Na,K-ATPase mutations in familial hemiplegic migraine lead to functional inactivation

The Na,K-ATPase is an ion-translocating transmembrane protein that actively maintains the electrochemical gradients for Na⁺ and K⁺ across the plasma membrane. The functional protein is a heterodimer comprising a catalytic α-subunit (four isoforms) and an ancillary β-subunit (three isoforms). Mutations in the α₂-subunit have recently been implicated in familial hemiplegic migraine type 2, but almost no thorough studies of the functional consequences of these mutations have been provided. We investigated the functional properties of the mutations L764P and W887R in the human Na,K-ATPase α₂-subunit upon heterologous expression in Xenopus oocytes. No Na,K-ATPase-specific pump currents could be detected in cells expressing these mutants. The binding of radiolabelled [³H]ouabain to intact cells suggested that this could be due to a lack of plasma membrane expression. However, plasma membrane isolation showed that the mutated pumps are well expressed at the plasma membrane. ⁸⁶Rb⁺-flux and ATPase activity measurements demonstrated that the mutants are inactive. Therefore, the primary disease-causing mechanism is loss-of-function of the Na,K-ATPase α₂-isoform.     

Na,K-ATPase mutations in familial hemiplegic migraine lead to functional inactivation

The Na,K-ATPase is an ion-translocating transmembrane protein that actively maintains the electrochemical gradients for Na+ and K+ across the plasma membrane. The functional protein is a heterodimer comprising a catalytic alpha-subunit (four isoforms) and an ancillary beta-subunit (three isoforms). Mutations in the alpha2-subunit have recently been implicated in familial hemiplegic migraine type 2, but almost no thorough studies of the functional consequences of these mutations have been provided. We investigated the functional properties of the mutations L764P and W887R in the human Na,K-ATPase alpha2-subunit upon heterologous expression in Xenopus oocytes. No Na,K-ATPase-specific pump currents could be detected in cells expressing these mutants. The binding of radiolabelled [3H]ouabain to intact cells suggested that this could be due to a lack of plasma membrane expression. However, plasma membrane isolation showed that the mutated pumps are well expressed at the plasma membrane. 86Rb+-flux and ATPase activity measurements demonstrated that the mutants are inactive. Therefore, the primary disease-causing mechanism is loss-of-function of the Na,K-ATPase alpha2-isoform.     

Lipid-protein interactions with the Na,K-ATPase

Studies of lipid interactions with membranous Na,K-ATPase by using electron spin resonance spectroscopy in conjunction with spin-labelled lipids are reviewed. The lipid stoichiometry, selectivity and exchange dynamics at the lipid-protein interface can be determined, in addition to information on the configuration and rotational dynamics of the protein-associated lipid chains. These parameters, particularly the stoichiometry and selectivity, are related directly to the intramembranous structure of the Na,K-ATPase, and can be used to check the integrity of extensively trypsinised preparations.     

Stabilization of Na,K-ATPase by ionic interactions

The effect of ions on the thermostability and unfolding of Na,K-ATPase from shark salt gland was studied and compared with that of Na,K-ATPase from pig kidney by using differential scanning calorimetry (DSC) and activity assays. In 1 mM histidine at pH 7, the shark enzyme inactivates rapidly at 20 degrees C, as does the kidney enzyme at 42 degrees C (but not at 20 degrees C). Increasing ionic strength by addition of 20 mM histidine, or of 1 mM NaCl or KCl, protects both enzymes against this rapid inactivation. As detected by DSC, the shark enzyme undergoes thermal unfolding at lower temperature (Tm approximately 45 degrees C) than does the kidney enzyme (Tm approximately 55 degrees C). Both calorimetric endotherms indicate multi-step unfolding, probably associated with different cooperative domains. Whereas the overall heat of unfolding is similar for the kidney enzyme in either 1 mM or 20 mM histidine, components with high mid-point temperatures are lost from the unfolding transition of the shark enzyme in 1 mM histidine, relative to that in 20 mM histidine. This is attributed to partial unfolding of the enzyme due to a high hydrostatic pressure during centrifugation of DSC samples at low ionic strength, which correlates with inactivation measurements. Addition of 10 mM NaCl to shark enzyme in 1 mM histidine protects against inactivation during centrifugation of the DSC sample, but incubation for 1 h at 20 degrees C prior to addition of NaCl results in loss of components with lower mid-point temperatures within the unfolding transition. Cations at millimolar concentration therefore afford at least two distinct modes of stabilization, likely affecting separate cooperative domains. The different thermal stabilities and denaturation temperatures of the two Na,K-ATPases correlate with the respective physiological temperatures, and may be attributed to the different lipid environments.     

Stabilization of Na,K-ATPase by ionic interactions

The effect of ions on the thermostability and unfolding of Na,K-ATPase from shark salt gland was studied and compared with that of Na,K-ATPase from pig kidney by using differential scanning calorimetry (DSC) and activity assays. In 1 mM histidine at pH 7, the shark enzyme inactivates rapidly at 20 °C, as does the kidney enzyme at 42 °C (but not at 20 °C). Increasing ionic strength by addition of 20 mM histidine, or of 1 mM NaCl or KCl, protects both enzymes against this rapid inactivation. As detected by DSC, the shark enzyme undergoes thermal unfolding at lower temperature (T_m ≈ 45 °C) than does the kidney enzyme (T_m ≈ 55 °C). Both calorimetric endotherms indicate multi-step unfolding, probably associated with different cooperative domains. Whereas the overall heat of unfolding is similar for the kidney enzyme in either 1 mM or 20 mM histidine, components with high mid-point temperatures are lost from the unfolding transition of the shark enzyme in 1 mM histidine, relative to that in 20 mM histidine. This is attributed to partial unfolding of the enzyme due to a high hydrostatic pressure during centrifugation of DSC samples at low ionic strength, which correlates with inactivation measurements. Addition of 10 mM NaCl to shark enzyme in 1 mM histidine protects against inactivation during centrifugation of the DSC sample, but incubation for 1 h at 20 °C prior to addition of NaCl results in loss of components with lower mid-point temperatures within the unfolding transition. Cations at millimolar concentration therefore afford at least two distinct modes of stabilization, likely affecting separate cooperative domains. The different thermal stabilities and denaturation temperatures of the two Na,K-ATPases correlate with the respective physiological temperatures, and may be attributed to the different lipid environments.     

Kinetic alterations due to a missense mutation in the Na,K-ATPase alpha2 subunit cause familial hemiplegic migraine type 2

A number of missense mutations in the ATP1A2 gene, which encodes the Na,K-ATPase alpha2 subunit, have been identified in familial hemiplegic migraine with aura. Loss of function and haploinsufficiency have been the suggested mechanisms in mutants for which functional analysis has been reported. This paper describes a kinetic analysis of mutant T345A, recently identified in a detailed genetic analysis of a large Finnish family (Kaunisto, M. A., Harno, H., Vanmolkot, K. R., Gargus, J. J., Sun, G., Hamalainen, E., Liukkonen, E., Kallela, M., van den Maagdenberg, A. M., Frants, R. R., Farkkila, M., Palotie, A., and Wessman, M. (2004) Neurogenetics 5, 141-146). Introducing T345A into the conserved rat alpha2 enzyme does not alter cell growth or catalytic turnover but causes a substantial decrease in apparent K+ affinity (2-fold increase in K0.5(K+)). In view of the location of Thr-345 in the cytoplasmic stalk domain adjacent to transmembrane segment 4, the 2-fold increase in K0.5(K+) is probably due to T345A replacement altering K+ occlusion/deocclusion. Faster K+ deocclusion of the mutant via the E2(K) + ATP --> E1.ATP + K+ partial reaction is evidenced in (i) a marked increase (300%) in K+ stimulation of Na-ATPase at micromolar ATP, (ii) a 4-fold decrease in KATP, and (iii) only a modest increase (approximately 3-fold) in I50 for vanadate, which was used as a probe of the steady state E1/E2 conformational equilibrium. We suggest that the decreased apparent K+ affinity is the basis for a reduced rate of extracellular K+ removal, which delays the recovery phase of nerve impulse transmission in the central nervous system and, thereby, the clinical picture of migraine with aura. This is the first demonstration of a mutation that leads to a disease associated with a kinetically altered but fully functional Na,K-ATPase, refining the molecular mechanism of pathogenesis in familial hemiplegic migraine.     

Biochemical characterization of sporadic/familial hemiplegic migraine mutations

Sporadic hemiplegic migraine type 2 (SHM2) and familial hemiplegic migraine type 2 (FHM2) are rare forms of hemiplegic migraine caused by mutations in the Na⁺,K⁺-ATPase α2 gene. Today, more than 70 different mutations have been linked to SHM2/FHM2, randomly dispersed over the gene. For many of these mutations, functional studies have not been performed. Here, we report the functional characterization of nine SHM2/FHM2 linked mutants that were produced in Spodoptera frugiperda (Sf)9 insect cells. We determined ouabain binding characteristics, apparent Na⁺ and K⁺ affinities, and maximum ATPase activity. Whereas membranes containing T345A, R834Q or R879W possessed ATPase activity significantly higher than control membranes, P796S, M829R, R834X, del 935–940 ins Ile, R937P and D999H membranes showed significant loss of ATPase activity compared to wild type enzyme. Further analysis revealed that T345A and R879W showed no changes for any of the parameters tested, whereas mutant R834Q possessed significantly decreased Na⁺ and increased K⁺ apparent affinities as well as decreased ATPase activity and ouabain binding. We hypothesize that the majority of the mutations studied here influence interdomain interactions by affecting formation of hydrogen bond networks or interference with the C-terminal ion pathway necessary for catalytic activity of Na⁺,K⁺-ATPase, resulting in decreased functionality of astrocytes at the synaptic cleft expressing these mutants.     

Role of the transmembrane domain of FXYD7 in structural and functional interactions with Na,K-ATPase

Members of the FXYD family are tissue-specific regulators of the Na,K-ATPase. Here, we have investigated the contribution of amino acids in the transmembrane (TM) domain of FXYD7 to the interaction with Na,K-ATPase. Twenty amino acids of the TM domain were replaced individually by tryptophan, and combined mutations and alanine insertion mutants were constructed. Wild type and mutant FXYD7 were expressed in Xenopus oocytes with Na,K-ATPase. Mutational effects on the stable association with Na,K-ATPase and on the functional regulation of Na,K-ATPase were determined by co-immunoprecipitation and two-electrode voltage clamp techniques, respectively. Most residues important for the structural and functional interaction of FXYD7 are clustered in a face of the TM helix containing the two conserved glycine residues, but others are scattered over two-thirds of the FXYD TM helix. Ile-35, Ile-43, and Ile-44 are only involved in the stable association with Na,K-ATPase. Glu-26, Met-30, and Ile-44 are important for the functional effect and/or the efficient association of FXYD7 with Na,K-ATPase, consistent with the prediction that these amino acids contact TM domain 9 of the alpha subunit (Li, C., Grosdidier, A., Crambert, G., Horisberger, J.-D., Michielin, O., and Geering, K. (2004) J. Biol. Chem. 279, 38895-38902). Several amino acids that are not implicated in the efficient association of FXYD7 with the Na,K-ATPase are specifically involved in the functional effect of FXYD7. Leu-32 and Phe-37 influence the apparent affinity for external K+, whereas Val-28 and Ile-42 are implicated in the apparent affinity for both external K+ and external Na+. These amino acids act in a synergistic way. These results highlight the important structural and functional role of the TM domain of FXYD7 and delineate the determinants that mediate the complex interactions of FXYD7 with Na,K-ATPase.     

ATP-induced conformational changes of the nucleotide-binding domain of Na,K-ATPase

The Na,K-ATPase hydrolyzes ATP to drive the coupled extrusion and uptake of Na+ and K+ ions across the plasma membrane. Here, we report two high-resolution NMR structures of the 213-residue nucleotide-binding domain of rat alpha1 Na,K-ATPase, determined in the absence and the presence of ATP. The nucleotide binds in the anti conformation and shows a relative paucity of interactions with the protein, reflecting the low-affinity ATP-binding state. Binding of ATP induces substantial conformational changes in the binding pocket and in residues located in the hinge region connecting the N- and P-domains. Structural comparison with the Ca-ATPase stabilized by the inhibitor thapsigargin, E2(TG), and the model of the H-ATPase in the E1 form suggests that the observed changes may trigger the series of events necessary for the release of the K+ ions and/or disengagement of the A-domain, leading to the eventual transfer of the gamma-phosphate group to the invariant Asp369.     

The Na,K-ATPase

The energy dependent exchange of cytoplasmic Na⁺ for extracellular K⁺ in mammalian cells is due to a membrane bound enzyme system, the Na,K-ATPase. The exchange sustains a gradient for Na⁺ into and for K⁺ out of the cell, and this is used as an energy source for creation of the membrane potential, for its de- and repolarisation, for regulation of cytoplasmic ionic composition and for transepithelial transport. The Na,K-ATPase consists of two membrane spanning polypeptides, an -subunit of 112-kD and a -subunit, which is a glycoprotein of 35-kD. The catalytic properties are associated with the -subunit, which has the binding domain for ATP and the cations. In the review, attention will be given to the biochemical characterization of the reaction mechanism underlying the coupling between hydrolysis of the substate ATP and transport of Na⁺ and K⁺.     

FXYD proteins stabilize Na,K-ATPase: amplification of specific phosphatidylserine-protein interactions

FXYD proteins are a family of seven small regulatory proteins, expressed in a tissue-specific manner, that associate with Na,K-ATPase as subsidiary subunits and modulate kinetic properties. This study describes an additional property of FXYD proteins as stabilizers of Na,K-ATPase. FXYD1 (phospholemman), FXYD2 (γ subunit), and FXYD4 (CHIF) have been expressed in Escherichia coli and purified. These FXYD proteins associate spontaneously in vitro with detergent-soluble purified recombinant human Na,K-ATPase (α1β1) to form α1β1FXYD complexes. Compared with the control (α1β1), all three FXYD proteins strongly protect Na,K-ATPase activity against inactivation by heating or excess detergent (C₁₂E₈), with effectiveness FXYD1 > FXYD2 ≥ FXYD4. Heating also inactivates E₁ ↔ E₂ conformational changes and cation occlusion, and FXYD1 protects strongly. Incubation of α1β1 or α1β1FXYD complexes with guanidinium chloride (up to 6 m) causes protein unfolding, detected by changes in protein fluorescence, but FXYD proteins do not protect. Thus, general protein denaturation is not the cause of thermally mediated or detergent-mediated inactivation. By contrast, the experiments show that displacement of specifically bound phosphatidylserine is the primary cause of thermally mediated or detergent-mediated inactivation, and FXYD proteins stabilize phosphatidylserine-Na,K-ATPase interactions. Phosphatidylserine probably binds near trans-membrane segments M9 of the α subunit and the FXYD protein, which are in proximity. FXYD1, FXYD2, and FXYD4 co-expressed in HeLa cells with rat α1 protect strongly against thermal inactivation. Stabilization of Na,K-ATPase by three FXYD proteins in a mammalian cell membrane, as well the purified recombinant Na,K-ATPase, suggests that stabilization is a general property of FXYD proteins, consistent with a significant biological function.     

Anion interactions with Na,K-ATPase: simultaneous binding of nitrate and eosin

Nucleotide binding affinity to Na,K-ATPase is reduced by a number of anions such as nitrate and perchlorate in comparison with affinity in the presence of chloride (all with sodium as the cation). The reduction correlates with the position of these anions in the Hofmeister series. Transient kinetic experiments using the fluorescent dye eosin—which binds to the nucleotide site of the Na,K-ATPase—show that simultaneous anion binding, exemplified with nitrate, and eosin binding is possible. The effect of nitrate on eosin binding is reflected in a decreased binding-rate constant and an increased dissociation rate constant, leading to a decreased equilibrium binding constant for eosin. Since eosin binding is analogous with nucleotide binding to Na,K-ATPase, the results suggest the simultaneous presence of nucleotide and anion binding sites.Abbreviations E₁ the protein conformation in Na⁺ - E₂ the enzyme conformation in K⁺ - Eo eosin (tetrabromofluorescein) - F fluorescence - I ionic strength - k_i rate constant - K_i equilibrium dissociation constant - K_i,0 equilibrium dissociation constant at zero ionic strength - N nitrate - z_i net charge - charge product z_i·z_j     

Phosphorylation of the Na,K-ATPase and the H,K-ATPase

Phosphorylation is a widely used, reversible means of regulating enzymatic activity. Among the important phosphorylation targets are the Na⁺,K⁺- and H⁺,K⁺-ATPases that pump ions against their chemical gradients to uphold ionic concentration differences over the plasma membrane. The two pumps are very homologous, and at least one of the phosphorylation sites is conserved, namely a cAMP activated protein kinase (PKA) site, which is important for regulating pumping activity, either by changing the cellular distribution of the ATPases or by directly altering the kinetic properties as supported by electrophysiological results presented here. We further review the other proposed pump phosphorylations.     

Diverse functional consequences of mutations in the Na+/K+-ATPase alpha2-subunit causing familial hemiplegic migraine type 2

Mutations in ATP1A2, the gene coding for the Na(+)/K(+)-ATPase alpha(2)-subunit, are associated with both familial hemiplegic migraine and sporadic cases of hemiplegic migraine. In this study, we examined the functional properties of 11 ATP1A2 mutations associated with familial or sporadic hemiplegic migraine, including missense mutations (T263M, T376M, R383H, A606T, R763H, M829R, R834Q, R937P, and X1021R), a deletion mutant (del(K935-S940)ins(I)), and a frameshift mutation (S966fs). According to the Na(+)/K(+)-ATPase crystal structure, a subset of the mutated residues (Ala(606), Arg(763), Met(829), and Arg(834)) is involved in important interdomain H-bond networks, and the C terminus of the enzyme, which is elongated by the X1021R mutation, has been implicated in voltage dependence and formation of a third Na(+)-binding site. Upon heterologous expression in Xenopus oocytes, the analysis of electrogenic transport properties, Rb(+) uptake, and protein expression revealed pronounced and markedly diverse functional alterations in all ATP1A2 mutants. Abnormalities included a complete loss of function (T376M), impaired plasma membrane expression (del(K935-S940)ins(I) and S966fs), and altered apparent affinities for extracellular cations or reduced enzyme turnover (R383H, A606T, R763H, R834Q, and X1021R). In addition, changes in the voltage dependence of pump currents and the increased rate constants of the voltage jump-induced redistribution between E(1)P and E(2)P states were observed. Thus, mutations that disrupt distinct interdomain H-bond patterns can cause abnormal conformational flexibility and exert long range consequences on apparent cation affinities or voltage dependence. Of interest, the X1021R mutation severely impaired voltage dependence and kinetics of Na(+)-translocating partial reactions, corroborating the critical role of the C terminus of Na(+)/K(+)-ATPase in these processes.     

Identification of the cofilin-binding sites in the large cytoplasmic domain of Na,K-ATPase

Na,K-ATPase, an alpha, beta heterodimer, is found in the plasma membrane of all animal cells. The alpha chain is believed to have 10 transmembrane regions and a large cytoplasmic domain between the 4th and 5th transmembrane regions (H4-H5). In our previous report, the large (3rd) cytoplasmic domains of the alpha1 and alpha2 isoform were found to interact with cofilin, an actin-modulating protein, by the yeast two-hybrid system. Here we show that cofilin interacts only with the 3rd cytoplasmic domain of the alpha2 subunit but not with the 2nd, 4th, and 5th cytoplasmic domains or the cytoplasmic region of the beta subunit of Na,K-ATPase. We also demonstrate that cofilin interacts with the large cytoplasmic domains of the alpha1, alpha2 and alpha3 isoforms of Na,K-ATPase, but not with those of glucose transporter 1, glucose transporter 4, cystic fibrosis transmembrane conductance regulator and plasma membrane Ca-ATPase. We introduced 10 mutations into the 3rd cytoplasmic domain of Na,K-ATPase to identify the binding sites with cofilin. Eight of these mutants were single amino acid substitutions (R417Q, K470Q, K654G, D672A, K691A, R700G, R700A and D710G) and two were double mutant (K654GR700G and K719AK720A). Analysis of the activity of the reporter gene of these mutants shows that residues D672 and R700 of the 3rd cytoplasmic domain of Na,K-ATPase are involved in the interaction with cofilin.     

Evidence for essential carboxyls in the cation-binding domain of the Na,K-ATPase.

Treatment of isolated canine renal Na,K-ATPase with a stable diazomethane analog, 4-(diazomethyl)-7-(diethylamino)-coumarin (DEAC), results in enzyme inactivation. The inactivation rate was dramatically increased when the enzyme was treated with DEAC in the presence of ATP and Mg2+ (in imidazole buffer) or Pi and Mg2+, conditions which produce enzyme phosphorylation. Inactivation in the presence of Pi and Mg2+ could be partially prevented by Na+ and almost completely prevented by K+. The quantity of DEAC covalently bound to the Na,K-ATPase was determined spectrophotometrically. The extent of inactivation was linearly related to the amount of K-protectable DEAC incorporation. Complete inactivation of ATPase activity occurred with 2.14 +/- 0.18 nmol of DEAC covalently bound/mg of protein. This suggests that only 1 or 2 carboxyl residues/catalytic center (estimated by high affinity ADP binding) are involved in the modification leading to inactivation. The modified enzyme exhibited normal levels of high affinity [3H]ADP (and hence ATP) binding, thus, the nucleotide-binding domain of the enzyme seems unaffected by the modification. In contrast, under conditions where native enzyme was able to occlude 3.82 nmol of K+ ions/mg of protein, DEAC-modified enzyme occluded only 0.33 nmol of K+ ions. Na+ occlusion by the enzyme (in the presence of oligomycin) was also reduced (by 80%) following treatment with DEAC. Phosphorylation by [32P]inorganic phosphate and Na(+)-activated phosphorylation of the modified enzyme with [32P]ATP yielded reduced levels of phosphoenzyme (about 36%) compared to native enzyme. The DEAC-modified [32P]phosphoenzyme formed from [32P]ATP was insensitive to the addition of K+ ions, under conditions which led to the rapid hydrolysis of native phosphoenzyme. Gel electrophoresis of modified protein revealed strong fluorescence labeling of the alpha-subunit, which was substantially reduced if treatment with DEAC was performed in the presence of K+ ions. Partial tryptic digestion and electrophoretic analysis revealed normal degradation patterns in the presence of ADP (E1 form) but the typical patterns, seen with K+ ions (E2K) or Na+ ions (E1Na) in native enzyme, were absent. A typical E2-like tryptic degradation pattern was seen, however, in the presence of vanadate ions and ouabain, suggesting that the modification does not freeze the enzyme in an E1 conformation and that the enzyme is still able to undergo the E1E2 conformational transition after modification. Our results suggest that a small number of carboxyl residues in the sodium pump alpha-subunit (perhaps one) are essential for K+ and Na+ binding and stabilizing the occluded enzyme cation forms. Esterification of the carboxyl groups by DEAC inactivates the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Familial hemiplegic migraine type 1 mutations W1684R and V1696I alter G protein-mediated regulation of CaV2.1 voltage-gated calcium channels

Familial hemiplegic migraine type 1 (FHM-1) is a monogenic form of migraine with aura that is characterized by recurrent attacks of a typical migraine headache with transient hemiparesis during the aura phase. In a subset of patients, additional symptoms such as epilepsy and cerebellar ataxia are part of the clinical phenotype. FHM-1 is caused by missense mutations in the CACNA1A gene that encodes the pore-forming subunit of Ca_V2.1 voltage-gated Ca^2 + channels. Although the functional effects of an increasing number of FHM-1 mutations have been characterized, knowledge on the influence of most of these mutations on G protein regulation of channel function is lacking. Here, we explored the effects of G protein-dependent modulation on mutations W1684R and V1696I which cause FHM-1 with and without cerebellar ataxia, respectively. Both mutations were introduced into the human Ca_V2.1α₁ subunit and their functional consequences investigated after heterologous expression in human embryonic kidney 293 (HEK‐293) cells using patch-clamp recordings. When co-expressed along with the human μ-opioid receptor, application of the agonist [d‐Ala2, N‐MePhe4, Gly‐ol]‐enkephalin (DAMGO) inhibited currents through both wild-type (WT) and mutant Ca_V2.1 channels, which is consistent with the known modulation of these channels by G protein-coupled receptors. Prepulse facilitation, which is a way to characterize the relief of direct voltage-dependent G protein regulation, was reduced by both FHM-1 mutations. Moreover, the kinetic analysis of the onset and decay of facilitation showed that the W1684R and V1696I mutations affect the apparent dissociation and reassociation rates of the Gβγ dimer from the channel complex, suggesting that the G protein-Ca^2 + channel affinity may be altered by the mutations. These biophysical studies may shed new light on the pathophysiology underlying FHM-1.     

Two-dimensional crystalline arrays of Na,K-ATPase with new subunit interactions induced by cobalt-tetrammine-ATP

Purified membrane-bound Na,K-ATPase incubated with cobalt-tetrammine-ATP [Co(NH₃)₄ATP], which is a stable MgATP complex analog, shows two new types of membrane crystals, a new p21 form and a p4 form. The building blocks of the crystalline arrays correspond to (αβ)₂ dimers of the enzyme protein suggesting that α-α interaction may be important in the pumping process.