E2P State Stabilization by the N-terminal Tail of the H,K-ATPase β-Subunit Is Critical for Efficient Proton Pumping under in Vivo Conditions*

The catalytic α-subunits of Na,K- and H,K-ATPase require an accessory β-subunit for proper folding, maturation, and plasma membrane delivery but also for cation transport. To investigate the functional significance of the β-N terminus of the gastric H,K-ATPase in vivo, several N-terminally truncated β-variants were expressed in Xenopus oocytes, together with the S806C α-subunit variant. Upon labeling with the reporter fluorophore tetramethylrho da mine-6-maleimide, this construct can be used to determine the voltage-dependent distribution between E1P/E2P states. Whereas the E1P/E2P conformational equilibrium was unaffected for the shorter N-terminal deletions βΔ4 and βΔ8, we observed significant shifts toward E1P for the two larger deletions βΔ13 and βΔ29. Moreover, the reduced ΔF/F ratios of βΔ13 and βΔ29 indicated an increased reverse reaction via E2P → E1P + ADP → E1 + ATP, because cell surface expression was completely unaffected. This interpretation is supported by the reduced sensitivity of the mutants toward the E2P-specific inhibitor SCH28080, which becomes especially apparent at high concentrations (100 μm). Despite unaltered apparent Rb+ affinities, the maximal Rb+ uptake of these mutants was also significantly lowered. Considering the two putative interaction sites between the β-N terminus and α-subunit revealed by the recent cryo-EM structure, the N-terminal tail of the H,K-ATPase β-subunit may stabilize the pump in the E2P conformation, thereby increasing the efficiency of proton release against the million-fold proton gradient of the stomach lumen. Finally, we demonstrate that a similar truncation of the β-N terminus of the closely related Na,K-ATPase does not affect the E1P/E2P distribution or pump activity, indicating that the E2P-stabilizing effect by the β-N terminus is apparently a unique property of the H,K-ATPase.

The gastric H,K-ATPase fulfills the remarkable task of pumping protons against a more than 10 6 -fold concentration gradient. H ϩ extrusion is coupled to countertransport of an equal number of K ϩ ions for each ATP molecule hydrolyzed, resulting in an electroneutral overall process (1). Characteristic for all P-type ATPases, the enzyme cycles between the two principal conformational states (E 1 and E 2 ) and the corresponding phosphointermediates (E 1 P and E 2 P), which are formed by reversible phosphorylation of an aspartate residue in the highly conserved DKTGTLT motif. According to a Post-Albers-like reaction scheme (see Fig. 1A), the conformational E 1 P 3 E 2 P transition converts the high H ϩ /low K ϩ affinity of the cation binding pocket into a low H ϩ /high K ϩ affinity binding site, hence enabling proton release into the stomach lumen and subsequent binding of extracellular K ϩ . Because the pump faces a lumenal proton concentration of ϳ150 mM (2), proton release is probably the energetically most demanding step in the reaction cycle. Thus, during the conformational E 1 P 3 E 2 P transition, enormous pK a changes of the H ϩ -coordinating residues have to occur that most likely involve the rearrangement of a positively charged lysine side chain (Lys-791 in rat H,K-ATPase) (3).
All P 2 -type ATPases share a common catalytic ␣-subunit, composed of 10 transmembrane domains harboring the ionbinding sites and a large cytoplasmic loop with the nucleotidebinding domain, the phosphorylation domain (P-domain), 2 and the actuator domain (A-domain) (4). However, a unique feature of K ϩ -transporting Na,K-and H,K-ATPase enzymes is the requirement for an accessory ␤-subunit, which is indispensable for proper folding, maturation, and plasma membrane delivery (5,6). Despite only 20 -30% overall sequence identity between the H,K-ATPase ␤-subunit and the Na,K ␤-isoforms, the topogenic structure is similar: a short N-terminal cytoplasmic tail, followed by a single transmembrane segment and a large extracellular C-terminal domain with glycosylation sites and disulfide-bridging cysteines. Numerous studies have demonstrated that the ␤-subunit of the Na,K-ATPase is more than just a chaperone for the ␣-subunit, being also required for proper ion transport activity of the holoenzyme. In fact, it has been discovered that different cell-and tissue-specific ␤-isoforms have distinct effects on the cation affinities (7)(8)(9). Furthermore, it was shown that mutational changes in all three topogenic domains of the Na,K-ATPase ␤-subunit (10 -19) as well as chemical interference with disulfide-forming cysteines in the Na,K-ATPase ␤-subunit ectodomain (20 -22) affect the cation transport properties of the sodium pump. Finally, conformational changes in the ␤-subunit during the Na,K-ATPase reaction cycle were demonstrated by proteolytic digestion studies (23) and voltage clamp fluorometry (24).
Far less is known about the functional significance of the single H,K-ATPase ␤-isoform, especially about its potential impact on cation transport (reviewed in Refs. 25 and 26). We have proven recently that E 2 P state-specific transmembrane interactions between residues in ␣TM7 and two highly conserved tyrosines in the ␤TM of both Na,Kand H,K-ATPase significantly stabilize the E 2 P conformation (19). Mutational disruptions of this interaction resulted in substantial shifts toward E 1 P and severely affected H ϩ secretion, which highlighted the physiological relevance of this E 2 P state stabilization. Notably, according to the recently published cryo-EM structure of pig gastric H,K-ATPase in the pseudo-E 2 P state (27), the N-terminal tail of the ␤-subunit makes direct contact with the phosphorylation domain of the ␣-subunit (see Fig. 1B), thus indicating an additional E 2 P state stabilization mediated by the ␤-N terminus. Although this idea was further supported by biochemical studies on N-terminally truncated mutants, direct evidence for this putative E 2 P-stabilizing interaction and its potential significance for ion transport in intact cells is still lacking.
Here, we demonstrate for the first time the functional importance of the gastric H,K-ATPase ␤-subunit N terminus in living cells under in vivo conditions: voltage clamp fluorometry, Rb ϩ flux, and SCH28080 sensitivity measurements revealed E 1 P-shifted, ion transport-impaired phenotypes for two N-terminally truncated H,K ␤-variants, thus substantiating the E 2 P-stabilizing effect of the ␤-N terminus suggested by the recent cryo-EM structure.

Molecular Biology and Oocyte
Preparation-The cDNAs of the rat gastric H,K-ATPase ␤-subunit and a modified form of the ␣-subunit with a single cysteine replacement in the M5/M6 extracellular loop (S806C; Fig. 1B) were subcloned into vector pTLN (28). This cysteine replacement enables environmentally sensitive TMRM labeling of gastric H,K-ATPase (29) without affecting its transport properties (30). For Na,K-ATPase studies, an ouabain-insensitive sheep Na,K-ATPase ␣ 1 -mutant (carrying mutations Q111R and N122D (31)) without extracellularly exposed cysteines (mutations C911S and C964A (32)) and a Na,K-ATPase ␤ 1 -subunit variant (␤ 1 S62C) that can be site-specifically labeled with TMRM (24) were used as templates for mutagenesis and cRNA preparation. N-terminally truncated ␤-constructs were generated by recombinant PCR and verified by DNA sequencing. Oocyte preparation, cRNA synthesis, and injection were performed as described previously (30). Prior to experiments that were usually carried out 2 or 3 days after injection at 21-24°C, the oocytes were preincubated in solutions containing 100 M ouabain (Sigma-Aldrich) to inhibit the endogenous Xenopus Na,K-ATPase.
Rb ϩ Uptake Measurements-The oocytes were incubated for 15 min in Rb ϩ flux buffer (5 mM RbCl, 85 mM tetramethylammonium chloride, 20 mM tetraethylammonium chloride, 5 mM BaCl 2 , 5 mM NiCl 2 , 10 mM MES, pH 5.5, 100 M ouabain). After three washing steps in Rb ϩ -free washing buffer (90 mM tetramethylammonium chloride or NaCl, 20 mM tetraethylammonium chloride, 5 mM BaCl 2 , 5 mM NiCl 2 , 10 mM MES, pH 5.5) and one wash in water, each individual oocyte was homogenized in 1 ml of Millipore water. For SCH28080 inhibition experiments, the K ϩ -competitive inhibitor SCH28080 (Sigma-Aldrich) was added to the preincubation solution and Rb ϩ flux buffer (to a final concentration of either 10 M or 100 M), respectively. To determine the apparent constant K 0.5 for halfmaximal activation of the H,K-ATPase by rubidium, the sum [tetramethylammonium chloride] plus [RbCl] in the Rb ϩ flux buffer was kept constant at 90 mM, e.g. 1 mM RbCl ϩ 89 mM tetramethylammonium chloride. After subtraction of the average Rb ϩ uptake into control oocytes from the same batches at a given [RbCl], the data were fitted to a Michaelis-Menten type Aliquots of 20 l from oocyte homogenates were analyzed by atomic absorption spectroscopy using an AAnalyst800 TM spectrometer (PerkinElmer Life Sciences), equipped with a transversely heated graphite furnace using a temperature protocol according to the manufacturer's procedures (conditions available on request). Absorption was measured at 780 nm using a rubidium hollow cathode lamp (Photron, Melbourne, Australia). After Zeeman background correction, the Rb ϩ contents were calculated by comparison with standard calibration curves (measured between 0 and 50 g/liter Rb ϩ ).
Western Blot Analysis of Isolated Plasma Membranes-The procedures for isolation of plasma membranes and total cellular membranes from Xenopus oocytes, gel electrophoresis, and immunoblotting were performed according to the protocols in Ref. 19. The polyclonal antibody HK12.18 (Merck) (6) and the monoclonal antibody 2B6 (MBL, Woburn, MA) (33) were used for detection of gastric H,K-ATPase ␣and ␤-subunits, respectively.

E 1 P/E 2 P Conformational Distribution of N-terminally
Deleted H,K-ATPase ␤-Mutants-To investigate whether the N-terminal domain of the ␤-subunit indeed contributes to E 2 P state stabilization of the gastric H,K-ATPase, we expressed the wild type ␤-subunit and four N-terminally deleted H,K ␤-mutants ( Fig. 2A) together with the ␣-subunit variant S806C in Xenopus oocytes. After site-specific labeling of this cysteine with TMRM (Fig. 1B), monoexponential fluorescence changes were observed upon voltage jumps from a holding potential of Ϫ40 mV to values between Ϫ180 and ϩ60 mV (Fig. 2B). Hence, this mutant ␣-subunit directly monitors the voltage-dependent distribution between the E 1 P/E 2 P states of H,K-ATPase enzymes, as demonstrated previously (19,30). Fig. 2C shows the resulting voltage dependence of steady-state fluorescence amplitudes (1 Ϫ ⌬F/F), which follow a Boltzmann-type distribution for both wild type and ␤-N-terminally deleted H,K-ATPase enzymes. Whereas these curves were not changed for the shorter N-terminal deletions ␤⌬4 and ␤⌬8 compared with ␤WT, the distributions for the ␤⌬13 and ␤⌬29 truncated variants were significantly shifted toward more positive potentials (see Table 1 for Boltzmann parameters), indicating a relative destabilization of the E 2 P state in favor of E 1 P. According to these distributions, ϳ65% of the ␤WT and ␤⌬4and ␤⌬8-containing H,K-ATPase molecules occur in the E 2 P state at Ϫ60 mV (which is the physiologically relevant membrane potential determined for parietal cells (34)), but only 60 and 50% of the ␤⌬29 and ␤⌬13 mutant enzymes are present in E 2 P, respectively. Furthermore, the E 1 P shift is also reflected by the voltagedependent reciprocal time constants obtained from monoexponential fits to the fluorescence changes (Fig. 2D); of note, these are significantly smaller for the ␤⌬29 and ␤⌬13 mutants than for the wild type only at positive potentials, which favor the forward reaction from E 1 P to E 2 P, but not at negative potentials, at which the enzyme is driven into the E 1 P state (via the backward reaction described by k rev in Figs. 1A and 2D). Therefore, these N-terminal deletions apparently cause a shift toward E 1 P by reducing the "forward" rate constant (designated as k fw ) of the E 1 P/E 2 P transition without changing the rate constant k rev for the "reverse" reaction.
However, if only the results shown in Fig. 2 (C and D) are considered, the conformational effect caused by the N-terminal deletions is probably underestimated because compared with ␤WT, the two variants ␤⌬13 and ␤⌬29 also showed substantially smaller values for the enzyme-specific fluorescence changes ⌬F/F (Fig. 2E). Western blot analysis of isolated plasma membranes excluded the possibility that the lower ⌬F/F values observed for the ␤⌬13 and ␤⌬29 constructs were simply due to a reduced cell surface delivery of the ␣-subunits that are avail-able for labeling with TMRM ( Fig. 2F and supplemental Fig. S1). Therefore, it is safe to assume that the shown ⌬F/F ratio is directly proportional to the number of H,K-ATPase molecules that can be shifted between E 1 P/E 2 P states by voltage jumps (according to the reaction sequence highlighted in gray in Fig.  1A). The ϳ2-fold lower ⌬F/F values observed for the ␤⌬13 and ␤⌬29 mutants (compared with ␤WT, ␤⌬4, and ␤⌬8) most likely reflect a higher tendency of the mutant H,K-ATPase molecules (that accumulate in the E 1 P state because of the reduced k fw ) to undergo further backward reactions via E 1 P (H ϩ ) 3 E 1 P ϩ H ϩ ϩ ADP 3 E 1 ϩ H ϩ ϩ ATP. The resulting substantial depletion in the sum of E 1 P/E 2 P states would therefore explain the observed smaller fluorescence changes. This interpretation is  Table 1 for fit parameters). D, reciprocal time constants obtained by monoexponential fits of the voltage jumpinduced fluorescence changes for wild type (f) and N-terminally deleted H,K-ATPase ␤-variants ␤⌬13 (E) and ␤⌬29 (ƒ). The data are the means Ϯ S.E. of 10 -16 oocytes. E, ⌬F/F values (in %) for TMRM-labeled oocytes expressing ␣S806C and either the wild type H,K-ATPase ␤-subunit or N-terminally truncated ␤-variants. The data are the means Ϯ S.E. of 13-32 oocytes from three different oocyte batches. Inset, ⌬F/F is calculated from the difference ⌬F (represented by a black arrow) between the fluorescence at the most hyperpolarizing voltage (Ϫ180 mV, upper black trace) and the most depolarizing voltage (ϩ60 mV, lower black trace), normalized to the background fluorescence F at Ϫ40 mV (gray trace). F, Western blot analysis of plasma membrane (PP, upper panel) and total membrane (TP, lower panel) preparations from H,K-ATPase-expressing oocytes, using anti-H,K␣ antibody HK12.18 or anti-H,K␤ antibody 2B6 (see supplemental Fig. S1). The equivalent of two oocytes was loaded per lane; detection of the endogenous Xenopus Na,K-ATPase ␣ 1 -isoform served as a loading standard, which is shown in supplemental Fig. S1. ␤-mutants (Fig. 2C) The values are the means Ϯ S.E. of 15-30 oocytes from two or three different oocyte batches. P-phosphoenzymes with ADP to form [␥-32 P]ATP via the aforementioned reverse reaction. Of note, in these chase experiments, already the ␤⌬8 mutant exhibited a significantly reduced amount of 32 P-phosphoenzyme after a 5-s chase with ADP. However, care has to be taken when comparing these and our voltage clamp fluorometry results because of the differences in experimental conditions. The pulse-chase experiments have been performed on membrane fractions at a ubiquitous pH of 6.4, lower ionic strength, and 0°C, whereas voltage clamp fluorometry probes the enzyme within an intact membrane environment with physiological ion concentrations (and neutral intracellular pH) at room temperature. Differences in the intracellular ion concentration or pH may well affect intramolecular interactions, especially if these interactions involve potentially charged residues (amino acids 6 -8 in the N-terminal sequence MAALQEKKSC…). Furthermore, the intracellular ATP and Mg 2ϩ concentrations are by far higher in Xenopus oocytes than in the pulse-chase experiments (2.3 mM ATP (35), 0.5 mM Mg 2ϩ versus 10 M ATP, 20 M Mg 2ϩ ), thus favoring the forward phosphorylation reaction. Therefore, small effects on the E 1 P/E 2 P distribution that would reflect enhanced ADP-dependent dephosphorylation of the E 1 P state of the ␤⌬8 mutant are possibly obscured under the conditions in living cells.

TABLE 1 Parameters from fits of a Boltzmann function to (1 ؊ ⌬F/F)/V distributions of N-terminally deleted H,K-ATPase
Rb ϩ Uptake and SCH28080 Sensitivity of N-terminally Truncated H,K-ATPase ␤-Subunits-To assess the impact on ion transport activity potentially caused by the conformational shifts of the N-terminally truncated ␤-variants, we determined Rb ϩ uptake at saturating extracellular concentrations. Whereas Rb ϩ uptake for ␤⌬4and ␤⌬8-expressing oocytes was comparable with ␤WT, it was ϳ20% lower for ␤⌬13 and ␤⌬29 (Fig. 3A and Table 2). This can be interpreted as a reduced turnover number (lowered v max ) because the apparent affinity for extracellular Rb ϩ was unaffected by the trunca-3 K. Abe, unpublished results.  (Table 2) were obtained from a fit of a Michaelis-Menten type function to the data (superimposed lines).

TABLE 2
Rb ؉ uptake activity, SCH28080 sensitivity, and apparent Rb ؉ affinity of N-terminally deleted H,K-ATPase ␤-mutants (see Fig. 3 tions (see Fig. 3B and Table 2 for apparent K 0.5 values). The affected turnover numbers of the ␤⌬13 and ␤⌬29 mutants demonstrate that obviously the transition that is characterized by the reduced rate constant k fw of these variants directly affects the rate-limiting step of the catalytic cycle. This illustrates how already small shifts in conformational equilibria can have significant functional consequences for the transport activity of the gastric H,K-ATPase.
In the presence of 10 M SCH28080, an E 2 P-specific inhibitor of the H,K-ATPase (36 -38), the Rb ϩ uptake of ␤WT-, ␤⌬4-, and ␤⌬8-expressing oocytes was reduced to ϳ20% (Fig.  3A, inset, and Table 2), in agreement with the data of Mathews et al. (39). In contrast, the inhibition of ATPase complexes containing ␤⌬13 and ␤⌬29 was less efficient, resulting in significantly higher residual activities of ϳ30% at 10 M SCH28080. Notably, 100 M SCH28080 resulted in a suppression of Rb ϩ uptake to ϳ6% residual activity for HK␣S806C/␤WT, whereas the effect was much smaller for any of the N-terminally deleted ␤-variants. As a possible consequence of the E 1 P-shifted phenotypes of the mutants, the mean dwell time of molecules in the SCH28080-sensitive E 2 P state may be substantially shorter. Thus, increasing the inhibitor concentration would not result in enhanced binding of the compound because the dwell time in E 2 P is not sufficient to reach binding equilibrium under turnover conditions. In contrast, the wild type H,K-ATPase, which stays longer in E 2 P, is able to bind more inhibitor molecules if the SCH28080 concentration is increased. Interestingly, at the higher inhibitor concentration, a significantly reduced SCH28080 sensitivity was also observed for ␤⌬4and ␤⌬8-expressing oocytes, which showed a more than 2-fold higher residual activity compared with ␤WT (13-14% versus 6%). This suggests that already the shorter deletions cause an elevated preference for E 1 P, which raises the question of why no effect on the conformational distribution (Fig. 2C) was seen for these constructs. Two possibilities may account for this: (i) minute shifts in the voltage-dependent E 1 P/E 2 P distribution may be difficult to resolve by voltage clamp fluorometry experiments because low slope factors z q of the Boltzmann curves limit the accuracy of V 0.5 determination, and (ii) E 2 -destabilizing effects that act on the relative distribution of pump molecules over all reaction intermediates may not be effective during the partial reaction sequence studied in pre-steady-state experiments (voltage clamp fluorometry) but rather become apparent under steady-state conditions (Rb ϩ uptake), in which the enzyme undergoes the full reaction cycle.
Although the reduction in Rb ϩ transport activity of ␤⌬13 and ␤⌬29 truncated mutants appears moderate, it is important to note that these subtle effects were observed already at a relatively mild proton gradient (pH int ϳ 7.4 versus pH ext ϭ 5.5). However, in vivo the H,K-ATPase pumps protons against a 10,000-fold higher gradient of ϳ10 6 . Under these physiological conditions (which are unfortunately not applicable to Xenopus oocytes), the high lumenal H ϩ concentration would even more favor H ϩ reverse binding at the extracellular sites, thereby also stimulating the E 2 P 3 E 1 P "backward" reaction (i.e. increasing k rev ). An enhanced k rev in addition to the reduced forward rate constant k fw (as observed for the N-terminally deleted mutants ␤⌬13 and ␤⌬29) is thus expected to have even more drastic effects on the turnover number. Therefore, although causing only small effects at the rather shallow pH gradient applied here, the E 2 P state destabilization by the ␤-N-terminal truncations will almost certainly slow down H ϩ secretion under the pH conditions in the stomach.
A surprising finding from the current study is that the more extensive deletion ␤⌬29 caused a smaller shift toward the E 1 P state than ␤⌬13 (Fig. 2C and Table 1). The two putative interaction sites between the ␤-N terminus and the ␣-subunit proposed by the recently published cryo-EM structure of the pig gastric H,K-ATPase (27) may provide a rationale to explain these rather unexpected effects; apart from the aforementioned contact between the ␤-N terminus and the P-domain (probably near Arg-716 to Ala-719; see red arrow in Fig. 1B), a stretch of the ␤-N terminus located closer to the transmembrane domain also approaches the cytoplasmic stalk of ␣TM3, which is connected to the A-domain (Fig. 1B, black arrow). Therefore, truncation of the first 12 amino acids in ␤⌬13 may only disrupt the interaction with the P-domain and thereby cause the strong E 1 P shift, whereas the more extensive deletion in ␤⌬29 most likely affects both critical contacts, which might involve an additional compensatory mechanism (see below).
As outlined by Abe et al. (27), the contact between the ␤-subunit N terminus and the P-domain probably stabilizes the enzyme in E 2 P, thereby minimizing the reverse reaction with ADP via E 2 P 3 E 1 P ϩ ADP 3 E 1 ϩ ATP in terms of a "ratchet"like mechanism. In the pseudo-E 2 P state revealed by the cryo-EM structure, the nucleotide-binding domain of the ␣-subunit is retracted far from the P-domain (supplemental Fig. S2). Therefore, reverse transfer of the phosphate (bound to Asp-385 in the P-domain) to ADP (bound to the nucleotidebinding domain) would require a large movement of the P-domain, which is presumably prohibited by the tethering between the P-domain and ␤-N terminus in the E 2 state. In contrast, in a putative E 1 P state derived from a SERCA-E 1 AlF 4 -ADP structure (40), the ADP at the nucleotide-binding domain is in close proximity to the phosphate analogue AlF 4 Ϫ (shown in supplemental Fig. S2), which could thus explain the enhanced ADP rephosphorylation of the E 1 P-shifted N-terminally deleted ␤-variants (27).
The interaction of the ␤-N terminus with the A-domain via the movement of ␣TM3 may also be functionally significant because the protrusion of the TGES loop from the A-domain into the gap between the N-and P-domains may have a segregating effect, too (supplemental Fig. S2). Although the influence of the ␤-N/␣TM3 contact on positioning of the A-domain is not resolved yet, it seems possible that the missing contact to the ␤-N terminus via ␣TM3 (in ␤⌬29) may lead to unrestricted movement of the A-domain. This may promote the segregating effect of the TGES loop, which could partially compensate for the destabilization of E 2 P that arises from the missing interaction with the P-domain, thus explaining the less E 1 P-shifted phenotype of ␤⌬29 compared with ␤⌬13.
N-terminal Truncation of the Na,K-ATPase ␤-Subunit-Notably, according to the recently published crystal structure of the closely related Na,K-ATPase in the Rb ϩ -occluded E 2 state (41), there is no indication for similar interactions between the ␤-N terminus and the ␣-subunit of the sodium pump. Although the ␤-N terminus of the Na,K-ATPase was not completely resolved in this structure so that evidence for a similar interaction in the Na,K-ATPase is absent so far, we tried to explore whether the intersubunit interaction mediated by the H,K-ATPase ␤-N terminus is conserved among P 2C -type ATPases.
To determine the impact of ␤-N-terminal truncation on sheep Na,K-ATPase, the eight most N-terminal amino acids of reporter construct ␤ 1 S62C (24) of the ␤ 1 -subunit were deleted (Fig. 4A). Because the cytoplasmic ␤-N terminus of the Na,K-ATPase is shorter than that of the gastric H,K-ATPase, this truncation results in a N-terminal domain comparable in length to the ␤⌬13 variant of the H,K-ATPase. This N-terminally truncated ␤-variant, NaK␤ 1 S62C⌬8, was coexpressed with the wild type sheep ␣ 1 -subunit, labeled by TMRM, and subjected to voltage jumps in a K ϩ -free solution containing 100 mM Na ϩ . The resulting voltage-dependent distribution of fluorescence amplitudes is not significantly different from the one observed for full-length ␤ 1 S62C-containing Na,K-ATPase complexes ( Fig. 4B and Table 3). Furthermore, as inferred from the stationary pump currents at 10 mM K ϩ , the ion transport activity of the Na,K-ATPase is completely unaffected by the ␤-N-terminal truncation (Fig. 4B, inset). Regarding the comparably shallow Na ϩ gradient under physiological conditions, an analogous ratchet-like E 2 P stabilization is probably not necessary to assist Na ϩ transport of the sodium pump. This further supports our hypothesis that the E 2 P state stabilization mediated by the H,K-ATPase ␤-N terminus is unique to facilitate proton transport against the steep proton gradient across the parietal cell membrane, which is ϳ10,000-fold higher than the typical transmembrane gradient for Na ϩ ions. Therefore, the mechanism of E 2 P state stabilization by the gastric H,K-ATPase ␤-N terminus appears to be a distinctive property of this enzyme.
Conclusions-As a key observation, the recently published cryo-EM structure of the gastric H,K-ATPase highlighted the close proximity between the P-domain of the ␣-subunit and the short N-terminal tail of the ␤-subunit, suggesting an E 2 P-stabilizing interaction of the two subunits. The current study on N-terminally truncated ␤-variants provides direct evidence that the ␤-N terminus assists in E 2 P state stabilization and that this is critical for the transport efficiency of the enzyme under in vivo conditions. Because the effects of the mutants were already significant under the relatively shallow H ϩ gradient (⌬pH Ϸ2), they may even be more relevant for the enzyme in situ, where a strong E 2 P preference is essential for efficient H ϩ release against a ⌬pH of ϳ6 units. Moreover, because we recently demonstrated an E 2 P-stabilizing effect of interactions between residues in the transmembrane domain of the H,K-ATPase ␤-subunit and TM7 of the ␣-subunit, the two effects exerted by different regions of the ␤-subunit may synergistically contribute to the functional requirement of E 2 P state stabilization.

TABLE 3
Stationary pump currents and parameters from fits of a Boltzmann function to (1 ؊ ⌬F/F)/V distributions of ␤-N-terminally truncated Na,K-ATPase (Fig. 4) The values are the means Ϯ S.D. of 10 -15 oocytes.