Transmembrane Segment M2 Research Articles

A threonine residue in the M2 region of the β 1 subunit is needed for expression of functional α 1β 1 GABA A receptors

Although there is a high degree of homology in the M2 transmembrane segments of α 1 and β 1 subunits, subunit-specific effects were observed in α 1β 1 GABA A receptors expressed in Spodoptera frugipedra ( Sf9) cells when the conserved 13′ threonine residue in the M2 transmembrane region was mutated to alanine. When threonine 263 (13′) was mutated to alanine in the β 1 subunit, high-affinity muscimol binding and the response to GABA were abolished. This did not occur when the threonine 263 (13′) was mutated to alanine in the α 1 subunit, but the rate of desensitisation increased and the effect of bicuculline, a competitive inhibitor, was reduced. The results show differential effects of subunits on receptor function and support a role for M2 in desensitisation.

Evidence for a Salt Bridge between Transmembrane Segments 5 and 6 of the Yeast Plasma-membrane H+-ATPase

The plasma-membrane H+-ATPase of Saccharomyces cerevisiae, which belongs to the P2 subgroup of cation-transporting ATPases, is encoded by the PMA1 gene and functions physiologically to pump protons out of the cell. This study has focused on hydrophobic transmembrane segments M5 and M6 of the H+-ATPase. In particular, a conserved aspartate residue near the middle of M6 has been found to play a critical role in the structure and biogenesis of the ATPase. Site-directed mutants in which Asp-730 was replaced by an uncharged residue (Asn or Val) were abnormally sensitive to trypsin, consistent with the idea that the proteins were poorly folded, and immunofluorescence confocal microscopy showed them to be arrested in the endoplasmic reticulum. Similar defects are known to occur when either Arg-695 or His-701 in M5 is replaced by a neutral residue (Dutra, M. B., Ambesi, A., and Slayman, C. W. (1998) J. Biol. Chem. 273, 17411-17417). To search for possible charge-charge interactions between Asp-730 and Arg-695 or His-701, double mutants were constructed in which positively and negatively charged residues were swapped or eliminated. Strikingly, two of the double mutants (R695D/D730R and R695A/D730A) regained the capacity for normal biogenesis and displayed near-normal rates of ATP hydrolysis and ATP-dependent H+ pumping. These results demonstrate that neither Arg-695 nor Asp-730 is required for enzymatic activity or proton transport, but suggest that there is a salt bridge between the two residues, linking M5 and M6 of the 100-kDa polypeptide.

Separate domains for desensitization of GABA rho 1 and beta 2 subunits expressed in Xenopus oocytes.

Desensitization of ligand-gated receptor channels is an intrinsic feedback mechanism and prevents the receptor/channels from becoming overly activated thereby maintaining biological function of the nervous system. Desensitization also plays an important role in neuronal plasticity. By taking advantage of biophysical and pharmacological diversities of GABA beta2 subunits from the brain and rho1 subunits from the retina, structural determinants that confer agonist-induced desensitization were identified. A synthetic chimeric receptor/channel was created from the beta2 and rho1 subunits for this investigation. The chimera was constructed from the extracellular N-domain of the beta2 subunit, extending from the amino terminus to the beginning region of the M1 transmembrane segment, and from the C-domain of the rho1 subunit extending from the M1 transmembrane segment to the carboxyl terminus. The C-domain region included the M1 to M4 transmembrane regions and the large intracellular loop between the M3 and M4 transmembrane segments. Homo-oligomeric GABA beta2, rho1, and beta2/rho1 chimeric receptor/channels were individually expressed in Xenopus oocytes, and the desensitization characteristics attributable to each type of subunit were compared. Results from the present study reveal that motifs in the amino-terminal and carboxyl-terminal domains of the beta2 subunit conferred the agonist-induced desensitization; chloroform modulation was linked to specific phases of the GABA-activated current decay.

Spatial structure of the M3 transmembrane segment of the nicotinic acetylcholine receptor alpha subunit.

The three-dimensional structure of a synthetic peptide corresponding to the putative transmembrane segment M3 (amino acid residues 277-301) of the alpha subunit of the nicotinic acetylcholine receptor from Torpedo californica has been studied by means of two-dimensional 1H-NMR spectroscopy in a chloroform/methanol (1:1) mixture containing 0.1 M LiClO4. Complete resonance assignment has been performed using double-quantum-filtered COSY (DQF-COSY), TOCSY and NOESY spectra. The spatial structure has been calculated using the Diana program on the basis of integrated intensities of NOESY spectra. HN-C(alpha)H and HC(alpha)-C(beta)H spin-spin coupling constants. Residues 279-297 of M3 form a right-handed helix (root mean square deviation is 0.032 nm for backbone atoms and 0.088 nm for all heavy atoms). The conformations of the 17 side chains have been unambiguously determined. The obtained structure is in accord with the photolabeling pattern of the membrane nicotinic acetylcholine receptor (nAChR) which suggests alpha-helical structure of M3 in the labeled portion [Blanton, M. P. & Cohen, J. B. (1994) Biochemistry 33, 2859-2872].

Structural determinants of the blocker binding site in glutamate and NMDA receptor channels

Glutamate receptor channels of the NMDA-type ( N-methyl- d-aspartate) and non-NMDA-type (GluR) differ in their pore properties. The N-site in the M2 transmembrane segment of NMDA receptors (NMDAR), or the corresponding Q/R-site in GluRs, is a pivotal structural determinant of their permeation and blockade characteristics. Substitutions at a second site in M2, the L-site (L577) in GluR1, drastically alter the receptor selectivity to divalent cations. Here we report that M2 mutants carrying an asparagine or a threonine residue at the Q-site of GluR1, along with a tryptophan residue at the L-site, form homomeric GluR1 channels that are highly sensitive to structurally diverse, uncompetitive NMDA antagonists such as arylcyclohexylamines, dibenzocycloheptenimines, and to morphinian and adamantane derivatives. Analysis of the voltage dependence of channel blockade locates the blocker binding site ∼0.65 partway into the transmembrane electric field in both GluR1 mutants and NMDAR channels. Our results suggest that the homomeric GluR1 double mutants, L577W/Q582N and L577W/Q582T, fairly approximate the pore properties of the heteromeric NMDA receptor and support the structural kinship of their permeation pathways.

Role of the putative transmembrane segment M3 in gating of neuronal nicotinic receptors.

The involvement of some structural domains in the gating of the neuronal nicotinic acetylcholine receptor (AChR) was studied by expressing functional alpha7/alpha3 chimeric subunits in Xenopus oocytes. Substitution of the M3 transmembrane segment in the alpha7 subunit modifies the kinetic properties of the chimeric AChRs as follows: (a) a 6-fold reduction in the maximal current evoked by nicotinic agonists, (b) a 10-fold decrease in the macroscopic desensitization rate, (c) an increase of almost 1 order of magnitude in the apparent affinity for acetylcholine and nicotine, and (d) a decrease in the affinity for alpha-bungarotoxin. Computer simulations showed that the first three effects could be accounted for by a simple kinetic model in which chimeric AChRs presented a smaller ratio of the gating rates, beta/alpha, and a slightly slower desensitization rate. It is concluded that the M3 domain influences the gating of neuronal AChRs.

Analysis of the glycine binding domain of the NMDA receptor channel ζ1 subunit

In an attempt to examine glycine binding domain of the zeta 1 subunit of the mouse N-methyl-D-aspartate (NMDA) receptor channel, we constructed N-terminal or C-terminal deletion mutants of the zeta 1 subunit cDNA and expressed them in Spodoptera frugiperda cells using a baculovirus system. Analysis of binding of a glycine binding site antagonist, 5,7-[3(-3)H]dichlorokynurenate ([3H]DCKA) to the deleted zeta 1 mutants provided the first direct experimental evidence that the regions of N-terminal 282 and C-terminal 102 amino acid residues do not significantly affect glycine binding, and that both the region of approximately 260 amino acid residues preceding the putative transmembrane segment M1 and the region between the segments M3 and M4 are required to form the glycine binding domain.

Acetylcholine receptor subunit homomer formation requires compatibility between amino acid residues of the M 1 and M2 transmembrane segments

The neuronal nicotinic acetylcholine receptor (nAChR) subunits a3 and 0 have different assembly behavior when expressed in heterologous expression systems: α3 subunits require other subunits to assemble functional nAChRs, whereas α7 subunits can produce homomeric nAChRs. A previous analysis of α7 α3 chimeric constructs identified a domain comprising the first putative membrane-spanning segment, M1, as essential to homomeric assembly. The present study dissected further this domain, identifying three amino acid residues, which are located at the most intracellular third of the M1 transmembrane segment, as important in the assembly of homomers. Moreover, formation of homooligomeric complexes seems to require a compatible accommodation between this region and certain residues of the second transmembrane segment, M2. Thus, compatibility between defined domains of the M1 and M2 transmembrane segments appears as a determinant factor governing homomer association of nAChR subunits.

Regions Responsible for the Assembly of Inwardly Rectifying Potassium Channels

Inwardly rectifying potassium channels have an important role in determining the resting potential of the cell. They are tetrameric proteins with two transmembrane segments (M1 and M2), a pore-forming loop (H5), a cytoplasmic N-terminal, and longer C-terminal domain. We have used biochemical and electrophysiological methods to identify regions required for homotypic interactions and those responsible for the incompatibility between IRK1 and two other members of the same subfamily (IRK2 and IRK3) and two members from other subfamilies (ROMK1 and 6.1\\\\uK ATP). The data indicate that, in contrast to the voltage-gated class of potassium channel, the proximal C-terminus and the transmembrane segment M2 determine homo- and heteromultimerization and that heteromultimerization between members of the same or different subfamilies is case specific.

A single tryptophan on M2 of glutamate receptor channels confers high permeability to divalent cations

Ionotropic glutamate receptors (iGluRs) of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate subtype display lower permeability to Ca2+ than the N-methyl-D-aspartate (NMDA) subtype. The well-documented N/Q/R site on the M2 transmembrane segment (M2) is an important determinant of the distinct Ca2+ permeability exhibited by members of the non-NMDA receptor subfamily. This site, however, does not completely account for the different permeation properties displayed by non-NMDA and NMDA receptors, suggesting the involvement of other molecular determinants. We have identified additional molecular elements on M2 of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate receptor GluR1 that specify its permeation properties. Higher permeability to divalent over monovalent cations is conferred on GluR1 by a tryptophan at position 577, whereas blockade by external divalent cations is imparted by an asparagine at position 582. Hence, the permeation properties of ionotropic glutamate receptors appear to be primarily specified by two distinct determinants on M2, the well-known N/Q/R site and the newly identified L/W site. These findings substantiate the notion that M2 is a structural component of the pore lining.

Site-directed mutagenesis studies of energy coupling in the sarcoplasmic reticulum Ca 2+-ATPase

Site-directed mutagenesis studies identifying residues important to energy transduction in the sarcoplasmic reticulum Ca 2+-ATPase are reviewed. Mutations blocking the crucial E1P to E2P transition are located in the small and the large cytoplasmic domains, in the stalk segment S4 linking transmembrane segment M4 with the catalytic site, as well as in transmembrane segments M4 and M8. Mutations that block the dephosphorylation of the E2P phosphoenzyme intermediate are located in transmembrane segments M4, M5, and M6, i.e., in the same domain as the Ca 2+-binding sites. Removal of the sidechain of Tyr 763 located at the boundary between transmembrane segment M5 and the corresponding stalk segment S5 linking M5 with the catalytic site leads to uncoupling of ATP hydrolysis from Ca 2+ uptake. Uncoupling may be due to efflux through the Ca 2+-ATPase of Ca 2+ that has been transported, and may thus be caused by a defective gating process in the late part of the catalytic cycle. A nearby located residue Lys 758 is also involved in energy coupling, since its substitution with Ile activates dephosphorylation at high pH and slows the E2 to E1 transition.

Tryptophan substitutions at the lipid-exposed transmembrane segment M4 of Torpedo californica acetylcholine receptor govern channel gating.

Our previous amino acid substitutions at the postulated lipid-exposed transmembrane segment M4 of the Torpedo californica acetylcholine receptor (AChR) focused on the alpha C418 position. A tryptophan substitution on the alpha C418 produced a 3-fold increase in normalized macroscopic response to acetylcholine in voltage-clamped Xenopus laevis oocytes (Lee et al., 1994). This result was explained by a 23-fold decrease in the closing rate constant measured from single-channel analysis (Ortiz-Miranda et al., 1996). In this study, we introduce more tryptophan substitutions at different positions of this postulated lipid-exposed segment M4 in order to examine functional consequences at the single-channel level. From a series of amino acid substitutions at alpha G421, only phenylalanine and tryptophan produced a substantial increase in the open time constant. The lack of response from a tyrosine substitution at the alpha G421 suggests that the side chain volume is not the main structural element responsible for the effect of tryptophan on the stabilization of the open state of the channel. Three multiple mutants, alpha C418W/G421A, alpha C418W/G421W, and alpha C418W/beta C447W, were constructed in order to establish the correlation between the number of lipid-exposed tryptophans and the channel open time constant. The alpha C418W/G421A double mutant demonstrated that when both previous mutations are combined the open time constant was increased 1.5-fold relative to the alpha C418W. When the two mutants (alpha C418W and alpha G421W) were combined in a single mutation, a functional receptor was expressed and the open time constant of the new double mutant increased to 33.4 ms, an 80-fold increase relative to wild type. Estimations of free energy changes calculated from the rate constant for the opening transition suggest that each tryptophan contributes to the stabilization of the open state of the channel by about 0.8 kcal/mol, and the effect of tryptophan substitutions on the free energy is additive. This result suggests that in the channel gating mechanism of the AChR, each subunit contributes independently to the energy barrier between the open and closed state. At selected positions within the postulated lipid surface of the AChR, tryptophan substitutions could establish hydrophobic and perhaps dipole interactions that may play a dramatic role in the channel gating mechanism.

The sensitivity of AMPA-selective glutamate receptor channels to pentobarbital is determined by a single amino acid residue of the α2 subunit

Clinical concentrations of pentobarbital inhibit the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-selective glutamate receptor (GluR) channels. Recently, the AMPA-selective GluR channels that contained the α2 subunit were shown to be more sensitive to pentobarbital block than those without the α2 subunit. Here we demonstrated that replacement by glutamine of the arginine residue in putative transmembrane segment M2 of the α2 subunit (mutation α2-R586Q) drastically reduced the pentobarbital sensitivity of the α2 heteromeric channel to the level comparable to those of the α1 and α2-R586Q homomeric channels. These results suggest that the arginine residue in segment M2 of the α2 subunit is the critical determinant of the sensitivities of the AMPA-selective GluR channels to pentobarbital.

A single point mutation decreases picrotoxinin sensitivity of the human GABA receptor rho 1 subunit.

The GABA receptor rho subunits are thought to form bicuculline-insensitive and picrotoxinin-sensitive GABAC receptors. We have investigated the role of the amino acid at position 309 in transmembrane segment M2 of the human rho 1 subunit as a determinant for picrotoxinin sensitivity. The mutant rho 1P309S was constructed by exchanging proline 309 for serine, the corresponding amino acid of the human rho 2 subunit. Whole-cell recordings from HEK-293 cells transfected with rho 1P309S cDNA revealed that the sensitivity of the rho 1P309S channels for picrotoxinin was four-fold lower than that of the wild type rho 1 subunit. The affinity of the mutant receptor for GABA was only slightly changed. These results provide direct evidence that the amino acid at position 309 is an important determinant for the picrotoxinin sensitivity of GABA receptors formed by the rho subunits.

Kinetics of thapsigargin-Ca(2+)-ATPase (sarcoplasmic reticulum) interaction reveals a two-step binding mechanism and picomolar inhibition.

Thapsigargin is a high affinity inhibitor of sarco- and endoplasmic reticulum (SERCA) type ATPases. We have used kinetics to determine the dissociation constant of thapsigargin-sarcoplasmic reticulum Ca(2+)-ATPase interaction in the absence and presence of non-ionic detergent. The observed "off" rate constant was measured as 0.0052 s-1 at 26 degrees C by the kinetics of inhibition of ATPase activity following transfer from an inactivated thapsigargin-ATPase complex to native ATPase. Inactive ATPase was produced by cross-linking the active site with glutaraldehyde. The observed dissociation rate constant was increased 7-fold by 0.1% Triton X-100, indicating that perturbation of the transmembrane and stalk region by detergent altered the binding parameters of the inhibitor. In addition, thapsigargin stabilized the ATPase against inactivation caused by detergent in the absence of Ca2+. The observed "on" rate constant of thapsigargin was measured at 26 degrees C as 25 s-1 irrespective of thapsigargin concentration, by the kinetics of thapsigargin- induced change in intrinsic fluorescence. An Arrhenius plot showed a temperature dependence of this rate constant, indicative of a conformational change in the protein with an activation energy of 9.5 kcal/mol for thapsigargin binding. The affinity of the Ca(2+)-ATPase for thapsigargin was calculated to be greater than 2 pM at pH 7.0 and 26 degrees C.

Calmodulin Binding Sites of the Skeletal, Cardiac, and Brain Ryanodine Receptor Ca2+ Channels: Modulation by the Catalytic Subunit of cAMP-Dependent Protein Kinase?

In this study, we define calmodulin binding sites of skeletal, cardiac, and brain ryanodine receptor (RYR) Ca2+ channels. Cardiac and brain RYR peptides corresponding to the calmodulin binding sites present in the skeletal RYR [Menegazzi, P., et al. (1994) Biochemistry 33, 9078-9084] were synthesized, and their interaction with calmodulin was monitored by fluorescent techniques. The central portions of the skeletal, cardiac, and brain RYR protomers display one high (CaM1; Kd ranging between 2.7 and 10.2 nM) and one low affinity (CaM2; Kd ranging between 116 and 142 nM) calmodulin binding site. Depending on the RYR model having 4 or 12 transmembrane segments, a third calmodulin binding site (CaM3) was identified a few residues upstream from the putative transmembrane segment M1 or M5. Its affinity for calmodulin varied between the RYR isoforms: the cardiac RYR CaM3 displays a high affinity (9.09 +/- 1.0 nM, n = 5), while the skeletal and brain RYR CaM3 have low affinity, the lowest affinity being displayed by the brain isoform (234 +/- 39 nM, n = 3). The RYRs calmodulin binding site CaM1 encompasses the sequence Arg-His-Arg-Val(Ile)-Ser-Leu, which is phosphorylated in vitro by the catalytic subunit of the cAMP-dependent protein kinase. Phosphorylation of RYR PM1 peptides occurs on the Ser, corresponding to amino acid number 2919, 3020, and 3055 of the brain, cardiac, and skeletal RYR protomers, respectively. We found that phosphorylation of the RYR PM1 peptides was inhibited by calmodulin binding and that the formation of the PM1 peptide-calmodulin complex was inhibited by peptide phosphorylation. These data indicate that the effect of calmodulin binding to RYR CaM1 may be regulated by the phosphorylation state of the Ser residue localized within the sequence Arg-His-Arg-Val(Ile)-Ser-Leu.

Functional consequences of mutation Asn 326→Leu in the 4th transmembrane segment of the a-subunit of the rat kidney Na +,K +-ATPase

Site-specific mutagenesis was used to replace Asn 326 in transmembrane segment M4 of the ouabain-insensitive α 1-isoform of rat kidney Na +,K +-ATPase. Mutant Asn 326 → Leu was functional as demonstrated by the ability of COS cells expressing the mutant enzyme to grow in the presence of ouabain. In three independent assays encompassing Na + titrations of Na +,K +-ATPase activity, Na +-ATPase activity, and phosphorylation from ATP, the Asn 326 → Leu mutant displayed a reduced apparent affinity for Na +. By contrast, this mutant exhibited a slightly increased apparent affinity for K + relative to the wild-type enzyme. In the presence of Na +. without K +, the Asn 326 → Len mutant hydrolyzed ATP at a high rate corresponding to 32% of the maximal Na +,K +-ATPase activity, and the rate of dephosphorylation of the phosphoenzyme intermediate was enhanced in the mutant relative to that of the wild-type enzyme. Oligomycin, known to stabilize the Na +-occluded phosphoenzyme intermediate, reduced the dephosphorylation rate of the mutant and increased the steady-state phosphoenzyme level formed by the mutant at least 3-fold, whereas an increase in the steady-state phosphoenzyme level of only 10–15% was determined for the wild-type enzyme. The molecular turnover number for the Na +,K +-ATPase reaction, calculated when the steady-state phosphoenzyme level obtained in the presence of oligomycin was taken as a measure of the concentration of active sites, was slightly reduced relative to that of the wild-type enzyme. The data are discussed in terms of a role for Asn326 in binding of cytoplasmic Na + and in mediation of inhibition of dephosphorylation.

Alpha-Bungarotoxin-sensitive nicotinic receptors on bovine chromaffin cells: molecular cloning, functional expression and alternative splicing of the alpha 7 subunit.

Chromaffin cells from the bovine adrenal medulla express alpha-bungarotoxin-sensitive acetylcholine receptors whose subunit composition is unknown. Northern blot analysis showed that the alpha 7 subunit, a main component of these alpha-bungarotoxin-sensitive acetylcholine receptors in avian and rat brain, is expressed in chromaffin cells. The cDNA of this bovine alpha 7 subunit was cloned by polymerase chain reaction amplification of adrenal medulla RNA for detailed characterization of structure and function. The protein-coding region revealed 92% amino acid sequence identity to rat alpha 7 and 89% to chicken alpha 7 subunits. The alpha-bungarotoxin affinity of alpha 7 homomers expressed in Xenopus oocytes was similar to that observed previously with native chromaffin alpha-bungarotoxin-sensitive acetylcholine receptors. Cross-linking and sucrose gradient experiments suggested that, like the muscular and neuronal acetylcholine receptors; the alpha 7 receptor has a pentameric structure. Upon activation with nicotinic agonists the alpha 7 receptor exhibited rapidly desensitizing cation currents that were blocked by nicotinic antagonists and showed inward rectification. The amplification of adrenal medulla RNA by reverse transcription-polymerase chain reaction methods revealed an alternatively spliced isoform of the bovine alpha 7 subunit, where the exon that codes for the M2 transmembrane segment was skipped during mRNA processing. Oocyte expression of this isoform does not yield functional channels. However, this alternative mRNA exhibits dose-dependent inhibition of alpha 7 homomer expression when coinjected with the undeleted isoform.

Structure-function relationships of cation translocation by Ca 2+- and Na +,K +-ATPases studied by site-directed mutagenesis

Site-directed mutagenesis studies of the sarcoplasmic reticulum Ca 2+-ATPase have pinpointed five amino acid residues that are essential to Ca 2+ occlusion, and these residues have been assigned to different parts of a Ca 2+ binding pocket with channel-like structure. Three of the homologous Na +,K +-ATPase residues have been shown to be important for binding of cytoplasmic Na + at transport sites. In addition, three of the above mentioned Ca 2+-ATPase residues appear to participate in the countertransport of H +, and two of the Na +,K +-ATPase residues to participate in the countertransport of K +. Residues involved in energy transducing conformational changes have also been identified by mutagenesis. In the Ca 2+-ATPase, ATP hydrolysis is uncoupled from Ca 2+ transport following mutation of a tyrosine residue located at the top of transmembrane segment M5. This tyrosine, present also in the Na +,K +-ATPase, may play a critical role in closing the gate to a transmembrane channel.

Pharmacological aspects of acid secretion

The secretion of gastric acid is regulated both centrally and peripherally. The finding that H2-receptor antagonists are able to reduce or abolish acid secretion due to vagal, gastrinergic, and histaminergic stimulation shows that histamine plays a pivotal role in stimulation of the parietal cell. In the rat, the fundic histamine is released from the ECL cell, in response to gastrin, acetylcholine, or epinephrine, and histamine release is inhibited by somatostatin or by the H3-receptor ligand, R-alpha-methyl histamine. The parietal cell has a muscarinic, M3, receptor responsible for [Ca]i regulation. Blockade of muscarinic receptors by atropine can be as effective as H2-receptor blockade in controlling acid secretion. However, general effects on muscarinic receptors elsewhere produce significant side effects. The different receptor pathways converge to stimulate the gastric H+,K(+)-ATPase, the pump responsible for acid secretion by the stomach. This enzyme is an alpha,beta heterodimer, present in cytoplasmic membrane vesicles of the resting cell and in the canaliculus of the stimulated cell. It has been shown that acid secretion by the pump depends on provision of K+Cl- efflux pathway becoming associated with the pump. As secretion occurs only in the canaliculus, this K+Cl- pathway is activated only when the pump inserts into the canalicular membrane. Transport by the enzyme involves reciprocal conformational changes in the cytoplasmic and extracytoplasmic domain. These result in changes in sidedness and affinity for H3O+ and K+, enabling active H+ for K+ exchange. The acid pump inhibitors of the substituted benzimidazole class, such as omeprazole, are concentrated in the canaliculus of the secreting parietal cell and are activated there to form sulfenamides. The omeprazole sulfenamide, for example, reacts covalently with two cysteines in the extracytoplasmic loops between the fifth and sixth transmembrane and the seventh and eighth transmembrane segments of the alpha subunit of the H+,K(+)-ATPase, forming disulfide derivatives. This inhibits ATP hydrolysis and H+ transport, resulting in effective, long-lasting regulation of acid secretion. Therefore, this class of acid pump inhibitor is significantly more effective and faster acting than the H2 receptor antagonists. K+ competitive antagonists bind to the M1 and M2 transmembrane segments of the alpha subunit of the acid pump and also abolish ATPase activity. These drugs should also be able to reduce acid secretion more effectively than receptor antagonists and provide shorter acting but complete inhibition of acid secretion.