: 84%

Section: Namentioning

confidence: 99%

Replacement of Several Single Amino Acid Side Chains Exposed to the Inside of the ATP-binding Pocket Induces Different Extents of Affinity Change in the High and Low Affinity ATP-binding Sites of Rat Na/K-ATPase

Teramachi

Imagawa

Kaya

et al. 2002

“…The coexistence of both high and low affinity sites for ATP in time and in different locations was proposed (26). Other experimental data that are consistent with multiple active sites have been also reported (18, 19, 25-30, 32, 40 -45).…”

Section: Relevance Of Modification Of Atp-protectable Lys Residues Wimentioning

confidence: 67%

Half-site Modification of Lys-480 of the Na+,K+-ATPase α-Chain with Pyridoxal 5′-Diphospho-5′-adenosine Reduces ATP-dependent Phosphorylation Stoichiometry from Half to a Quarter

Tsuda¹,

Kaya²,

Yokoyama³

et al. 1998

Pig and dog kidney Na؉ ,K ؉ -ATPase preparations, irrespective of specific activity, showed ϳ0.5 mol of maximum phosphorylation/mol ␣-chain for ATP or acetyl phosphate (AcP) at steady state conditions. Pyridoxal 5-diphospho-5-adenosine (AP 2 PL)-treated pig kidney enzymes containing ϳ0.5 mol of AP 2 PL probe at Lys-480/ mol (Tsuda, T., Kaya, S., Funatsu, H., Hayashi, Y., and Taniguchi, K. (1998) J. Biochem. (Tokyo) 123, 169 -174) showed a quarter-site phosphorylation by ATP and halfsite phosphorylation from AcP. The addition of 10 M ATP to the Mg 2؉ -Na ؉ -bound AP 2 PL enzyme induced rapid quarter-site phosphorylation (47/s), followed by two different AP 2 PL fluorescence changes, a rapid decrease (29/s) and a slow increase (1.1/s). The addition of 1 mM AcP to the Mg 2؉ -Na ؉ -bound AP 2 PL enzyme induced a slow half-site phosphorylation (3/s), followed by a monophasic AP 2 PL fluorescence increase (1.2/s). After treatment of the AP 2 PL enzyme with fluorescein 5-isothiocyanate to modify Lys-501 fully, the Mg 2؉ -Na ؉ -dependent phosphorylation capacity from ATP of the resulting AP 2 PL-fluorescein 5-isothiocyanate enzyme was reduced to ϳ6% without significant changes in half-site phosphorylation capacity with respect to AcP, dynamic AP 2 PL fluorescence change by ATP and change by AcP. These data and others support the hypothesis that the functional membrane-bound Na ؉ ,K ؉ -ATPase has tetrameric properties.The transport of sodium and potassium ions coupled with the hydrolysis of ATP is performed by Na ϩ ,K ϩ -ATPase (1-6), which shows high affinity ATP binding for phosphorylation in the presence of Mg 2ϩ and Na ϩ and low affinity binding for the deocclusion of K ϩ (2, 4). To understand the mechanism of energy transduction for this enzyme, a detailed knowledge of ATP-induced conformational changes is essential. ATP hydrolysis is accompanied by conformational changes in the enzyme, and several conformational states in the reaction cycle have been characterized using an N-(p-(2-benzimidazolyl)phenyl)-maleimide (BIPM) 1 probe at Cys-964 (7-12), which seems to be present near the extracellular border of transmembrane segment M9 (13). An ATP protectable modification of the enzyme by FITC (14) at Lys-501 (15) and pyridoxal 5Ј-phosphate or AP 2 PL at 17), respectively, caused a decrease in Na ϩ ,K ϩ -ATPase activity. The loss of Na ϩ ,K ϩ -ATPase activity is generally thought to be due to the loss of ATP binding to the enzyme. However, such preparations retain both K ϩ -dependent p-nitrophenylphosphatase activity (14, 17) as well as phosphorylation capacity derived from acetyl phosphate (AcP) in the presence of Mg 2ϩ and Na ϩ (10,11,17). It has been shown that ATP is capable of inducing pyridoxal 5Ј-phosphate and AP 2 PL fluorescence changes (17) and that the ATP or trinitrophenyl-ADP (TNP-ADP) inhibits the K ϩ -p-nitrophenylphosphatase activity of the enzyme when modified with FITC at 19).These data suggest that both probes in the vicinity of the ATP binding domain of the enzyme are affected by ATP binding or ATP in...

“…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). The existence of such oligomerization of the Na,KATPase in native membranes provides a mechanism that can explain the recent observation of dominant-negative ␣-mutants in Drosophila (43).…”

Section: Resultsmentioning

confidence: 99%

Oligomerization of the Na,K-ATPase in Cell Membranes

Laughery

Todd

Kaplan

2004

The higher order oligomeric state of the Na,K-ATPase ␣␤ heterodimer in cell membranes is the subject of controversy. We have utilized the baculovirus-infected insect cell system to express Na,K-ATPase with ␣-subunits bearing either His 6 or FLAG epitopes at the carboxyl terminus. Each of these constructs produced functional Na,K-ATPase ␣␤ heterodimers that were delivered to the plasma membrane (PM). Cells were simultaneously co-infected with viruses encoding ␣-His/␤ and ␣-FLAG/␤ Na,K-ATPases. Co-immunoprecipitation of the Histagged ␣-subunit in the endoplasmic reticulum (ER) and PM fractions of co-infected cells by the anti-FLAG antibody demonstrates that protein-protein associations exist between these heterodimers. This suggests the Na,KATPase is present in cell membranes in an oligomeric state of at least (␣␤) 2 composition. Deletion of 256 amino acid residues from the central cytoplasmic loop of the ␣-subunit results in the deletion ␣-4,5-loop-less (␣-4,5LL), which associates with ␤ but is confined to the ER. Co-immunoprecipitation demonstrates that when this inactive ␣-4,5LL/␤ heterodimer is co-expressed with wild-type ␣␤, oligomers of wild-type ␣␤ and ␣-4,5LL/␤ form in the ER, but the ␣-4,5LL mutant remains retained in the ER, and the wild-type protein is still delivered to the PM. We conclude that the Na,K-ATPase is present as oligomers of the monomeric ␣␤ heterodimer in native cell membranes.