Comprehensive Physiology || Gastric H + ,K + ...

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Gastric H+,K+-ATPaseJai Moo Shin,*1 Keith Munson,1 and George Sachs1

ABSTRACTThe gastric H+,K+-ATPase is responsible for gastric acid secretion. This ATPase is composed of twosubunits, the catalytic α subunit and the structural β subunit. The α subunit with molecular mass ofabout 100 kDa has 10 transmembrane domains and is strongly associated with the β subunit witha single transmembrane segment and a peptide mass of 35 kDa. Its three-dimensional structureis based on homology modeling and site-directed mutagenesis resulting in a proton extrusionand K+ reabsorption model. There are three conserved H3O+-binding sites in the middle of themembrane domain and H3O+ secretion depends on a conformational change involving Lys791

insertion into the second H3O+ site enclosed by E795, E820, and D824 that allows export ofprotons at a concentration of 160 mM. K+ countertransport involves binding to this site after therelease of protons with retrograde displacement of Lys791 and then K+ transfer to E343 and exitto the cytoplasm. This ATPase is the major therapeutic target in treatment of acid-related diseasesand there are several known luminal inhibitors allowing analysis of the luminal vestibule. Oneclass contains the acid-activated covalent, thiophilic proton pump inhibitors, the most effectiveof current acid-suppressive drugs. Their binding sites and trypsinolysis allowed identification ofall ten transmembrane segments of the ATPase. In addition, various K+-competitive inhibitors ofthe ATPase are being developed, with the advantage of complete and rapid inhibition of acidsecretion independent of pump activity and allowing further refinement of the structure of theluminal vestibule of the E2 form of this ATPase. C© 2011 American Physiological Society. ComprPhysiol 1:2141-2153, 2011.

IntroductionAbout 160-mM HCl acid is secreted by the parietal cells of thestomach. Two acid-stimulatory receptors, the H2-receptor andthe muscarinic M3 receptor, are present on the parietal cell.After histamine or acetylcholine binds to the correspondingreceptor, the morphology of parietal cell changes. This mor-phological change drives gastric H+,K+-ATPase and vesi-cles containing a K+ conductance pathway from cytoplas-mic membranes to the (apical) membrane of the secretorycanaliculus, where the pump exchanges cytoplasmic protonsfor extracytoplasmic potassium ions in an electroneutral man-ner. This activity is responsible for the elaboration of HClby the parietal cell of the gastric mucosa. The transport ofH+ across the apical membrane is by means of conforma-tional changes in the protein driven by cyclic phosphoryla-tion and dephosphorylation of the catalytic subunit of theATPase. This mechanism places the gastric H+,K+-ATPasein the P2 ATPase family, containing the Ca2+- and the Na+,K+-ATPases.

The gastric H+,K+-ATPase is composed of two subunits,the α subunit and the β subunit. The α subunit with molecularmass of about 100 kDa has the catalytic site and the β subunitwith peptide mass of 35 kDa is strongly but noncovalentlyassociated with the α subunit. The β subunit has six or sevenN-glycosylated sites exposed to the extracytoplasmic surface(Figure 1).

Since the gastric H+,K+-ATPase is responsible for thefinal step of acid secretion, this enzyme has been a therapeutic

target in treatment of acid-related disease for the last 20 years.The first class of the pump enzyme inhibitor contains thesubstituted benzimidazoles which are the most effective classof anti-ulcer drugs used in the treatment of a variety of acid-related diseases of the upper gastrointestinal tract. A secondclass of inhibitor which competes with extracytoplasmic K+

is being developed to get better inhibition.

Structure of the GastricH+,K+-ATPaseThe primary sequences of the α subunits deduced fromcDNA have been defined for rat (82), pig (39), and rabbit(5). The gene sequence for human H+,K+-ATPase α sub-units have also been determined (41, 55). The human gastricH+,K+-ATPase gene has 22 exons and encodes a proteinof 1035 residues including the initiator methionine residue(Mr = 114,047). These H+,K+-ATPase α subunits show highhomology (∼60 % identity) with the Na+,K+-ATPase cat-alytic α subunits and lesser homology with the sarcoplamic

*Correspondence to [email protected] of Physiology and Medicine, University of California atLos Angeles, and VA Greater Los Angeles Healthcare System, LosAngeles, California

Published online, October 2011 (comprehensivephysiology.com)

DOI: 10.1002/cphy.c110010

Copyright C© American Physiological Society

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Gastric H+,K+-ATPase Comprehensive Physiology

N domain A domain

Membrane domain

P domain

Ion-binding site

Beta subunit (pink)

Cytoplasmic domain

Luminal domainN146

N193

N130 N221

N161

N99

M5/M6 loop

Figure 1 The structure of the gastric H+,K+-ATPase. The modeledthree-dimensional structure of the H+,K+-ATPase, HKzxe based on theNa+,K+-ATPase in the E2P state. The α subunit has three lobes, N(ATP binding), P (phosphorylation), and A (activation) domains in thecytoplasmic domain, and ten transmembrane segments in the mem-brane domain. The gastric β subunit has short cytoplasmic region,one transmembrane segment, and a heavily glycosylated extracellularregion. In this model, the backbone of protein with carbohydrate at-tached Asn sites is shown. Amino acid sequence number is based onpig H+,K+-ATPase.

reticulum Ca2+-ATPase (SERCA) (22%) but more structuresare known for the latter hence more homology modeling hasbeen done with this pump than with the Na+,K+-ATPase.The hog gastric H+,K+-ATPase α subunit sequence deducedfrom its cDNA consists of 1034 amino acids and has a Mrof 114,285. The rat gastric H+,K+-ATPase consists of 1033amino acids and has a Mr of 114,012, and the rabbit gastricH+,K+-ATPase consists of 1035 amino acids, with a Mr of114,201. The sequence based on the known N-terminal aminoacid sequence is one less than the cDNA-derived sequence dueto loss of the N-terminal methionine and this numbering isadopted in this review (34). The homology of the H+,K+-ATPase α subunits among the animal species is extremelyhigh (over 97% identity).

The gastric α subunit has conserved sequences along withthe other P2-type ATPases, the SERCA and the Na+,K+-ATPase, for the ATP-binding site and the phosphorylationsite. The phosphorylation site was observed to be at Asp386

(101), which is well conserved in other P-type ATPases. Themembrane topology of the α subunit has been shown to con-tain 10 membrane spanning segments (5, 6).

The β subunit was first identified in work analyzing thecarbohydrates of gastric membranes (19). Postembeddingelectron microscopic staining techniques showed that wheatgerm agglutinin staining occurred on the inside face of the

inside out gastric vesicles. The same β subunit was found bydetermining the nature of the antigen in autoimmune gastri-tis (90). Using a lectin affinity chromatography, the H+,K+-ATPase α subunit was copurified with the β subunit, showingthat the α subunit interacts with the β subunit (10, 59). Bycross-linking with low concentrations of glutaraldehyde, theα subunit was shown to be closely associated with the β

subunit (20, 65).The primary sequences of the β subunits have been

reported for rabbit (68), hog (90), rat (12, 40, 56, 81),mouse (11, 48), and human (38). The β subunit con-sists of about 290 amino acids with a single transmem-brane segment, which is located at the region near the N-terminus. There are three disulfide bridges in the luminalregion of the β subunit. Seven putative N-glycosylated sites(AsnXaaSer and AsnXaaThr) are found in rabbit (68), thatare conserved in rat and human, but only six putative N-glycosylation sites are present in the hog gastric β subunit.The structure of N-linked oligosaccharides of the β sub-unit of rabbit gastric H+,K+-ATPase was identified (95, 96).All seven N-linked AsnX(Ser/Thr) sites at positions 99,103, 130, 146, 161, 193, and 222 were fully glycosylated.Asn99 was modified exclusively with oligomannosidic-typestructures, Man6GlcNAc2-Man8GlcNAc2, and Asn193 hasMan5GlcNAc2-Man8GlcNAc2 and lactosamine-type struc-tures. Asn 103, 146, 161, and 222 contain lactosamine-typestructures. All the branches of the lactosamine-type structureterminate with Galα-Galβ-GlcNAc extensions (95). The roleof the carbohydrate chains in membrane trafficking has beenstudied in HEK-293 cells and in polarized cells such as LillyLaboratories Cell – Porcine Kidney (LLC-PK) and Madin-Darby canine kidney (MDCK) cells (3, 97-100). In polarizedcells, the steady-state distribution of the H+,K+-ATPase β

subunit depends on the balance between (i) direct sorting fromthe trans-Golgi network (TGN), (ii) secondary associativesorting with a partner protein, and (iii) selective trafficking.

Studies on the expression of the β subunit of the gas-tric H+,K+-ATPase in LLC-PK1 cells showed that mutationof each of the seven N-glycosylation sites of rabbit gastricH+,K+-ATPase provided differential distribution of the sub-unit in endoplasmic reticulum (ER), Golgi, or plasma mem-brane. For example, the β subunit of wild type expressed inLLC-PK1 cells is distributed between the plasma membraneand the intracellular compartments even in the absence ofthe α subunit of the H+,K+-ATPase. At steady state, ∼40%of the protein is detected in the ER, and the remaining 60%is distributed between the plasma membrane, Golgi, TGN,and endosomes. Removal of the seventh N-glycosylation site,Asn222, leads to virtually total retention of the subunit inthe ER. Mutation of N1 increases the amount retained inthe ER to 75%. Similarly, mutation of Asn130 and Asn161also significantly increases the relative amount of the ER-resident fraction, but to a lesser extent. However, mutation ofAsn103, Asn146, or Asn193 does not appreciably alter thequantity of protein retained in the ER. These findings indicatethat N-glycans at Asn99, Asn130, Asn161, and Asn222 are

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Comprehensive Physiology Gastric H+,K+-ATPase

important for apical membrane trafficking of this β subunit. Itis remarkable that the same N-glycans (Asn99, Asn130, andAsn161) of the H+,K+-ATPase β subunit that are importantfor ER-to-Golgi trafficking are also important for apical de-livery and membrane retention of the protein. These resultssuggest that, similar to ER-to-Golgi trafficking, both apicaldelivery and apical membrane retention of the H+,K+-ATPaseβ subunit are lectin dependent and only specific N-glycansare accessible to these lectins (99).

Another role of the H+,K+-ATPase β subunit is stabi-lizing the E2 P conformation. The N-terminal cytoplasmicregion of the β subunit is known to be responsible for this(16). Two-dimensional crystals of the enzyme at 6.5 A res-olution showed that the N-terminal tail of the β subunit isin direct contact with the phosphorylation domain of the α

subunit. This interaction may hold the phosphorylation do-main in place, thus stabilizing the enzyme conformation andpreventing the reverse reaction of the transport cycle. Indeed,truncation of the β subunit N-terminus did allow reversal oftransport. These results suggest that the β subunit N-terminusprevents the reverse reaction from E2P to E1P, which is likelyto be relevant for the generation of the large in vivo protongradient (2).

Regions of Association Between theα and β SubunitsThe α subunit of the H+,K+-ATPase is strongly associatedwith the β subunit (76). Specifically, the region of the se-quence Arg898 to Arg922 in the α subunit has strong inter-action with the extracytoplasmic domain of the β subunit.By using yeast two-hybrid analysis, two different sequencesin the β subunit Gln64 to Asn130 and Ala156 to Arg188 wereidentified as associated with the extracytoplasmic sequenceof the β subunit. These data enabled identification of majorassociative regions of the α-β subunits of the H+,K+-ATPase(43, 77). There has been much suggestive evidence that theα-β heterodimeric H+,K+-ATPase exists in the membrane asan (α-β)2 oligomer. Such evidence includes target size (63) inthe membrane and unit cell size of the enzyme in membraneimbedded two-dimensional crystals (22). More recent evi-dence showed that the gastric H+,K+-ATPase may functionas an (α-β)2 dimeric heterodimer (79). The key observationin these studies was that the membrane preparation showedfull stoichiometry with respect to ATP binding (1 mol/molα-β) and half stoichiometry with respect to inhibitor bind-ing and phosphorylation suggesting an (α-β)2 oligomer. Theactual oligomeric state of the Na+,K+- and H+,K+-ATPasesnevertheless remains controversial. It was shown that the α-βprotomer of the Na+,K+-ATPase is active in its detergent-solubilized form but the membrane oligomeric form is un-proven (13). It should be noted that the SERCA pump crys-tallizes as a monomer after detergent solubilization but thisof course is unrelated to the oligomeric state in the membrane(92).

Kinetics of the gastric H+,K+-ATPaseThe H+,K+-ATPase exchanges intracellular hydrogen ionsfor extracellular potassium ions by consuming ATP. TheH+/ATP ratio was independent of external KCl and ATP con-centration (64). However, the stoichiometry of H+ per ATPwas different depending on luminal pH. The H+ for K+ stoi-chiometry of the H+,K+-ATPase per ATP hydrolyzed was one(42, 67, 86) at low pH, and two (64, 85) at neutral pH or nearneutral pH. This could be explained by retained protonationat one of the ion binding site carboxylic acids at a pH wellbelow the pKa of the carboxylic amino acid in this site.

The H+,K+-ATPase has many reaction steps, similar tothose of the Na+,K+-ATPase (104). The H+,K+-ATPasebinds the hydronium ion on the cytoplasmic side at highaffinity (E1 conformation). The initial step is the reversiblebinding of ATP to the enzyme in the absence of added K+

ion, followed by a Mg2+ (and hydronium)-dependent trans-fer of γ-phosphate of ATP to Asp386 of the catalytic sub-unit (E1P•H+). Following phosphorylation, the conformationchanges from E1P•H3O+ to the E2P•H30+ form, which hashigh affinity for K+ and low affinity for H3O+ allowing re-lease of H3O+ and binding of K+ from the extracytoplasmicsurface of the enzyme. Breakdown of the E2P form requiresK+ or its congeners on the outside face of the enzyme. Withdephosphorylation, the E1K+ conformation is produced witha low affinity for K+, releasing K+ to the cytoplasmic side,allowing rebinding of H3O+ (Figure 2).

The addition of K+ to the enzyme-bound acyl phosphateresults in a biphasic dephosphorylation. The faster initial stepis dependent on the concentration of K+, whereas the slowerstep is not affected by K+ concentration. The second phase

E1•Mg

E1•Mg•(K+) E1[ATP]•Mg•(H+)

E1P•Mg•(H+)

E2P•Mg•(H+)H+ K+

E2•Mg•(Kocc+)

E2P•Mg•(K+)

K+ ATP

Pi

ADP

Figure 2 The catalytic cycle of the gastric H+,K+-ATPase. A hydro-nium ion binds to the cytoplasmic surface of the enzyme, and MgATPphosphorylates the protein at Asp386 to form the first ion transportintermediate in the E1P form. The E1P form then converts by a confor-mational change to the second ion transport form, E2P, with the ion sitenow exposed to the exterior and hydronium is released at pH ∼1.0. Tothis form, K+ binds from the outside surface to the same region fromwhich the hydronium was released, and the enzyme dephosphorylates,and then K+ is trapped within the membrane domain in the occludedform (and a similar form is postulated for the hydronium in the outwardstep of the cycle). The K+ is then deoccluded allowing reformation ofthe E1 form of the enzyme with the ion site now again facing the cy-toplasm and K+ is displaced when ATP is bound. In this model, thenaked H+ is used instead of the hydronium ion (H3O+). This modelreflects a stoichiometry of 1H/1K/1ATP.




of EP breakdown is accelerated in the presence of K+ but,at K+ concentrations exceeding 500 μM, the rate becomesindependent of K+ concentration. This shows that two formsof EP exist. The first form, presumably E1P is K+ insensitiveand converts spontaneously in the rate-limiting step to E2P, theK+ sensitive form. ATP binding to the H+,K+-ATPase occursin both the E1 and the E2 state, but with a lower affinity in E2

state (9). The Mg2+ remains occluded in the P domain nearAsp730 (74) until dephosphorylation (44, 45, 66) in contrastto the Na+,K+-ATPase, where the Mg ion is released fromE2P (62).

The sequence and mechanistic similarity among the P2-type ATPases, the H+,K+-ATPase, the Na+,K+-ATPase, andthe Ca-ATPase allows extension of the structural results foundfor any individual pump to the other family members. Becausemany crystal structures of the SERCA ATPase have beensolved (27, 60, 61, 87, 89, 91-94), the conformational interme-diates of the catalytic cycle of Ca-ATPase have become wellunderstood at a molecular level. Like the H+,K+-ATPase al-pha subunit, SERCA1a is composed of 10 transmembrane he-lices (M1-M10) and 3 cytosolic domains: N (nucleotide bind-ing), P (phosphorylation), and A (actuator). In its E1 form,

SERCA1a binds two Ca ions with high affinity in the middleof the 10 transmembrane helices, whereas in the E2 form thecation binding sites are associated with protons. Transitionstates of phosphorylation and dephosphorylation are coupledto occlusion of bound calcium and protons, respectively. Inthe forward direction of the functional cycle, the Ca2 E1∼Pstate, formed by reaction of Ca2 E1 with ATP, is converted toan E2P state, accompanied by translocation of Ca2+ from thecytosolic to luminal orientation of the ion-binding site. TheE2P state is then dephosphorylated along with counter trans-port of protons. All steps in the reaction cycle are reversible;thus ADP stimulates the dephosphorylation of the Ca2 E1∼Pstate through regeneration of ATP, and Ca2+/Ca2+ exchangeis observed at high vesicular levels of Ca2+ (60).

A similar set of steps was generated for the H+,K+-ATPase using this ATPase as the basis for homology modeling(35, 49-51).

A detailed model of H+ and K+ transport was proposed asfollows. In panel 1 of Figure 3, Lys-NH3

+-791 is above the hy-dronium site H2. With phosphorylation and formation of E1P,K791 moves into the second hydronium site containing E795,E820, and D824 forming E2P and the lys-NH3

+ displaces the

Figure 3 A model of the H+,K+ ion exchange at acidic luminal pH based on the three-dimensional structure of the srCa-ATPase. In panel1 the 3 hydronium ion-binding sites at the carboxylates in TM 5, 6 and TM3 with the exported ion at acidic pH outlined in yellow. In panel 2after phosphorylation, lys791 displaces the hydronium from site 2 in the E1P form (arrow). Then in panel 3 K+ binds to site 2 displacing lys791generating the E2P form and in panel 4 K+ moves into site 3 allowing exit to the cytoplasm along TM3.




H3O+ outward (Fig. 3, panel 2). At pH < 3.0, the hydroniumat H1 remains bound due to the higher pKa of this site con-taining so the K+ binds only at H2, displacing K791 upward(Fig. 3, panel 3) to form the E2P·K+

occ occluded state. Withdephosphorylation, K+ moves into H3, where E820, E343,and the carbonyls of V337 and V341 are reoriented to allowexit of K+ along TM3 (arrow, Fig. 3, panel 4). Critical tothe coupling of ATP binding and subsequent phosphorylationis the conformational change, where the A domain rotatesclockwise to bring the N and P domains into apposition withthe bound ATP acting like a hook to retain this conformationuntil dephosphorylation (1, 58, 92, 94).

Recently, two-dimensional crystals of the H+,K+-ATPasewere analyzed in the presence of the phosphate analog BeFand an inhibitor SCH28080 (1). This structure of the E2Pconformation was similar to that of Ca-ATPase. The overallstructure of the present EM map (Fig. 4) shows characteristicfeatures of the ADP-insensitive E2P conformation, to whichSCH28080 is preferentially bound. The BeF-bound phospho-rylation site (Asp385) at the P domain is covered by the A

domain, and the ADP-bound N domain is separated from theP domain, thus the bound phosphate analog seems to be iso-lated from both ADP and the bulk solution. At this resolution,the side chains cannot be identified and their orientation wasbased on the homology model above.

Potassium ion pathway

The model generated for the E2P conformation showed achannel for the passage of K+ from the luminal vestibule tothe ion occlusion site near the middle of the membrane. TheM5/M6 loop presents the first protein segment encounteredfor passage of the ion into the channel. The only pathway tothe site accessed by the ion is between the carbonyl oxygenatoms of L811 and G812 and the sulfur of C813. This leadsto apparent binding to these two carbonyls and two moleculesof water. This appears from the model to be the initial entrysite into the channel.

At close to neutral pH with deprotonation of both E820and D824 at sites 1 and 2, two K+ can bind to provide equal

A-M2

A domain

SCH28080

β-subunit

βNt

αNt

(A) (B)

N

N domain

P

P domain

TM

AADP

ADP

linker

Cytoplasm

Membrane

Lumen

Figure 4 SCH28080-bound E2P structure of the gastric H+,K+-ATPase analyzed at 7 A resolution. (A) The structure of the H+,K+-ATPaseshowing cytoplasmic-side up. Surface represents the EM density map with superimposed homology model of H+,K+-ATPase (ribbon). BoundSCH28080 at the luminal cavity, and ADP at the N domain are shown as sticks. A wheat box indicates approximate location of the lipidbilayer. Color code: A domain, blue; P domain, green; N domain, yellow; TM domain, light blue (surface); and β subunit, red. Color for theTM helices of the homology model gradually changes from M1 (blue) to M10 (red). (B) Conformational change of the cytoplasmic domainseen from the top. Surface shows EM density map of (SCH)E2BeF and orange mesh represents E2AlF EM map of the gastric H+,K+-ATPase.The intersubunit interaction between the P domain and the N-terminal tail of the β subunit is observed in E2AlF structure, but not present in(SCH)E2BeF structure. White arrows indicate observation of the conformational changes during E2AlF → (SCH)E2BeF transition. This figureis cited, with permission, from the work of Abe et al. (1) and is similar to the homology model of the H+,K+-ATPase shown above andbelow.




Figure 5 K+ pathway in the H+,K+-ATPase. The image illustrates theentry path for K+ (illustrated as a series of violet spheres) between M4,M5, M6, and M8 in the E2P conformation as determined by moleculardynamics simulation (M3 in the foreground is omitted for clarity) (35,50). The arrival of K+ at the top of this path is predicted to destabilizethe interaction of K791 with E820 and E795, and initiate the confor-mational changes leading to release of phosphate at the active siteand conversion back to E1.

stoichiometry to the two extruded protons.## With the absenceof deprotonation of D824 due to its higher pKa, the stoichiom-etry will drop to 1K+/1ATP at low pH, with the initial siteof K+ occupancy at E820 and E795 and then movement toE343 with reprotonation of E820. Such a model would beconsistent with the K+ independence of turnover of the E820mutant (88).

A notable feature of the luminal face of the enzyme mod-eled in the E2 form is the presence of a luminal vestibulebounded by TM4, TM5, and TM6 and the connecting exo-plasmic loops between TM3-TM4 and TM5-TM6 containingcysteine 813 as part of the loop between TM5 and TM6.This vestibule is a natural exit and entry point for transportedcations and also provides access to the different classes ofinhibitors of the gastric H+,K+-ATPase and is illustrated inthe model of Figure 5. Analysis of the SERCA Ca2+-ATPaseshows that this vestibule is closed in the E1 form and open inthe E2 form (47).

A three-dimensional (3D) model of the K+ pathway fromlumen to cytoplasm based on molecular dynamics is shownin Figure 6.

Inhibitors of the H+,K+-ATPaseTwo types of H+,K+ ATPase inhibitors have been devel-oped that react exclusively with the extracytoplasmic surface

Figure 6 Proposed K+ pathway through the pump srCa-ATPase ho-mology model. Red to pink is area of restriction including the occlusionsite. White to blue less occlusion. Arrows highlight entry and exit path-ways for K+.

of the gastric H+,K+-ATPase. One commercially availableclass of inhibitors is the substituted benzimidazoles such asomeprazole, lansoprazole, rabeprazole, pantoprazole, and es-omeprazole, the proton pump inhibitor class (PPI). The otherclass contains a series of K+-competitive reagents, includingimidazo-pyridines that are also known to react on the outsidesurface of the pump competing with potassium ion. This typeis called either the acid pump antagonist (APA) or potassium-competitive acid blocker (P-CAB) class.

The substituted benzimidazole inhibitorsThe first antisecretory inhibitor of this class was 2-(pyridylmethylsulfinyl)benzimidazole (17). This compoundis a weak base ∼pKa 4. The H+,K+-ATPase in the parietalcell secretes acid into the secretory canaliculus generating apH of <1.0 in lumen of this structure. The acidity of thisspace allows accumulation of weak bases of this pKa. Weakbases of a pKa less than 4.0 can be accumulated only in thisacidic space and no other acidic space in the body. Then, thiscompound is rapidly activated by the high acidity and inhibitsacid secretion by binding to the cysteines accessible to theactivated form.

Since a substituted benzimidazole was first reportedto inhibit the H+,K+-ATPase (17), many inhibitors of theH+,K+-ATPase have been synthesized (Figure 7). The




Figure 7 Proton pump inhibitors.

first pump inhibitor used clinically was 2-[[3,5-dimethyl-4-methoxypyridin-2-yl]methylsulfinyl]-5-methoxy-1H-benzimidazole, omeprazole (102), and this was followed byother covalent binding inhibitors all belonging to the substi-tuted benzimidazole family (18, 54, 83). These reagents areweak base, acid activated compounds, which form cationicsulfenamides or sulfenic acids in highly acidic environments.The thiophilic compounds formed react with the SH groupof cysteines in the ATPase to form relatively stable disulfides(Figure 8) (7, 8, 71-73). Since the pump generates acid onits extracytoplasmic surface, only those cysteines availablefrom that surface are accessible to these reagents if labelingis carried out under acid transporting conditions.

Omeprazole has a stoichiometry of 2 mol inhibitor boundper mol phosphoenzyme under acid transporting conditionsand is bound only to the α subunit even in vivo (31, 37, 73).The binding sites of omeprazole are cysteine 813 and 892(7). The sites of reaction of the different PPIs on the enzymediffer according to the particular PPI. However, all PPIs reactwith cysteine 813 in the loop between TM5 and TM6 thatfixes the enzyme in the E2 configuration. Lansoprazole reactswith cysteine 813 and cysteine 321, these being in the luminalvestibule (70), whereas pantoprazole and tenatoprazole reactwith cysteines 813 and 822 (72,73, 75, 78). The reaction withcysteine 822 confers a rather special property to the covalentlyinhibited enzyme, namely, irreversibility to reducing agentsin vitro and in vivo due to lack of accessibility of Cys822.

An innovation was introduced by the S-enantiomer ofomeprazole, esomeprazole, which has a slightly delayedmetabolism compared to the R-enantiomer. In comparativestudies, 40 mg esomeprazole was compared to 20 mg of theracemic omeprazole and some improvement in outcome was

observed (69). However, the active compound generated inacid from esomeprazole has no chiral center and is identicalto that formed from omeprazole.

The key issue in efficacy of this class of drug is their short60-90 min plasma half-life so that there is no drug presentat night even with twice a day meal time-related adminis-tration. Recently, the R-enantiomer of lansoprazole has beenintroduced with a delayed release formulation (30) and a sim-ilar strategy has been adopted for rabeprazole (32) to try toprovide night time drug levels.

There are several conceptual means of improving acid in-hibition with the H+,K+-ATPase as the target. Firstly, a newcore structure can be generated with slower metabolism toimprove the residence time in the blood hence exposure to ac-tive pumps. A delayed release formulation can be establishedalso with the aim of increasing exposure. A prodrug of a PPIcan be synthesized so as to delay absorption and thus also toincrease exposure. Or, a novel mechanism can be used wherepump inhibition is reversible.

Recently a novel formulation of the R-enantiomer of lan-soprazole has been introduced. This is a dual release formula-tion of 60 mg of the PPI with normal enteric coating releasingat around pH 5.0 and a coating labile at pH 7 releasing dexlan-soprazole some hours later.

A biphasic PK profile for this formulation is observed forfirst day treatment that is the same as day 5, showing clearsuperiority 4 to 8 h after dose but not during the night. Thisprofile might explain the small increase in median 24 h pHsince this reflects mainly the low pH at night.

There was no statistically significant difference in healingbetween the 60 mg dose and regular 30 mg lansoprazole.However, there was an improvement in the time above pH




R1 R2

R4Bz-Py

Bz-PyH+

BzH+

Sulfenic acid Sulfenamide

A1

-PyH+

-PyBzH+

R3

N

+H+

+H+

-H+

-H+

NH

SON

R1 R2

R4

R3

N

NH

H

S

ON

R1H2O

H3OR2

R4

R3NNH

HS O

N +

+

-

-

+H+

-H+

R1

R2

R4 R3

N

NH

H

SON

+

+

R1

R1

H2O

R2

R4

R3

N

NH

Enz–SS

Enz-SH Enz-SH

N+

R2

R4

R3NN

S

N +

R1 R2

R4

R3

N

N

H

H

H

S

O

N

++

+

+

R1 R2

R4

R3

NNH

H

H

SON

+

+

Figure 8 The mechanism of activation of the PPIs shown in general struc-tural form. The substituent R1, R2, R3, and R4 of the general structure (Bz-Py)represent a substituent chosen from hydrogen, methoxy, methyl, and substi-tuted alkoxy group. Top part shows the protonation of the pyridine ring andthe second row of structures shows protonation also of the benzimidazolering. The bis-protonated forms are in equilibrium with the protonated ben-zimidazole and unprotonated pyridine. In brackets is shown the mechanismof activation whereby the 2C of the protonated benzimidazole reacts with theunprotonated fraction of the pyridine moiety that results in rearrangement toa permanent cationic tetracyclic sulfenic acid which in aqueous solute dehy-drates to form a cationic sulfenamide. Either of these thiophilic species canreact with the enzyme to form disulfides with one or more enzyme cysteinesaccessible from the luminal surface of the enzyme.




4.0, 71% relative to 60% but mean pH was 4.55 relative tolansoprazole at 4.13 (30). This improvement is similar to whathas been claimed for esomeprazole.

Another method, not requiring alternative formulation, isto make a slowly absorbed derivative of a PPI that is absorbedthroughout the small intestine and not just the duodenumlike all the PPIs. Of various derivatives tested, a sulfonamidederivative, the phenoxyacetic acid sodium salt derivative ofomeprazole seems to be a candidate drug with several de-sirable properties (80). This compound is slowly absorbedthroughout the small intestine but then rapidly hydrolyzed inthe blood to omeprazole and the sulfonic acid. Only tracequantities of the intact molecule are found in humans henceits safety profile should resemble that of omeprazole. The ef-fect of various doses in man showed a linear dose relationshipwith no evidence for saturation hence movement across thegut is by simple diffusion.

There is great prolongation of the blood level of omepra-zole and as found for omeprazole there is increased bioavail-ability after 5 days dosing. But significantly, there is prolon-gation of the residence time of omeprazole in the blood sothat there is drug present at sufficient levels over 24 h after 5days administration (25).

Acid Pump Antagonists (orPotassium-Competitive Acid Blockers)This class of the inhibitor blocks acid pumping by inhibit-ing K+ competitively, so this class is called either APAsor P-CABs. The first core structure of an APA developedin 1980s was an imidazo-pyridine (28). A typical struc-ture of this class is SCH28080. Following SCH28080, manydifferent core structures were made such as piperidinopy-ridines (23), substituted 4-phenylaminoquinolines (26),pyrrolo[3,2-c]quinolines (36), guanidino-thiazoles (33), sub-stituted pyrimidines (21), and scopadulcic acid (4). Some nat-ural products such as cassigarol A (53) and naphthoquinone(14) also showed inhibitory activity.

SCH28080 was the first compound extensively studiedfor its inhibitory mechanism. SCH28080 inhibits the H+,K+-ATPase activity by competing with K+ (103). SCH28080binds to free enzyme extracytoplasmically in the absence of

Figure 9 The amino acids lining the luminal entrance (vestibule) tothe ion-binding sites of the H+,K+-ATPase that is the basis for dockingpyrrolo-pyridines suggesting critical amino acid residues responsiblefor high affinity and slow dissociation.

substrate to form E2 (SCH28080) complexes. SCH28080 in-hibits ATPase activity with high affinity in the absence ofK+. SCH28080 has no effect on spontaneous dephospho-rylation but inhibits K+-stimulated dephosphorylation, pre-sumably by forming a E2−P•[I] complex. Hence SCH28080inhibits K+-stimulated ATPase activity by also competingwith K+ for binding to E2P (46). SCH28080 does notinhibit the Na+,K+-ATPase though there are high similari-ties of the cation-binding region between the Na+,K+- andthe H+,K+-ATPase.

Generally speaking, APA compounds show similar in-hibitory mechanism to that shown by SCH28080. They de-pend for their action on binding to the open form of thevestibule in the H+,K+-ATPase as shown in Figure 9.

Vestibule structureThe structural characteristics of this space are viewed in theorientation of Figure 10 with the M1/M2 segments to the right,

Figure 10 Acid pump antagonists.




M5 to the left, M4 in the foreground, M6 in the background,the ion site above, and the aqueous lumen to the bottom. Be-tween the ion site above and the lumen is the loop connectingM5 and M6. The left and right sides of the vestibule are linedwith hydrophobic side chains which provide the surface forhydrophobic groups of inhibitors to shed their water of hydra-tion before binding thus providing much of the driving forcefor binding. Above the center of the vestibule is a cluster ofnegative charge density given by carboxylate side chains ofthe ion-binding/transport site. This provides through-spacecharge/charge attraction for any positive charge density pro-vided by an inhibitor, either in the form of a formal chargegroup such as an amine or guanidinium or as in the delocal-ized positive charge ring surrounding an aromatic ring systemsuch as the imidazopyridines. These charge interactions aremade more significant by the displacement of water in thispart of the vestibule by specific groups attached appropriatelyto the inhibitor core such as a pyrrolo-pyridine.

The vestibule is narrow in the space between M4 andM6, specifically between A335 and C813, and mutationalanalysis has shown the inhibitors have to fit between theseside chains. C813 is known to be the side chain to which PPIsbind covalently to inhibit the H+,K+-ATPase (8, 73) showingthat these bulky inhibitors (e.g., omeprazole) can enter thevestibule up to the point directly below the M5M6 loop nextto C813. On the left side of the vestibule the space is restrictedby the close interaction of M5 with M4 and a high densityof bulky side chains whereas on the right side there is moreavailable volume. These conclusions confirm earlier workusing models of the H+,K+-ATPase derived from the firstavailable molecular structures of the srCa-ATPase (Fig. 9).

Many of the side chains lining the vestibules are the samein the Na,K and H+,K+-ATPases. A set of side chain muta-tions that are mostly conservative substitutions from the Na,K-ATPase confer ouabain sensitivity to the H+,K+-ATPasehave been identified, namely, Y799F, A335G, C813T, I331V,M330I, R328E, and E820D (15). All but E820D are in thevestibule region. We have shown that the nonconserved Y799,A335, C813, and additionally, M334 contribute to the bindingaffinity (and specificity) of SCH28080 to the H+,K+-ATPasewhile other conserved residues also make strong contributionsto binding affinity especially L809, P810, and L811. Subtledifferences in this region of the two pumps determine thedifferences between efficacies of pump selective inhibitors.

Binding of planar structuresThe best characterized reversible inhibitor family has a coreplanar imidazo-pyridine structure of which SCH28080 is theprototypical example. Early work with derivatives in this classestablished the extended conformation of the phenyl groupwith respect to the imidazopyridine ring as a requirement forhigh-affinity binding (28, 29). Later it was shown that fixingthis conformation with an ethylene bridge in the homolo-gous naphthyridine (e.g., Byk99) increased binding affinity5- to 10-fold (105). Hence, a model for the binding of these

compounds must provide this conformation for the bound in-hibitor. Also, in early work with a photo affinity derivative ofSCH28080 containing a reactive azido group in the para po-sition of the phenyl ring showed specific labeling only in theM1/M2 segments of the membrane domain (52). This resultput the phenyl ring on the right side of the vestibule towardM1/M2. The observed labeling by omeprazole at C813 estab-lished the existence of a space adjacent to this position foran elongated inhibitor structure. Across from C813 is A335and this orientation was established when we showed that theA335C mutation gave a disulfide linkage between the twoside chains and complete loss of SCH28080 inhibition. TheC813T mutation also resulted in a 10-fold decrease in affinity(50). These results showed that there is a narrow space be-tween C813 and A335 and that at least part of the inhibitorwas bound between them. Positioning the flat imidazopyri-dine in the slot between these side chains then accounted notonly for the decrease in SCH28080 affinity seen in A335Cand C813T but also in the L809F, P810G, L811F, M334I, andY799S mutants.

Some of these APAs are shown in Figure 10.The inhibition by the APAs is expected to be fast and

effective since they do not require acid activation. Furtherthe pump is inhibited at half cycle without any proton se-cretion occurring. Data in human show the expected rapidand virtually complete inhibition by APAs (or P-CABs). Forexample, in healthy volunteers, high doses of AZD0865 re-sulted in over 95% inhibition of acid secretion within 1 hafter oral dosing (57). This inhibitor exhibit a classical (sig-moid) dose-response profile, with the magnitude and durationof effect being determined by dose, pKa, and plasma half-life. AZD0865 demonstrated a dose-effect relationship witha dose-dependent duration of inhibition of acid secretion.

A fused ring system of an imidazopyridine is soraprazan.Soraprazan inhibits the H+,K+-ATPase with an IC50 of 0.1μM, Ki of 6.4 nM, and a Kd of 26.4 nM (84). An APA struc-tural class, 5-aryl-1-(3-pyridinesulfonyl)pyrroles, was devel-oped (24). This member of a nonplanar class of compoundhas an IC50 value of 9 to 30 nM thus is the most potent ofthis type of inhibitor so far. It is noteworthy to mention thatthe 3-methylaminomethyl substituent is crucial to for the in-hibitory activity in this class of compound. One compound ofthis class, TAK-438 has a very slow dissociation rate produc-ing prolonged inhibition with a single dose lasting into thenight. It binds to the luminal domain in the open form whichthen closes to the predicted occluded binding configurationas shown in Figure 11.

ConclusionThe final step of gastric acid secretion in the stomach is per-formed by the gastric H+,K+-ATPase. The gastric H+,K+-ATPase exchanges hydronium ion with potassium using ATPhydrolysis. To transport the hydronium ion, the enzyme under-goes large conformational changes in the transition between




Figure 11 The vestibule structure predicted for bound TAK-438. Se-lected amino acids in the binding region are predicted to affect binding(white, bold). Also shown is H bonding to E795 -COO− and -C=Oand to Y799.

E1 and E2. The functional form of the gastric H+,K+-ATPaseis an oligomeric form, composed of the catalytic α subunitand the glycoprotein β subunit. This enzyme is the majortarget for anti-acid secretory drugs. PPIs are in wide spreadclinical use. Another type of acid blocker is being studied toget better inhibition of acid secretion.

AcknowledgementsThis work was supported by US VA Merit Grant, NIH/NIDDKgrant numbers DK053642 and DK058333.

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Further Reading1. Ogawa H, Shinoda T, Cornelius F, Toyoshima C. Crystal structure of

the sodium-potassium pump (Na+,K+-ATPase) with bound potassiumand ouabain. Proc Natl Acad Sci U S A 106: 13742-13747, 2009.

2. Shin JM, Munson K, Vagin O, Sachs G. The gastric HK-ATPase: Struc-ture, function, and inhibition. Pflugers Arch 457: 609-622, 2009.

3. Shin JM, Vagin O, Munson K, Kidd M, Modlin IM, Sachs G. Molecularmechanisms in therapy of acid-related diseases. Cell Mol Life Sci 65:264-281, 2008.

4. Shinoda T, Ogawa H, Cornelius F, Toyoshima C. Crystal structure ofthe sodium-potassium pump at 2.4 A resolution. Nature 459: 446-450,2009.

5. Vagin O, Tokhtaeva E, Yakubov I, Shevchenko E, Sachs G. Inversecorrelation between the extent of N-glycan branching and intercellularadhesion in epithelia. Contribution of the Na,K-ATPase beta1 subunit.J Biol Chem 283: 2192-2202, 2008.


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