Transmembrane Segment M2 Research Articles

Cosegregation of Permeability and Single-Channel Conductance in Chimeric Connexins

The physiological function of gap junction channels goes well beyond their initially discovered role in electrical synchronization of excitable cells. In most tissues, gap junction cells facilitate the exchange of second messengers and metabolites between cells. To test which parts of the channels formed by connexins determine the exclusion limit for the transit of molecules in the size range of second messengers and metabolites a domain exchange approach was used in combination with an accessibility assay for nonelectrolytes and flux measurements. The experimental results suggest that two open hemichannel forming connexins, Cx46 and Cx32E 143, differ in accessibility and permeability. Sucrose is at the exclusion limit for Cx46 channels whereas sorbitol is at the exclusion limit for Cx32E 143 channels. In chimeras between these connexins, where the first transmembrane segment M1 is exchanged, the exclusion limits correlate with those of the M1 donor. The same segregation was found in a separate study for the unitary conductance of the channels. Thus, conductance and permeability/accessibility of the channels cosegregate with M1.

Spontaneous Thermal Motion of the GABAA Receptor M2 Channel-lining Segments

The gamma-aminobutyric acid type A (GABA(A)) receptor channel opening involves translational and rotational motions of the five channel-lining, M2 transmembrane segments. The M2 segment's extracellular half is loosely packed and undergoes significant thermal motion. To characterize the extent of the M2 segment's motion, we used disulfide trapping experiments between pairs of engineered cysteines. In alpha1beta1 gamma2S receptors the single gamma subunit is flanked by an alpha and beta subunit. The gamma2 M2-14' position is located in the alpha-gamma subunit interface. Gamma2 13' faces the channel lumen. We expressed either the gamma2 14' or the gamma2 13' cysteine substitution mutants with alpha1 cysteine substitution mutants between 12' and 16' and wild-type beta1. Disulfide bonds formed spontaneously between gamma2 14'C and both alpha1 15'C and alpha1 16'C and also between gamma2 13'C and alpha1 13'C. Oxidation by copper phenanthroline induced disulfide bond formation between gamma2 14'C and alpha1 13'C. Disulfide bond formation rates with gamma2 14'C were similar in the presence and absence of GABA, although the rate with alpha1 13'C was slower than with the other two positions. In a homology model based on the acetylcholine receptor structure, alphaM2 would need to rotate in opposite directions by approximately 80 degrees to bring alpha1 13' and alpha1 15' into close proximity with gamma2 14'. Alternatively, translational motion of alphaM2 would reduce the extent of rotational motion necessary to bring these two alpha subunit residues into close proximity with the gamma2 14' position. These experiments demonstrate that in the closed state the M2 segments undergo continuous spontaneous motion in the region near the extracellular end of the channel gate. Opening the gate may involve similar but concerted motions of the M2 segments.

Theoretical Studies of the M2 Transmembrane Segment of the Glycine Receptor: Models of the Open Pore Structure and Current-Voltage Characteristics

The pentameric glycine receptor (GlyR), a member of the nicotinicoid superfamily of ligand-gated ion channels, is an inhibitory Cl − channel that is gated by glycine. Using recently published NMR data of the second transmembrane segment (M2) of the human α1 GlyR, structural models of pentameric assemblies embedded in a lipid bilayer were constructed using a combination of experimentally determined constraints coupled with all-atom energy minimization. Based on this structure of the pentameric M2 “pore”, Brownian dynamics simulations of ion permeation through this putative conducting open state of the channel were carried out. Simulated I-V curves were in good agreement with published experimental current-voltage curves and the anion/cation permeability ratio, suggesting that our open-state model may be representative of the conducting channel of the full-length receptor. These studies also predicted regions of chloride occupancy and suggested residues critical to anion permeation. Calculations of the conductance of the cation-selective mutant A251E channel are also consistent with experimental data. In addition, both rotation and untilting of the pore helices of our model were found to be broadly consistent with closing of the channel, albeit at distinct regions that may reflect alternate gates of the receptor.

Reconstruction of the Complete Ouabain-binding Pocket of Na,K-ATPase in Gastric H,K-ATPase by Substitution of Only Seven Amino Acids

Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na,K-ATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we started from the fact that a closely related enzyme, the gastric H,K-ATPase, does not bind glycosides like ouabain. Previously, we showed that a chimera of these two enzymes, in which only the M3-M4 and M5-M6 hairpins were of Na,K-ATPase, bound ouabain with high affinity (Koenderink, J. B., Hermsen, H. P. H., Swarts, H. G. P., Willems, P. H. G. M., and De Pont, J. J. H. H. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11209-11214). We also demonstrated that only three amino acids (Phe(783), Thr(797), and Asp(804)) present in the M5-M6 hairpin of Na,K-ATPase were sufficient to confer high affinity ouabain binding to a chimera which contained in addition the M3-M4 hairpin of Na,K-ATPase (Qiu, L. Y., Koenderink, J. B., Swarts, H. G., Willems, P. H., and De Pont, J. J. H. H. M. (2003) J. Biol. Chem. 278, 47240-47244). To further pinpoint the ouabain-binding site here we used a chimera-based loss-of-function strategy and identified four amino acids (Glu(312), Val(314), Ile(315), Gly(319)), all present in M4, as being important for ouabain binding. In a final gain-of-function study we showed that a gastric H,K-ATPase that contained Glu(312), Val(314), Ile(315), Gly(319), Phe(783), Thr(797), and Asp(804) of Na,K-ATPase bound ouabain with the same affinity as the native enzyme. Based on the E(2)P crystal structure of Ca(2+)-ATPase we constructed a homology model for the ouabain-binding site of Na,K-ATPase involving all seven amino acids as well as several earlier postulated amino acids.

Mutation of Gly-94 in transmembrane segment M1 of Na + ,K + -ATPase interferes with Na + and K + binding in E 2 P conformation

The importance of Gly-93 and Gly-94 in transmembrane segment M1 of the Na+,K+-ATPase for interaction with Na+ and K+ was demonstrated by functional analysis of mutants Gly-93-Ala and Gly-94-Ala. In the crystal structures of the Ca2+-ATPase, the corresponding residues, Asp-59 and Leu-60, are located exactly where M1 bends. Rapid kinetic measurements of K+-induced dephosphorylation allowed determination of the affinity of the E2P phosphoenzyme intermediate for K+. In Gly-94-Ala, the K+ affinity was reduced 9-fold, i.e., to the same extent as seen for mutation of the cation-binding residue Glu-329. Furthermore, Gly-94-Ala showed strongly reduced sensitivity of the E1P-E2P equilibrium to Na+, with accumulation of E2P even at 600 mM Na+, indicating that interaction of E2P with extracellular Na+ is impaired. On the contrary, in Gly-93-Ala, the affinity for K+ was slightly increased, and the E1P-E2P equilibrium was displaced in favor of E1P. In both mutants, the affinity of the cytoplasmically facing sites of E1 for Na+ was reduced, but this effect was relatively small compared with the effects seen for E2P in Gly-94-Ala. Comparison with Ca2+-ATPase mutagenesis data suggests that the role of M1 in binding of the transported ions is universal among P-type ATPases, despite the low sequence homology in this region. Structural modeling of Na+,K+-ATPase mutant Gly-94-Ala on the basis of the Ca2+-ATPase crystal structures indicates that the alanine side chain comes close to Ile-287 of M3, particularly in E2P, thus resulting in a steric clash that may explain the present observations.

A Systematic Mutagenesis Study of Ile-282 in Transmembrane Segment M4 of the Plasma Membrane H+-ATPase

Homology models of plasma membrane H(+)-ATPase (Bukrinsky, J. T., Buch-Pedersen, M. J., Larsen, S., and Palmgren, M. G. (2001) FEBS Lett. 494, 6-10) has pointed to residues in transmembrane segment M4 as being important for proton translocation by P-type proton pumps. To test this model, alanine-scanning mutagenesis was carried out through 12 residues in the M4 of the plant plasma membrane H(+)-ATPase AHA2. An I282A mutation showed apparent reduced H(+) affinity, and this residue was subsequently substituted with all other naturally occurring amino acids by saturation mutagenesis. The ability of mutant enzymes to substitute for the yeast proton pump PMA1 was found to correlate with the size of the side chain rather than its chemical nature. Thus, smaller side chains (Gly, Ala, and Ser) at this position resulted in lower H(+) affinity and lowered levels of H(+) transport in vivo, whereas substitution with side chains of similar and larger size resulted in only minor effects. Substitutions of Ile-282 had only minor effects on ATP affinity and sensitivity toward vanadate, ruling out an indirect effect through changes in the enzyme conformational equilibrium. These results are consistent with a model in which the backbone carbonyl oxygen of Ile-282 contributes directly to proton translocation.

Thr-774 (Transmembrane Segment M5), Val-920 (M8), and Glu-954 (M9) Are Involved in Na+ Transport, and Gln-923 (M8) Is Essential for Na,K-ATPase Activity

The highly conserved amino acids of rat Na,K-ATPase, Thr-774 in the transmembrane helices M5, Val-920 and Gln-923 in M8, and Glu-953 and Glu-954 in M9, the side chains of which appear to be in close proximity, were mutated, and the resulting proteins, T774A, E953A/K, and E954A/K, V920E and Q923N/E/D/L, were expressed in HeLa cells. Ouabain-resistant cell lines were obtained from T774A, V920E, E953A, and E954A, whereas Q923N/E/D/L, E953K, and E954K could only be transiently expressed as fusion proteins with an enhanced green fluorescent protein. The apparent K0.5 values for Na+, as estimated by the Na+-dependent phosphoenzyme formation (K0.5(Na,EP)) or Na,K-ATPase activity (K(0.5)(Na,ATPase)), were increased by around 2 approximately 8-fold in the case of T774A, V920E, and E954A. The apparent K0.5 values for K+, as estimated by the Na,K-ATPase (K0.5(K,ATPase)) or p-nitrophenylphosphatase activity (K0.5(K,pNPPase)), were affected only slightly by the 3 mutations, except that V920E showed a 1.7-fold increase in the K0.5(K,ATPase). The apparent K0.5 values for ATP (K0.5(EP)), as estimated by phosphorylation (a high affinity ATP effect), were increased by 1.6 approximately 2.6-fold in the case of T774A, V920E, and E954A. Those estimated by Na,K-ATPase activity (K0.5(ATPase)) and ATP-induced inhibition (K(i,0.5)(pNPPase)) of K-pNPPase activity (low affinity ATP effects) were, respectively, increased by 1.8-fold and unchanged in the case of T774A but decreased by 2- and 4.8-fold in the case of V920E and were slightly changed and increased by 1.7-fold in the case of E954A. The E953A showed little significant change in the apparent affinities. These results suggest that Gln-923 in M8 is crucial for the active transport of Na+ and/or K+ across membranes and that the side chain oxygen atom of Thr-774 in M5, the methyl group(s) of Val-920 in M8, and the carboxyl oxygen(s) of Glu-954 in M9 mainly play some role in the transport of Na+ and also in the high and low affinity ATP effects rather than the transport of K+.

Interaction between the Catalytic Site and the A-M3 Linker Stabilizes E2/E2P Conformational States of Na+,K+-ATPase

The consequences of mutations Ile(265) --> Ala, Thr(267) --> Ala, Gly(271) --> Ala, and Gly(274) --> Ala for the partial reaction steps of the Na(+),K(+)-ATPase transport cycle were analyzed. The mutated residues are part of the long loop ("A-M3 linker") connecting the cytoplasmic A-domain with transmembrane segment M3. It was found that mutation Ile(265) --> Ala displaces the E(1)-E(2) and E(1)P-E(2)P equilibria in favor of E(1)/E(1)P, whereas mutations Thr(267) --> Ala, Gly(271) --> Ala, and Gly(274) --> Ala displace these conformational equilibria in favor of E(2)/E(2)P. The mutations affect both the rearrangement of the cytoplasmic domains (seen by changes in phosphoenzyme properties and apparent ATP/vanadate affinities) and the membrane sector (indicated by change in K(+)/Rb(+) deocclusion rate). Destabilization of E(2)/E(2)P in Ile(265) --> Ala, as well as a direct effect on the intrinsic affinity of the E(2) form for vanadate, may be explained on the basis of the E(2) crystal structures of the Ca(2+)-ATPase, showing interaction of the equivalent isoleucine with conserved residues near the catalytic region of the P-domain. The rate of phosphorylation from ATP was unaffected in Ile(265) --> Ala, indicating a lack of interference with the catalytic function in E(1)/E(1)P. The effects of mutations Thr(267) --> Ala, Gly(271) --> Ala, and Gly(274) --> Ala provide the first evidence in the literature of a relative stabilization of E(2)/E(2)P resulting from perturbation of the A-M3 linker region. These mutations may lead to increased strain of the A-M3 linker in E(1)/E(1)P, increased stability of the A3 helix of the A-M3 linker in E(2)/E(2)P, and/or a change of the orientation of the A3 helix, facilitating its interaction with the P-domain.

Structural dynamics of the M4 transmembrane segment during acetylcholine receptor gating.

The transition state structures that link the stable end states of allosteric proteins are largely unresolved. We used single-molecule kinetic analysis to probe the dynamics of the M4 transmembrane segments during the closed<==>open isomerization of the neuromuscular acetylcholine receptor ion channel (AChR). We measured the slopes (phi) of the free energy relationships for 87 mutants, which reveal the open- versus closed-like characters of the mutated residues at the transition state and hence the sequence and organization of gating molecular motions. phi was constant throughout the length of the alpha subunit M4 segment with an average value of 0.54, suggesting that this domain moves as a unit, approximately midway through the reaction. Analysis of a hybrid construct indicates that the two alpha subunits move synchronously. Between subunits, the sequence of M4 motions is alpha-epsilon-beta. The AChR ion channel emerges as a dynamic nanomachine with many moving parts.

Importance of Transmembrane Segment M1 of the Sarcoplasmic Reticulum Ca2+-ATPase in Ca2+ Occlusion and Phosphoenzyme Processing

The functional consequences of a series of point mutations in transmembrane segment M1 of sarcoplasmic reticulum Ca2+-ATPase were analyzed in steady-state and transient kinetic experiments examining the partial reaction steps involved in Ca2+ interaction and phosphoenzyme turnover. Arginine or leucine substitution of Glu51, Glu55, or Glu58, located in the N-terminal third of M1, did not affect these functions. Arginine or leucine substitution of Asp59, located right at the bend of M1 seen in the crystal structure of the thapsigargin-bound form, caused a 10-fold increase of the rate of Ca2+ dissociation toward the cytoplasmic side. Mutation of Leu60 to alanine or proline and of Val62 to alanine also enhanced Ca2+ dissociation, whereas an 11-fold reduction of the rate of Ca2+ dissociation was observed upon alanine substitution of Leu65, thus providing evidence for a relation of the middle part of M1 to a gating mechanism controlling the dissociation of occluded Ca2+ from its membranous binding sites. Moreover, phosphoenzyme processing was affected by some of the latter mutations, in particular leucine substitution of Asp59, and alanine substitution of Leu65 accelerated the transition to ADP-insensitive phosphoenzyme and blocked its dephosphorylation, thus demonstrating that this part of M1, besides being important in Ca2+ interaction, furthermore, is a critical element in the long range signaling between the transmembrane domain and the cytoplasmic catalytic site.

Identification of the prokaryotic ligand-gated ion channels and their implications for the mechanisms and origins of animal Cys-loop ion channels

Acetylcholine receptor type ligand-gated ion channels are well known in animals. Homologs are identified in prokaryotes that may act as chemotactic receptors.

Mechanism of proton transport by plant plasma membrane proton ATPases.

The mechanism of proton translocation by P-type proton ATPases is poorly defined. Asp684 in transmembrane segment M6 of the Arabidopsis thaliana AHA2 plasma membrane P-type proton pump is suggested to act as an essential proton acceptor during proton translocation. Arg655 in transmembrane segment M5 seems to be involved in this proton translocation too, but in contrast to Asp684, is not essential for transport. Asp684 may participate in defining the E(1) proton-binding site, which could possibly exist as a hydronium ion coordination center. A model of proton translocation of AHA2 involving the side chains of amino acids Asp684 and Arg655 is discussed.

Conserved Asp684 in Transmembrane Segment M6 of the Plant Plasma Membrane P-type Proton Pump AHA2 Is a Molecular Determinant of Proton Translocation

The mechanism of proton pumping by P-type H(+)-ATPases is still unclear. In the plant P-type plasma membrane H(+)-ATPase AHA2, two charged residues, Arg(655) and Asp(684), are conserved in transmembrane segments M5 and M6, respectively, a region that has been shown be contribute to ion coordination in related P-type ATPases. Substitution of Arg(655) with either alanine or aspartate resulted in mutant enzymes exhibiting a significant shift in the P-type ATPase E(1)P-E(2)P conformational equilibrium. The mutant proteins accumulated in the E(1)P conformation, but were capable of conducting proton transport. This points to an important role of Arg(655) in the E(1)P-E(2)P conformational transition. The presence of a carboxylate moiety at position Asp(684) proved essential for coupling between initial proton binding and proton pumping. The finding that the carboxylate side chain of Asp(684) contributes to the proton-binding site and appears to function as an absolutely essential proton acceptor along the proton transport pathway is discussed in the context of a possible proton pumping mechanism of P-type H(+)-ATPases.

Mutagenesis of residues involved in control of the Ca2+ entry pathway and conformational changes associated with Ca2+ binding in the SR Ca2+-ATPase.

Rapid kinetic measurements were used to study the rate of Ca(2+) dissociation from the high-affinity Ca(2+) sites of the dephosphoenzyme (i.e., from the E(1)Ca(2) form toward the cytoplasmic side) as well as the rate of Ca(2+) binding with associated conformational changes (E(2) --> E(1)Ca(2) transition) in the wild type and mutants of the sarcoplasmic reticulum Ca(2+)-ATPase expressed in mammalian cells. Cluster mutations as well as single mutations in transmembrane segment M3 resulted in conspicuous effects on the rate of Ca(2+) migration. Furthermore, mutation of Asp(59) in transmembrane segment M1 to arginine exerted a profound effect on Ca(2+) interaction. The data demonstrate an important role for M3 residues in control of the Ca(2+) entry pathway and provide functional evidence in support of a close relationship between this pathway and the water-accessible channel leading between transmembrane segments M1 and M3 in the thapsigargin stabilized E(2) structure. In addition, rapid kinetic measurements demonstrated that the hydrogen bond network involving Asp(813) of loop L6-7 and Lys(758) of M5 is important for the E(2) --> E(1)Ca(2) transition.

Importance of transmembrane segment M3 of Na+,K+-ATPase for control of conformational changes and the cytoplasmic entry pathway for Na.

A series of mutations were introduced into the sequence Glu(282)-Ile-Glu-His-Phe-Ile-His(288) of the NH(2)-terminal part of M3 of the rat kidney Na(+),K(+)-ATPase, and the resulting mutant pumps were analyzed functionally. Several of the mutations affected the conformational transitions between E(1) and E(2) forms of dephospho- and phosphoenzyme. Mutations to Glu(282) and Phe(286) affected the E(1)-E(2) and E(1)P-E(2)P equilibria in parallel, indicating a role for these two residues in both conformational changes. Mutation to His(285) preferentially affected the E(1)P-E(2)P equilibrium, and mutation to Ile(283) affected only the E(1)-E(2) equilibrium of the dephosphoenzyme, demonstrating that the conformational changes of M3 in the phospho- and dephospho-forms are not identical. Several of the mutants showed a reduced apparent affinity for Na(+), pointing to an important role of the region in optimizing the Na(+) binding properties of the enzyme. It is possible that this part of M3 is closely associated with an entry pathway through which the Na(+) ions pass from the cytoplasmic surface to reach the cation-binding pocket. Some of the mutants also displayed an increased Na(+)-ATPase activity, and a good correlation was observed between the turnover rate for Na(+)-ATPase activity and the rate of dephosphorylation in the absence of K(+), indicating an increased ability of Na(+) to activate dephosphorylation of E(2)P by binding in place of K(+) at the extracellularly facing sites. Thus, M3 also seems to be a part of the signaling pathway between the external cation sites and the catalytic site.

Transmembrane segment M2 of glycine receptor as a model system for the pore-forming structure of ion channels.

The glycine receptor belongs to the ligand-gated ion channel superfamily. It is a chloride conducting channel composed of four transmembrane domains. It was previously shown that the second transmembrane domain (M2) of the glycine receptor forms an ion conduction pathway throughout lipid bilayers. The amino-acid sequence of the transmembrane segment M2 of the glycine receptor has a high homology to all receptors of the ligand-gated ion channel superfamily. In our report, we have used a synthetic M2 peptide. It was incorporated into a planar membrane of known lipid composition and currents induced by M2 were measured by the Black Lipid Membrane technique. When the planar lipid bilayer was composed of 75% phosphatidylethanolamine and 25% phosphatidylserine, the reversal potential measured in a 150/600 mM KCl (cis/trans) gradient was -19 mV suggesting that the examined &gt;pore was preferential to anions, P(K)/P(Cl) = 0.25. In contrast, when 75% phosphatidylserine and 25% phosphatidylethanolamine was used, the reversal potential was +20 mV and the &gt;pore was preferential to cations, P(K)/P(Cl) = 4.36. Single-channel currents were recorded with two predominant amplitudes corresponding to the main-conductance and sub-conductance states. Both conductance states (about 12 pS and 30 pS) were measured in a symmetric solution of 50 mM KCl. The observed single-channel properties suggest that the selectivity and conductance of the pore formed by the M2 peptide of the glycine receptor depend on the lipid composition of the planar bilayer.

Transmembrane segment M2 of glycine receptor as a model system for the pore-forming structure of ion channels.

The glycine receptor belongs to the ligand-gated ion channel superfamily. It is a chloride conducting channel composed of four transmembrane domains. It was previously shown that the second transmembrane domain (M2) of the glycine receptor forms an ion conduction pathway throughout lipid bilayers. The amino-acid sequence of the transmembrane segment M2 of the glycine receptor has a high homology to all receptors of the ligand-gated ion channel superfamily. In our report, we have used a synthetic M2 peptide. It was incorporated into a planar membrane of known lipid composition and currents induced by M2 were measured by the Black Lipid Membrane technique. When the planar lipid bilayer was composed of 75% phosphatidylethanolamine and 25% phosphatidylserine, the reversal potential measured in a 150/600 mM KCl (cis/trans) gradient was -19 mV suggesting that the examined >pore was preferential to anions, P(K)/P(Cl) = 0.25. In contrast, when 75% phosphatidylserine and 25% phosphatidylethanolamine was used, the reversal potential was +20 mV and the >pore was preferential to cations, P(K)/P(Cl) = 4.36. Single-channel currents were recorded with two predominant amplitudes corresponding to the main-conductance and sub-conductance states. Both conductance states (about 12 pS and 30 pS) were measured in a symmetric solution of 50 mM KCl. The observed single-channel properties suggest that the selectivity and conductance of the pore formed by the M2 peptide of the glycine receptor depend on the lipid composition of the planar bilayer.

Importance of Glu282 in Transmembrane Segment M3 of the Na+,K+-ATPase for Control of Cation Interaction and Conformational Changes

Glu(282) located in the NH(2)-terminal part of transmembrane helix M3 of the Na(+),K(+)-ATPase was replaced by alanine, glycine, leucine, lysine, aspartate, or glutamine, and the effects of the mutations on the overall and partial reactions of the enzyme were analyzed. The mutations affected at least 3 important functions of the Na(+),K(+)-ATPase: (i) the conformational transitions between E(1) and E(2) forms of dephospho- and phosphoenzyme, (ii) Na(+) binding at the cytoplasmically facing sites of E(1), and (iii) long-range interaction controlling dephosphorylation. In mutants Glu(282) --> Lys and Glu(282) --> Asp, the E(1) form was favored during ATP hydrolysis, whereas the E(2) form was favored in Glu(282) --> Ala and Glu(282) --> Gly. Regardless of the change of conformational equilibrium, all the mutants displayed a reduced apparent affinity for Na(+), at least 3-fold for Glu(282) --> Lys and Glu(282) --> Asp, suggesting a direct effect on the Na(+) binding properties of E(1). Glu(282) --> Ala and Glu(282) --> Gly exhibited an extraordinary high rate of ATP hydrolysis in the mere presence of Na(+) without K(+) ("Na(+)-ATPase activity"), because of an increased rate of dephosphorylation of E(2)P. These results are in accordance with the hypothesis that Glu(282) is involved in the communication between the cation binding pocket and the catalytic site and in control of the cytoplasmic entry pathway for Na(+).

Selective Fe2+-catalyzed Oxidative Cleavage of Gastric H+,K+-ATPase: IMPLICATIONS FOR THE ENERGY TRANSDUCTION MECHANISM OF P-TYPE CATION PUMPS

In the presence of ascorbate/H(2)O(2), Fe(2+) ions or the ATP-Fe(2+) complex catalyze selective cleavage of the alpha subunit of gastric H(+),K(+)-ATPase. The electrophoretic mobilities of the fragments and dependence of the cleavage patterns on E(1) and E(2) conformational states are essentially identical to those described previously for renal Na(+),K(+)-ATPase. The cleavage pattern of H(+),K(+)-ATPase by Fe(2+) ions is consistent with the existence of two Fe(2+) sites: site 1 within highly conserved sequences in the P and A domains, and site 2 at the cytoplasmic entrance to trans-membrane segments M3 and M1. The change in the pattern of cleavage catalyzed by Fe(2+) or the ATP-Fe(2+) complex induced by different ligands provides evidence for large conformational movements of the N, P, and A cytoplasmic domains of the enzyme. The results are consistent with the Ca(2+)-ATPase crystal structure (Protein Data Bank identification code; Toyoshima, C., Nakasako, M., Nomura, H., and Ogawa, H. (2000) Nature 405, 647-655), an E(1)Ca(2+) conformation, and a theoretical model of Ca(2+)-ATPase in an E(2) conformation (Protein Data Bank identification code ). Thus, it can be presumed that the movements of N, P, and A cytoplasmic domains, associated with the E(1) <--> E(2) transitions, are similar in all P-type ATPases. Fe(2+)-catalyzed cleavage patterns also reveal sequences involved in phosphate, Mg(2+), and ATP binding, which have not yet been shown in crystal structures, as well as changes which occur in E(1) <--> E(2) transitions, and subconformations induced by H(+),K(+)-ATPase-specific ligands such as SCH28080.

Proximity of transmembrane segments M3 and M1 of the alpha subunit of Na+,K+-ATPase revealed by specific oxidative cleavage mediated by a complex of Cu2+ ions and 4,7-diphenyl-1,10-phenanthroline.

This paper describes a novel approach to specific oxidative cleavage of Na(+),K(+)-ATPase, mediated by Cu(2+) ions and a hydrophobic phenanthroline, 4,7-diphenyl-1,10-phenanthroline (DPP), in the presence of ascorbate and H(2)O(2). The cleavage produces two major fragments of the alpha subunit, with apparent molecular masses of 96.5 and 76 kDa, and N-termini near the cytoplasmic entrance of transmembrane segments M1 and M3, respectively, The kinetics indicate that both cleavages are mediated by a single Cu(2+)-DPP complex. We infer that M3 and M1 are in proximity near the cytoplasmic surface. The yields of 96.5 and 76 kDa fragments are not significantly affected by ligands that stabilize different E(1) and E(2) conformations. In E(2)(K) and E(2)P conformations, a minor 5.5 kDa fragment with its N-terminus in M10 is also observed. The 96.5 and 76 kDa fragments are indistinguishable from two fragments near M3 and M1 produced by Fe(2+)-catalyzed cleavage described previously [Goldshleger, R., and Karlish, S. J. D. (1999) J. Biol. Chem. 274, 16213-16221], whereas other Fe(2+)-catalyzed cleavage fragments in the cytoplasmic P and A domains are not observed with the Cu(2+)-DPP complex. These findings provide experimental support for the concept of two separate Fe(2+) sites. A homology model, with Na(+),K(+)-ATPase residues within transmembrane segments and connecting loops substituted into the crystal structure of Ca(2+)-ATPase, shows the proximity between the sequences HFIH in M3 and EVWK in M1, near the cytoplasmic surface. Thus, the model strongly supports the conclusions based on cleavages mediated by the Cu(2+)-DPP complex (or Fe(2+) at site 2). As a corollary, the cleavages provide evidence for similar packing of M1 and M3 of Na(+),K(+)-ATPase and Ca(2+)-ATPase.