Purification and characterization of (Na+, K+)-ATPase. V. Conformational changes in the enzyme Transitions between the Na-form and the K-form studied with tryptic digestion as a tool.

1. Purified (Na+, K+)-ATPase consisting of membrane fragments was digested with trypsin. The time course of enzyme inactivation was related to the electrophoretic pattern of native and cleaved proteins remaining in the membrane. 2. Differences in both the inactivation kinetics and the cleavage of the large chain (mol. wt 98 000) allow distinction of two patterns of tryptic digestion of (Na+, K+)-ATPase seen with Na+ or K+ in the medium. 3. With K+, the inactivation of (Na+, K+)-ATPase is linear with time in semilogarithmic plots and the activity is lost in parallel with cleavage of the large chain to fragments with molecular weights 58 000 and 48 000. 4. With Na+, the inactivation curves are biphasic. In the initial phase of rapid inactivation, 50% of the activity is lost with minor changes in the composition of the large chain. In the final phase, the large chain is cleaved at a low rate to a fragment with a molecular weight of 78 000. 5. It is concluded that the regions of the large chain exposed in the presence of K+ are distinct from the regions exposed in presence of Na+ and that two conformations of (Na+, K+)-ATPase can be sensed with trypsin, a (t)K-form and a (t)Na-form. 6. Reaction of the (t)K-form with ATP cause transition to the (t)Na-form. Relatively high concentrations of ATP are required and Mg2+ is not necessary. Phosphorylation of (Na+, K+)-ATPase is accompanied by transition from the (t)Na-form to the (t)K-form. Previous kinetic data suggest that these conformational changes are accompanied by shifts in the affinities of the enzyme for Na+ and K+.     

H+ transport by reconstituted gastric (H+ + K+)-ATPase

Gastric (H+ + K+)-ATPase was reconstituted into artificial phosphatidylcholine/cholesterol liposomes by means of a freeze-thaw-sonication technique. Upon addition of MgATP, active H+ transport was observed, with a maximal rate of 2.1 mumol X mg-1 X min-1, requiring the presence of 100 mM K+ at the intravesicular site. However, in the absence of ATP an H+-K+ exchange with a maximal rate of 0.12 mumol X mg-1 X min-1 was measured, which could be inhibited by the well-known ATPase inhibitors vanadate and omeprazole, giving the first evidence of a passive K+-H+ exchange function of gastric (H+ + K+)-ATPase. An Na+-H+ exchange activity was also measured, which was fully inhibited by 1 mM amiloride. Simultaneous reconstitution of Na+/H+ antiport and (H+ + K+)-ATPase could explain why reconstituted ATPase appeared less cation-specific than the native enzyme (Rabon, E.C., Gunther, R.B., Soumarmon, A., Bassilian, B., Lewin, M.J.M. and Sachs, G. (1985) J. Biol. Chem. 260, 10200-10212).     

Transport ratios of reconstituted (H+ + K+)-ATPase

Gastric (H+ + K+)-ATPase was reconstituted into artificial phosphatidylcholine/cholesterol vesicles by means of a freeze-thaw-sonication procedure. The passive and active transport mediated by these vesicles were measured (Skrabanja, A.T.P., Asty, P., Soumarmon, A., De Pont, J.J.H.H.M. and Lewin, M.J.M. (1986) Biochim. Biophys. Acta 860, 131-136). To determine real initial velocities, the proteoliposomes were separated from non-incorporated enzyme, by means of centrifugation on a sucrose gradient. The purified proteoliposomes were used to measure active H+ and Rb+ transport, giving at room-temperature velocities of 46.3 and 42.5 mumol per mg per h, respectively. A transport ratio of two cations per ATP hydrolyzed was also measured. These figures indicate that the enzyme catalyzes an electroneutral H+-Rb+ exchange.     

Lack of immunological cross reactivity between the transport enzymes (Na+ + K+)-ATPase and (K+ + H+)-ATPase

Goat antisera against (Na⁺ + K⁺)-ATPase and its isolated subunits and against (K⁺ + H⁺)-ATPase have been prepared in order to test for immune cross-reactivity between the two enzymes, whose catalytic subunits show great chemical similarity. None of the (Na⁺ + K⁺)-ATPase antisera cross-reacted with (K⁺ + H⁺)-ATPase or inhibited its enzyme activity. The same was true for the (K⁺ + H⁺)-ATPase antiserum with regard to (Na⁺ + K⁺)-ATPase and its subunits and its enzyme activity. So not withstanding the chemical similarity of their subunits, there is no immunological cross-reactivity between these two plasma membrane ATPases.Number LIII in the series Studies on (Na⁺ + K⁺)-Activated ATPase.     

Conformational transitions between Na+-bound and K+-bound forms of (Na+ + K+)-ATPase, studied with formycin nucleotides.

1. Fluorescence measurements have shown that formycin triphosphate (FTP) or formycin diphosphate (FDP) bound to (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in Na+-containing media can be displaced by the following ions (listed in order of effectiveness): Tl+, K+, Rb+, NH4+, Cs+. 2. The differences between the nucleotide affinities displayed by the enzyme in predominantly Na+ and predominantly K+ media in the absence of phosphorylation, are thought to reflect changes in enzyme conformation. These changes can therefore be monitored by observing the changes in fluorescence that accompany net binding or net release of formycin nucleotides. 3. The transition from a K+-bound form (E2-(K)) to an Na+-bound form (E1-Na) is remarkably slow at low nucleotide concentrations, but is accelerated if the nucleotide concentration is increased. This suggests that the binding of nucleotide to a low-affinity site on E2-(K) accelerates its conversion to E1-Na; it supports the hypothesis that during the normal working of the pump, ATP, acting at a low affinity site, accelerates the conversion of dephosphoenzyme, newly formed by K+-catalysed hydrolysis of E2P, to a form in which it can be phosphorylated in the presence of Na+. 4. The rate of the reverse transformation, E1-Na to E2-(K), varies roughly linearly with the K+ concentration up to the highest concentration at which the rate can be measured (15 mM). Since much lower concentrations of K+ are sufficient to displace the equilibrium to the K-form, we suggest that the sequence of events is: (i) combination of K+ with low affinity (probably internal) binding sites, followed by (ii) spontaneous conversion of the enzyme to a form, E2-(K), containing occluded K+. 5. Mg2+ or oligomycin slows the rate of conversion of E1-Na to E2-(K) but does not significantly affect the rate of conversion of E2-(K) to E1-Na. 6. In the light of these and previous findings, we propose a model for the sodium pump in which conformational changes alternate with trans-phosphorylations, and the inward and outward fluxes of both Na+ and K+ each involve the transfer of a phosphoryl group as well as a change in conformation between E1 and E2 forms of the enzyme or phosphoenzyme.     

Conformational states of sarcoplasmic reticulum Ca2+-ATPase as studied by proteolytic cleavage

Summary Conformational states in sarcoplasmic reticulum Ca²⁺-ATPase have been examined by tryptic and chymotryptic cleavage. High affinity Ca²⁺ binding (E₁ state) exposes a peptide bond in the A fragment of the polypeptide chain to trypsin. Absence of Ca²⁺ (E₂ state) exposes bonds in the B fragment, which are protected by binding of Mg²⁺ or ATP. After phosphorylation from ATP the tryptic cleavage pattern depends on the predominant phosphoenzyme species present. ADP-sensitive E₁P and ADP-insensitive E₂P have cleavage patterns identical to those of unphosphorylated E₁ and E₂, respectively, indicating that two major conformational states are involved in Ca²⁺ translocation. The transition from E₁P to E₂P is inhibited by secondary tryptic splits in the A fragment, suggesting that parts of this fragment are of particular importance for the energy transduction process.The tryptic cleavage patterns of phosphorylated forms of detergent solubilized monomeric Ca²⁺-ATPase were similar to those of the membrane-bound enzyme, indicating that Ca²⁺ translocation depends mainly on structural changes within a single peptide chain. On the other hand, the protection of the second cleavage site as observed after vanadate binding to membranous Ca²⁺-ATPase could not be achieved in the soluble monomeric enzyme. Shielding of this peptide bond may therefore be due to protein-protein interactions in the semicrystalline state of the vanadate-bound Ca²⁺-ATPase in membranous form.     

Papain fragmentation of the gastric (H+ + K+)-ATPase

Membrane-bound (H+ + K+)-ATPase purified from hog gastric mucosa was exposed to limited papain digestion. Such treatment resulted in a rapid inhibition of the K+-stimulated adenosine triphosphatase and p-nitrophenyl phosphatase activities, with about 90% of these activities lost after 3 min incubation at 37 degrees C with 0.1 units of papain per mg of enzyme protein. Parallel to the inhibition of the enzyme activities, there was a production of a 77 kDa membrane-bound fragment containing the aspartyl phosphate residue of the phospho-intermediate. This fragment accounted for about 45% of the total enzyme protein after the 3 min papain treatment. The digestion barely affected the steady-state level of phosphorylation, allowed the aspartyl phosphate of the 77 kDa fragment to undergo the transition to the E2P form, and did not significantly alter the fraction of ADP-sensitive phosphoenzyme. The presence of KCl, however, depressed the steady-state level of phosphoenzyme formed from [gamma-32P]ATP considerably less than that of the control enzyme. With further exposure to papain the 77 kDa peptide became fragmented into a 28 kDa soluble peptide that retained the phosphorylating site. Binding of fluorescein 5'-isothiocyanate (FITC) to the native enzyme did not affect the sites of papain hydrolysis because the same peptide fragments were obtained. The FITC reaction site was also in the 28 kDa soluble peptide fragment.     

Solubilization of active (H+ + K+)-ATPase from gastric membrane

(H+ + K+)-ATPase-enriched membranes were prepared from hog gastric mucosa by sucrose gradient centrifugation. These membranes contained Mg2+-ATPase and p-nitrophenylphosphatase activities (68 +/- 9 mumol Pi and 2.9 +/- 0.6 mumol p-nitrophenol/mg protein per h) which were insensitive to ouabain and markedly stimulated by 20 mM KCl (respectively, 2.2- and 14.8-fold). Furthermore, the membranes autophosphorylated in the absence of K+ (up to 0.69 +/- 0.09 nmol Pi incorporated/mg protein) and dephosphorylated by 85% in the presence of this ion. Membrane proteins were extracted by 1-2% (w/v) n-octylglucoside into a soluble form, i.e., which did not sediment in a 100 000 X g X 1 h centrifugation. This soluble form precipitated upon further dilution in detergent-free buffer. Extracted ATPase represented 32% (soluble form) and 68% (precipitated) of native enzyme and it displayed the same characteristic properties in terms of K+-stimulated ATPase and p-nitrophenylphosphatase activities and K+-sensitive phosphorylation: Mg2+-ATPase (mumol Pi/mg protein per h) 32 +/- 9 (basal) and 86 +/- 20 (K+-stimulated); Mg2+-p-nitrophenylphosphatase (mumol p-nitrophenol/mg protein per h) 2.6 +/- 0.5 (basal) and 22.2 +/- 3.2 (K+-stimulated); Mg2+-phosphorylation (nmol Pi/mg protein) 0.214 +/- 0.041 (basal) and 0.057 +/- 0.004 (in the presence of K+). In glycerol gradient centrifugation, extracted enzyme equilibrated as a single peak corresponding to an apparent 390 000 molecular weight. These findings provide the first evidence for the solubilization of (H+ + K+)-ATPase in a still active structure.     

Interaction of fluorescein isothiocyanate with the (H+ + K+)-ATPase

Fluorescein isothiocyanate was used to covalently label the gastric (H+ + K+)-ATPase. FITC treatment of the enzyme inhibited the ATPase activity while largely sparing partial reactions such as the associated p-nitrophenylphosphatase activity. ATP protected against inhibition suggesting the ligand binds at or near an ATP binding site. At 100% inhibition the stoichiometry of binding was 1.5 nmol FITC per mg Lowry protein a value corresponding to maximal phosphoenzyme formation. Binding occurred largely to a peptide of 6.2 isoelectric point, although minor labelling of a peptide of pI 5.6 was also noted. Fluorescence was quenched by K+, Rb+ and Tl+ in a dose-dependent manner, and the K0.5 values of 0.28, 0.83 and 0.025 mM correspond rather well to the values required for dephosphorylation at a luminal site. Vanadate, a known inhibitor of the gastric ATPase produced a slow Mg2+-dependent fluorescent quench. Ca2+ reversed the K+-dependent loss of fluorescence and inhibited it when added prior to K+. This may relate to the slow phosphorylation in the presence of ATP found when Ca2+ was substituted for Mg2+ and the absence of K+-dependent dephosphorylation. The results with FITC-modified gastric ATPase provide evidence for a conformational change with K+ binding to the enzyme.     

Molecular cloning of the rat stomach (H+ + K+)-ATPase

We have isolated cDNA clones for the rat stomach (H+ + K+)-ATPase by employing a novel procedure involving the use of oligonucleotides corresponding to conserved amino acid sequences of related cation transport ATPase and a cross-hybridization with the sheep kidney (Na+ + K+)-ATPase alpha-subunit cDNA. The complete nucleotide sequence of the cDNA has been determined and the amino acid sequence of the protein deduced. The ATPase consists of 1,033 amino acids and has an Mr of 114,012. Amino acid homology and hydropathy plot comparisons between the gastric ATPase and the (Na+ + K+)-ATPase catalytic subunit demonstrate a striking similarity which suggests that their higher order structure and mechanism of action are virtually identical. The greatest homology occurs in the phosphorylation site region and in domains which may be involved in nucleotide binding and energy transduction. The most substantial differences occur in the N-terminal region and in the transmembrane domains. In addition, we report the presence of an open reading frame 5' to the translation initiation site of the gastric ATPase, which raises the possibility that the mRNA is polycistronic.     

Tryptophan fluorescence of (Na+ + K+)-ATPase as a tool for study of the enzyme mechanism.

1. The protein fluorescence intensity of (Na+ + K+)-ATPase is enhanced following binding of K+ at low concentrations. The properties of the response suggest that one or a few tryptophan residues are affected by a conformational transition between the K-bound form E2 . (K) and a Na-bound form E1 . Na. 2. The rate of the conformational transition E2 . (K) leads to E . Na has been measured with a stopped-flow fluorimeter by exploiting the difference in fluorescence of the two states. In the absence of ATP the rate is very slow, but it is greatly accelerated by binding of ATP to a low affinity site. 3. Transient changes in tryptophan fluorescence accompany hydrolysis of ATP at low concentrations, in media containing Mg2+, Na+ and K+. The fluorescence response reflects interconversion between the initial enzyme conformation, E1 . Na and the steady-state turnover intermediate E2 . (K). 4. The phosphorylated intermediate, E2P can be detected by a fluorescence increase accompanying hydrolysis of ATP in media containing Mg2+ and Na+ but no K+. 5. The conformational states and reaction mechanism of the (Na+ + K+)-ATPase are discussed in the light of this work. The results permit a comparison of the behaviour of the enzyme at both low and high nucleotide concentrations.     

Conformational changes of purified (Na+ + K+)-ATPase detected by a sulfhydryl fluorescence probe.

The fluorescent sulfhydryl reagent S-mercuric-N-dansyl cysteine (Dn-Cys-Hg+) has been used to label a purified preparation of the (Na+ + K+)-ATPase obtained from the electric organ of Electrophorous electricus. The labelled (Na+ +K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3), although reversibly inhibited, was capable of undergoing conformational changes associated with the active enzyme that could be monitored fluorometrically. The presence of ligands (Na+ + Mg2+ + ATP or Mg2+ + Pi) which are known to convert the enzyme from the E-1 state to the E-2-P state brought about large (97--100%) increases in fluorescence of the dimethylaminonaphthalene sulfonyl (Dn) label. An E-2 state could be achieved by the addition of Mg2+ which caused only a 32.3% increase in fluorescence over the E-1 state. Neither AMP nor TTP with or without Mg2+ or Na+ or Pi added without Mg2+ had any effect on the Dn fluorescence. If the enzyme was denatured, no fluorescence changes were observed. Small changes in the polarization of fluorescence of the Dn moiety were observed under all the conditions used. These small polarization changes and the large increases in the fluorescence intensity suggest that the enzyme can change conformational states in the presence of appropriate ligands and these conformational changes may take place in a relatively limited region of the protein's structure.     

Activation of (Na+ + K+)-ATPase

Enzymes catalyze essential chemical reactions needed for living processes. (Na+ +K+)-ATPase (NKA) is one of the key enzymes that control intracellular ion homeostasis and regulate cardiac function. Little is known about activation of NKA and its biological impact. Here we show that native activity of NKA is markedly elevated when protein-protein interaction occurs at the extracellular DVEDSYGQQWTYEQR (D-R) region in the alpha-subunit of the enzyme. The apparent catalytic turnover of NKA is approximately twice as fast as the controls for both ouabain-resistant and ouabain-sensitive enzymes. Activation of NKA not only markedly protects enzyme function against denaturing, but also directly affects cellular activities by regulating intracellular Ca2+ transients and inducing a positive inotropic effect in isolated rat cardiac myocytes. Immunofluorescent labeling indicates that the D-R region of NKA is not a conventional digitalis-binding site. Our findings uncover a novel activation site of NKA that is capable of promoting the catalytic function of the enzyme and establish a new concept that activating of NKA mediates cardiac contraction.     

The interaction of H+ and K+ with the partial reactions of gastric (H+ + K+)-ATPase

The influence of H+ and K+ on the partial reactions and transport of gastric (H+ + K+)-ATPase was studied. Using transient kinetics, the effects and sidedness of effects of H+ and K+ on formation and breakdown of phosphoenzyme were determined in intact and lyophilized reconstituted vesicles in the absence and presence of gramicidin. Whereas increasing H+ concentrations on the ATP-binding face of the vesicles accelerates phosphorylation, increasing K+ concentrations inhibits phosphorylation. Increasing H+ on this side reduces K+ inhibition of the phosphorylation rate. At low ATP/K+ ratios, the phosphorylation step can become rate-limiting for steady state hydrolysis. Decreasing H+ accelerates dephosphorylation in the absence of K+. K+ on the internal or luminal face of the vesicles accelerates dephosphorylation, and this rate is reduced with increasing H+ concentrations. At low internal pH, K+-dependent dephosphorylation may become rate-limiting. H+ transport measurements using fluorescence quenching of acridine orange show that whereas internal K+ is required for H+ transport, external K+ inhibits the rate of formation of a pH gradient, and the inhibition is reduced by decreasing medium pH. The pH optimum for ATPase activity and transport correlated in the vesicles, and the K0.5 of K+ for transport correlated with data for intact parietal cells.     

Modification of gastric (H+ + K+)-ATPase with pyridoxal 5'-phosphate

Pig gastric membrane vesicles enriched in (H+ + K+)-ATPase were covalently modified with pyridoxal 5'-phosphate (PLP). The modification resulted in inhibition of K+-dependent ATP hydrolysis, formation of phosphoenzyme and ATP-driven H+-uptake catalyzed by (H+ + K+)-ATPase. ATP, ADP, and adenyl-5'-yl imidodiphosphate were protective ligands, whereas Mg2+ and K+ were not. Specific PLP-binding of about 4.5 nmol/mg membrane protein was necessary for complete inhibition of the enzyme activity, indicating that the stoichiometry of PLP-binding to the enzyme was about 1:1. Limited proteolysis of the enzyme modified with [3H]PLP by trypsin suggests that PLP specifically modifies the lysine residue located in the 16-kDa fragment of the enzyme cleaved by trypsin. These results suggested that PLP binds to a specific lysine residue in the nucleotide-binding site or a region in its vicinity and inhibits the substrate binding or phosphorylation step of (H+ + K+)-ATPase.     

Direct evidence for an ADP-sensitive phosphointermediate of (K+ + H+)-ATPase

Direct evidence for the occurrence of an ADP-sensitive phosphoenzyme of (K+ + H+)-ATPase, the proton-pumping system of the gastric parietal cell is presented. The enzyme is phosphorylated with 5 microM [gamma-32P]ATP in 50 mM imidazole-HCl (pH 7.0) and in the presence of 7-15 microM Mg2+. Addition of 5 mM ADP to this preparation greatly accelerates its hydrolysis. We have been able to establish this by stopping the phosphorylation with radioactive ATP, by adding 1 mM non-radioactive ATP, which leads to a slow monoexponential process of dephosphorylation of 32P-labeled enzyme. The relative proportion of the ADP-sensitive phosphoenzyme is 22% of the total phosphoenzyme. Values for the rate constants of breakdown and interconversion of the two phosphoenzyme forms have been determined.     

ATP/ADP exchange activity of gastric (H+ +K+)-ATPase

The ATP/ADP exchange is shown to be a partial reaction of the (H+ +K+)-ATPase by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to P1, P5-di(adenosine-5') pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg2+ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration (K0.5=116 microM) required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg2+ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K+ as a function of pH and Mg2+.     

Defective conformational response in a selectively trypsinized (Na+ + K+)-ATPase studied with tryptophan fluorescence

1. Monitoring protein conformations of purified (Na+ + K+)-ATPase with intrinsic fluorescence we have examined if altered conformational responses accompany the defective catalytic and transport processes in selectively modified 'invalid' (Na+ + K+)-ATPase which is obtained by graded tryptic digestion of the Na+ form of the protein. 2. The protein fluorescence intensity of the K+ form (E2K) of both control and invalid (Na+ + K+)-ATPase is 2--3% higher than that of the Na+ form (E1Na). By varying the NaCl concentration we found evidence for different fluorescence intensities of the two phosphoenzymes; E2P has the same fluorescence intensity as E2K and the intensity of E1P is similar to that of E1Na. The fraction of phosphoenzyme present as E2P can therefore be determined as the amplitude of the fluorescence change accompanying phosphorylation in the absence of K+ divided by the amplitude of the full response to K+. 3. Titration of the fluorescence responses of the invalid (Na+ + K+)-ATPase shows that the tryptic split alters the noise of the equilibria between the cation-bound conformations, E1Na and E2K, and between the phosphoforms, E1P and E2P, in the direction of the E1 forms. 4. Vanadate binds to the Mg2+-bound form of E2K and prevents further changes in fluorescence intensity of the protein. The conformative responses of invalid (Na+ + K+)-ATPase are insensitive to vanadate in agreement with the reduced vanadate binding affinity of this enzyme. 5. The defective conformative response of the invalid (Na+ + K+)-ATPase in relation to its catalytic defects, reduced Na+ transport, and insensitivity to vanadate suggest that the transitions between Na+ forms (E1) and K+ forms (E2) of the protein are coupled to the catalytic and transport reactions of the (Na+ + K+)-pump.     

Photoaffinity labeling of the lumenal K+ site of the gastric (H+ + K+)-ATPase

A photoaffinity label for the lumenal K+ site of the gastric (H+ + K+)-ATPase has been identified. Seven azido derivatives based upon the reversible K+ site inhibitor SCH 28080 were studied, one of which, m-ATIP (8-(3-azidophenylmethoxy)-1,2,3-trimethylimidazo[1,2-a] pyridinium iodide), was subsequently synthesized in radiolabeled form. In the absence of UV irradiation, m-ATIP inhibited K+ -stimulated ATPase activity in lyophilized gastric vesicles competitively with respect to K+, with a Ki value of 2.4 microM at pH 7.0. Irradiation of lyophilized gastric vesicles at pH 7.0 with [14C]m-ATIP in the presence of 0.2 mM ATP resulted in a time-dependent inactivation of ATPase activity that was associated with an incorporation of radioactivity into a 100-kDa polypeptide representing the catalytic subunit of the (H+ + K+)-ATPase. Both inactivation and incorporation were blocked in the presence of 10 mM KCl but not with 10 mM NaCl, consistent with interaction at the K+ site. The level of incorporation required to produce complete inhibition of ATPase activity was 1.9 +/- 0.2 times the number of catalytic phosphorylation sites in the same preparation. Tryptic digestion of gastric vesicle membranes, labeled with [14C]m-ATIP, failed to release the radioactivity from the membranes suggesting that the site of interaction was close to or within the membrane-spanning sections of this ion pump.