A simple light-driven transmembrane proton pump.

Light-induced lipophilic porphyrin/aqueous acceptor charge separation across a single lipid-water interface can pump protons across the lipid bilayer when the hydrophobic weak acids, carbonylcyanide m-chlorophenylhydrazone and its p-trifluoromethoxyphenyl analogue, are present. These compounds act as proton carriers across lipid bilayers. In their symmetric presence across the bilayer, the positive currents and voltages produced by the photogeneration of porphyrin cations are replaced by larger negative currents and voltages. The maximum negative current and voltage occur at the pH of maximum dark conductance. The reversed larger current and voltage show a positive ionic charge transport in the same direction as the electron transfer. This transport can form an ion concentration gradient. The movement of protons is verified by an unusual D2O isotope effect that increases the negative ionic current by 2- to 3-fold. These effects suggest that an interfacial pK shift of the weak acid caused by the local electric field of photoformed porphyrin cations/acceptor anions functions as the driving force. The estimated pumping efficiency is 10-30%. Time-resolved results show that proton pumping across the bilayer occurs on the millisecond time scale, similar to that of biological pumps. This light-driven proteinless pump offers a simple model for a prebiological energy transducer.

Rapid activation of Na+/H+ exchange by aldosterone in renal epithelial cells requires Ca2+ and stimulation of a plasma membrane proton conductance.

There is increasing evidence for an additional acute, nongenomic action of the mineralocorticoid hormone aldosterone on renal epithelial cells, leading to a two-step model of mineralocorticoid action on electrolyte excretion. We investigated the acute effect of aldosterone on intracellular free Ca2+ and on intracellular pH in an aldosterone-sensitive Madin-Darby canine kidney cell clone. Within seconds of application of aldosterone, but not of the glucocorticoid hydrocortisone, there was a 3-fold sustained increase of intracellular Ca2+ at a half-maximal concentration of 10(-10) mol/liter. Omission of extracellular Ca2+ prevented this hormone response. In the presence of extracellular Ca2+ aldosterone led to intracellular alkalinization. The Na+/H+ exchange inhibitor ethyl-isopropanol-amiloride (EIPA) prevented the aldosterone-induced alkalinization but not the aldosterone-induced increase of intracellular Ca2+. Omission of extracellular Ca2+ also prevented aldosterone-induced alkalinization. Instead, aldosterone led to a Zn(2+)-dependent intracellular acidification in the presence of EIPA, indicative of an increase of plasma membrane proton conductance. Under control conditions, Zn2+ prevented the aldosterone-induced alkalinization completely. We conclude that aldosterone stimulated net-entry of Ca2+ from the extracellular compartment and a plasma membrane H+ conductance as prerequisites for the stimulation of plasma membrane Na+/H+ exchange which in turn modulates K+ channel acitivity. It is probable that the aldosterone-sensitive H+ conductance maintains Na+/H+ exchange activity by providing an acidic environment in the vicinity of the exchanger. Thus, genomic action of aldosterone determines cellular transport equipment, whereas the nongenomic action regulates transporter activity that requires responses within seconds or minutes, which explains the rapid effects on electrolyte excretion.

Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer.

The green fluorescent protein (GFP) of the jellyfish Aequorea Victoria has attracted widespread interest since the discovery that its chromophore is generated by the autocatalytic, posttranslational cyclization and oxidation of a hexapeptide unit. This permits fusion of the DNA sequence of GFP with that of any protein whose expression or transport can then be readily monitored by sensitive fluorescence methods without the need to add exogenous fluorescent dyes. The excited state dynamics of GFP were studied following photo-excitation of each of its two strong absorption bands in the visible using fluorescence upconversion spectroscopy (about 100 fs time resolution). It is shown that excitation of the higher energy feature leads very rapidly to a form of the lower energy species, and that the excited state interconversion rate can be markedly slowed by replacing exchangeable protons with deuterons. This observation and others lead to a model in which the two visible absorption bands correspond to GFP in two ground-state conformations. These conformations can be slowly interconverted in the ground state, but the process is much faster in the excited state. The observed isotope effect suggests that the initial excited state process involves a proton transfer reaction that is followed by additional structural changes. These observations may help to rationalize and motivate mutations that alter the absorption properties and improve the photo stability of GFP.

prlA suppressors in Escherichia coli relieve the proton electrochemical gradient dependency of translocation of wild-type precursors.

The SecY protein of Escherichia coli is an integral membrane component of the protein export apparatus. Suppressor mutations in the secY gene (prlA alleles) have been isolated that restore the secretion of precursor proteins with defective signal sequences. These mutations have never been shown to affect the translocation of wild-type precursor proteins. Here, we report that prlA suppressor mutations relieve the proton-motive force (pmf) dependency of the translocation of wild-type precursors, both in vivo and in vitro. Furthermore, the proton-motive force dependency of the translocation of a precursor with a stably folded domain in the mature region was suppressed by prlA mutations in vitro. These data show that prlA mutations cause a general relaxation of the export apparatus rather than a specific change that results in bypassing of the recognition of the signal sequence. In addition, these results are indicative for a mechanism in which the proton-motive force stimulates translocation by altering the conformation of the translocon.

Promotion of sleep mediated by the A2a-adenosine receptor and possible involvement of this receptor in the sleep induced by prostaglandin D2 in rats.

A 6-hr continuous infusion of 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenos ine (CGS21680), a selective A2a-adenosine agonist, into the subarachnoid space underlying the ventral surface region of the rostral basal forebrain, which has been defined as the prostaglandin (PG) D2-sensitive sleep-promoting zone, at rates of 0.02, 0.2, 2.0, and 12 pmol/min increased slow-wave sleep (SWS) and paradoxical sleep (PS) in a dose-dependent manner up to 183% and 202% of their respective baseline levels. The increments produced by the infusion of CGS21680 at 0.2 and 2.0 pmol/min were totally diminished when the rats had been pretreated with an i.p. injection of (E)-1,3-dipropyl-7-methyl-8-(3,4-dimethoxystyryl)xanthine (KF17837; 30 mg/kg of body weight), a selective A2-adenosine antagonist. In contrast, the infusion of N6-cyclohexyladenosine (CHA), a selective A1-adenosine agonist, at 2 pmol/min significantly suppressed SWS before causing an increase in SWS, and a decrease in PS was also markedly visible. Essentially the same effects of CGS21680 and CHA were observed when these compounds were administered to the parenchymal region of the rostral basal forebrain through chronically implanted microdialysis probes. Thus, we clearly showed that stimulation of A2a-adenosine receptors in the rostral basal forebrain promotes SWS and PS. Furthermore, i.p. injections of KF17837 at 30 and 100 mg/kg of body weight dose-dependently attenuated the magnitude of the SWS increase produced by the infusion of PGD2 into the subarachnoid space of the sleep-promoting zone, thus indicating that the A2a-adenosine receptors are crucial in the sleep-promoting process triggered by PGD2.

Potentiation of proton transfer function by electrostatic interactions in photosynthetic reaction centers from Rhodobacter sphaeroides: First results from site-directed mutation of the H subunit.

The x-ray crystallographic structure of the photosynthetic reaction center (RC) has proven critical in understanding biological electron transfer processes. By contrast, understanding of intraprotein proton transfer is easily lost in the immense richness of the details. In the RC of Rhodobacter (Rb.) sphaeroides, the secondary quinone (QB) is surrounded by amino acid residues of the L subunit and some buried water molecules, with M- and H-subunit residues also close by. The effects of site-directed mutagenesis upon RC turnover and quinone function have implicated several L-subunit residues in proton delivery to QB, although some species differences exist. In wild-type Rb. sphaeroides, Glu L212 and Asp L213 represent an inner shell of residues of particular importance in proton transfer to QB. Asp L213 is crucial for delivery of the first proton, coupled to transfer of the second electron, while Glu L212, possibly together with Asp L213, is necessary for delivery of the second proton, after the second electron transfer. We report here the first study, by site-directed mutagenesis, of the role of the H subunit in QB function. Glu H173, one of a cluster of strongly interacting residues near QB, including Asp L213, was altered to Gln. In isolated mutant RCs, the kinetics of the first electron transfer, leading to formation of the semiquinone, QB-, and the proton-linked second electron transfer, leading to the formation of fully reduced quinol, were both greatly retarded, as observed previously in the Asp L213 --> Asn mutant. However, the first electron transfer equilibrium, QA-QB <==> QAQB-, was decreased, which is opposite to the effect of the Asp L213 --> Asn mutation. These major disruptions of events coupled to proton delivery to QB were largely reversed by the addition of azide (N3-). The results support a major role for electrostatic interactions between charged groups in determining the protonation state of certain entities, thereby controlling the rate of the second electron transfer. It is suggested that the essential electrostatic effect may be to "potentiate" proton transfer activity by raising the pK of functional entities that actually transfer protons in a coupled fashion with the second electron transfer. Candidates include buried water (H3O+) and Ser L223 (serine-OH2+), which is very close to the O5 carbonyl of the quinone.

31P magnetic resonance spectroscopy of the Sherpa heart: a phosphocreatine/adenosine triphosphate signature of metabolic defense against hypobaric hypoxia.

Of all humans thus far studied, Sherpas are considered by many high-altitude biomedical scientists as most exquisitely adapted for life under continuous hypobaric hypoxia. However, little is known about how the heart is protected in hypoxia. Hypoxia defense mechanisms in the Sherpa heart were explored by in vivo, noninvasive 31P magnetic resonance spectroscopy. Six Sherpas were examined under two experimental conditions [normoxic (21% FiO2) and hypoxic (11% FiO2) and in two adaptational states--the acclimated state (on arrival at low-altitude study sites) and the deacclimating state (4 weeks of ongoing exposure to low altitude). Four lowland subjects were used for comparison. We found that the concentration ratios of phosphocreatine (PCr)/adenosine triphosphate (ATP) were maintained at steady-state normoxic values (0.96, SEM = 0.22) that were about half those found in normoxic lowlanders (1.76, SEM = 0.03) monitored the same way at the same time. These differences in heart energetic status between Sherpas and lowlanders compared under normoxic conditions remained highly significant (P < 0.02) even after 4 weeks of deacclimation at low altitudes. In Sherpas under acute hypoxia, the heart rate increased by 20 beats per min from resting values of about 70 beats per min, and the percent saturation of hemoglobin decreased to about 75%. However, these perturbations did not alter the PCr/ATP concentration ratios, which remained at about 50% of the values expected in healthy lowlanders. Because the creatine phosphokinase reaction functions close to equilibrium, these steady-state PCr/ATP ratios presumably coincided with about 3-fold higher free adenosine diphosphate (ADP) concentrations. Higher ADP concentrations (i.e., lower [PCr]/[ATP] ratios) were interpreted to correlate with the Km values for ADP-requiring kinases of glycolysis and to reflect elevated carbohydrate contributions to heart energy needs. This metabolic organization is postulated as advantageous in hypobaria because the ATP yield per O2 molecule is 25-60% higher with glucose than with free fatty acids (the usual fuels utilized in the human heart in postfasting conditions).

Cloning of human adenosine kinase cDNA: sequence similarity to microbial ribokinases and fructokinases.

Adenosine kinase catalyzes the phosphorylation of adenosine to AMP and hence is a potentially important regulator of extracellular adenosine concentrations. Despite extensive characterization of the kinetic properties of the enzyme, its primary structure has never been elucidated. Full-length cDNA clones encoding catalytically active adenosine kinase were obtained from lymphocyte, placental, and liver cDNA libraries. Corresponding mRNA species of 1.3 and 1.8 kb were noted on Northern blots of all tissues examined and were attributable to alternative polyadenylylation sites at the 3' end of the gene. The encoding protein consists of 345 amino acids with a calculated molecular size of 38.7 kDa and does not contain any sequence similarities to other well-characterized mammalian nucleoside kinases, setting it apart from this family of structurally and functionally related proteins. In contrast, two regions were identified with significant sequence identity to microbial ribokinase and fructokinases and a bacterial inosine/guanosine kinase. Thus, adenosine kinase is a structurally distinct mammalian nucleoside kinase that appears to be akin to sugar kinases of microbial origin.

Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter.

The presence of a proton-coupled electrogenic high-affinity peptide transporter in the apical membrane of tubular cells has been demonstrated by microperfusion studies and by use of brush border membrane vesicles. The transporter mediates tubular uptake of filtered di- and tripeptides and aminocephalosporin antibiotics. We have used expression cloning in Xenopus laevis oocytes for identification and characterization of the renal high-affinity peptide transporter. Injection of poly(A)+ RNA isolated from rabbit kidney cortex into oocytes resulted in expression of a pH-dependent transport activity for the aminocephalosporin antibiotic cefadroxil. After size fractionation of poly(A)+ RNA the transport activity was identified in the 3.0- to 5.0-kb fractions, which were used for construction of a cDNA library. The library was screened for expression of cefadroxil transport after injection of complementary RNA synthesized in vitro from different pools of clones. A single clone (rPepT2) was isolated that stimulated cefadroxil uptake into oocytes approximately 70-fold at a pH of 6.0. Kinetic analysis of cefadroxil uptake expressed by the transporter's complementary RNA showed a single saturable high-affinity transport system shared by dipeptides, tripeptides, and selected amino-beta-lactam antibiotics. Electrophysiological studies established that the transport activity is electrogenic and affected by membrane potential. Sequencing of the cDNA predicts a protein of 729 amino acids with 12 membrane-spanning domains. Although there is a significant amino acid sequence identity (47%) to the recently cloned peptide transporters from rabbit and human small intestine, the renal transporter shows distinct structural and functional differences.

Efflux pump of the proton antiporter family confers low-level fluoroquinolone resistance in Mycobacterium smegmatis.

Due to the resurgence of tuberculosis and the emergence of multidrug-resistant strains, fluoroquinolones (FQ) are being used in selected tuberculosis patients, but FQ-resistant strains of Mycobacterium tuberculosis have rapidly begun to appear. The mechanisms involved in FQ resistance need to be elucidated if the effectiveness of this class of antibiotics is to be improved and prolonged. By using the rapid-growing Mycobacterium smegmatis as a model genetic system, a gene was selected that confers low-level FQ resistance when present on a multicopy plasmid. This gene, lfrA, encodes a putative membrane efflux pump of the major facilitator family, which appears to recognize the hydrophilic FQ, ethidium bromide, acridine, and some quaternary ammonium compounds. It is homologous to qacA from Staphylococcus aureus, tcmA, of Streptomyces glaucescens, and actII and mmr, both from Streptomyces coelicoler. Increased expression of lfrA augments the appearance of subsequent mutations to higher-level FQ resistance.

Latent N-methyl-D-aspartate receptors in the recurrent excitatory pathway between hippocampal CA1 pyramidal neurons: Ca(2+)-dependent activation by blocking A1 adenosine receptors.

When performed at increased external [Ca2+]/[Mg2+] ratio (2.5 mM/0.5 mM), temporary block of A1 adenosine receptors in hippocampus [by 8-cyclopentyltheophylline (CPT)] leads to a dramatic and irreversible change in the excitatory postsynaptic current (EPSC) evoked by Schaffer collateral/commissural (SCC) stimulation and recorded by in situ patch clamp in CA1 pyramidal neurons. The duration of the EPSC becomes stimulus dependent, increasing with increase in stimulus strength. The later occurring component of the EPSC is carried through N-methyl-D-aspartate (NMDA) receptor-operated channels but disappears under either the NMDA antagonist 2-amino-5-phosphonovaleric acid (APV) or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). These findings indicate that the late component of the SCC-evoked EPSC is polysynaptic: predominantly non-NMDA receptor-mediated SCC inputs excite CA1 neurons that recurrently excite each other by predominantly NDMA receptor-mediated synapses. These recurrent connections are normally silent but become active after CPT treatment, leading to enhancement of the late component of the EPSC. The activity of these connections is maintained for at least 2 hr after CPT removal. When all functional NMDA receptors are blocked by dizocilpine maleate (MK-801), subsequent application of CPT leads to a partial reappearance of NMDA receptor-mediated EPSCs evoked by SCC stimulation, indicating that latent NMDA receptors are recruited. Altogether, these findings indicate the existence of a powerful system of NMDA receptor-mediated synaptic contacts in SCC input to hippocampal CA1 pyramidal neurons and probably also in reciprocal connections between these neurons, which in the usual preparation are kept latent by activity of A1 receptors.

Adenosine activates ATP-sensitive potassium channels in arterial myocytes via A2 receptors and cAMP-dependent protein kinase.

The mechanism by which the endogenous vasodilator adenosine causes ATP-sensitive potassium (KATP) channels in arterial smooth muscle to open was investigated by the whole-cell patch-clamp technique. Adenosine induced voltage-independent, potassium-selective currents, which were inhibited by glibenclamide, a blocker of KATP currents. Glibenclamide-sensitive currents were also activated by the selective adenosine A2-receptor agonist 2-p-(2-carboxethyl)-phenethylamino-5'-N- ethylcarboxamidoadenosine hydrochloride (CGS-21680), whereas 2-chloro-N6-cyclopentyladenosine (CCPA), a selective adenosine A1-receptor agonist, failed to induce potassium currents. Glibenclamide-sensitive currents induced by adenosine and CGS-21680 were largely reduced by blockers of the cAMP-dependent protein kinase (Rp-cAMP[S], H-89, protein kinase A inhibitor peptide). Therefore, we conclude that adenosine can activate KATP currents in arterial smooth muscle through the following pathway: (i) Adenosine stimulates A2 receptors, which activates adenylyl cyclase; (ii) the resulting increase intracellular cAMP stimulates protein kinase A, which, probably through a phosphorylation step, opens KATP channels.

Proton transport by a bacteriorhodopsin mutant, aspartic acid-85-->asparagine, initiated in the unprotonated Schiff base state.

At alkaline pH the bacteriorhodopsin mutant D85N, with aspartic acid-85 replaced by asparagine, is in a yellow form (lambda max approximately 405 nm) with a deprotonated Schiff base. This state resembles the M intermediate of the wild-type photocycle. We used time-resolved methods to show that this yellow form of D85N, which has an initially unprotonated Schiff base and which lacks the proton acceptor Asp-85, transports protons in the same direction as wild type when excited by 400-nm flashes. Photoexcitation leads in several milliseconds to the formation of blue (630 nm) and purple (580 nm) intermediates with a protonated Schiff base, which decay in tens of seconds to the initial state (400 nm). Experiments with pH indicator dyes show that at pH 7, 8, and 9, proton uptake occurs in about 5-10 ms and precedes the slow release (seconds). Photovoltage measurements reveal that the direction of proton movement is from the cytoplasmic to the extracellular side with major components on the millisecond and second time scales. The slowest electrical component could be observed in the presence of azide, which accelerates the return of the blue intermediate to the initial yellow state. Transport thus occurs in two steps. In the first step (milliseconds), the Schiff base is protonated by proton uptake from the cytoplasmic side, thereby forming the blue state. From the pH dependence of the amplitudes of the electrical and photocycle signals, we conclude that this reaction proceeds in a similar way as in wild type--i.e., via the internal proton donor Asp-96. In the second step (seconds) the Schiff base deprotonates, releasing the proton to the extracellular side.

Structure determination of murine mitochondrial carbonic anhydrase V at 2.45-A resolution: implications for catalytic proton transfer and inhibitor design.

The three-dimensional structure of murine mitochondrial carbonic anhydrase V has been determined and refined at 2.45-A resolution (crystallographic R factor = 0.187). Significant structural differences unique to the active site of carbonic anhydrase V are responsible for differences in the mechanism of catalytic proton transfer as compared with other carbonic anhydrase isozymes. In the prototypical isozyme, carbonic anhydrase II, catalytic proton transfer occurs via the shuttle group His-64; carbonic anhydrase V has Tyr-64, which is not an efficient proton shuttle due in part to the bulky adjacent side chain of Phe-65. Based on analysis of the structure of carbonic anhydrase V, we speculate that Tyr-131 may participate in proton transfer due to its proximity to zinc-bound solvent, its solvent accessibility, and its electrostatic environment in the protein structure. Finally, the design of isozyme-specific inhibitors is discussed in view of the complex between carbonic anhydrase V and acetazolamide, a transition-state analogue. Such inhibitors may be physiologically important in the regulation of blood glucose levels.