The barrier for proton transport in aquaporins as a challenge for electrostatic models: the role of protein relaxation in mutational calculations

The origin of the barrier for proton transport through the aquaporin channel is a problem of general interest. It is becoming increasingly clear that this barrier is not attributable to the orientation of the water molecules across the channel but rather to the electrostatic penalty for moving the proton charge to the center of the channel. However, the reason for the high electrostatic barrier is still rather controversial. It has been argued by some workers that the barrier is due to the so-called NPA motif and/or to the helix macrodipole or to other specific elements. However, our works indicated that the main reason for the high barrier is the loss of the generalized solvation upon moving the proton charge from the bulk to the center of the channel and that this does not reflect a specific repulsive electrostatic interaction but the absence of sufficient electrostatic stabilization. At this stage it seems that the elucidation and clarification of the origin of the electrostatic barrier can serve as an instructive test case for electrostatic models. Thus, we reexamine the free-energy surface for proton transport in aquaporins using the microscopic free-energy perturbation/umbrella sampling (FEP/US) and the empirical valence bond/umbrella sampling (EVB/US) methods as well as the semimacroscopic protein dipole Langevin dipole model in its linear response approximation version (the PDLD/S-LRA). These extensive studies help to clarify the nature of the barrier and to establish the "reduced solvation effect" as the primary source of this barrier. That is, it is found that the barrier is associated with the loss of the generalized solvation energy (which includes of course all electrostatic effects) upon moving the proton charge from the bulk solvent to the center of the channel. It is also demonstrated that the residues in the NPA region and the helix dipole cannot be considered as the main reasons for the electrostatic barrier. Furthermore, our microscopic and semimacroscopic studies clarify the problems with incomplete alternative calculations, illustrating that the effects of various electrostatic elements are drastically overestimated by macroscopic calculations that use a low dielectric constant and do not consider the protein reorganization. Similarly, it is pointed out that microscopic potential of mean force calculations that do not evaluate the electrostatic barrier relative to the bulk water cannot be used to establish the origin of the electrostatic barrier. The relationship between the present study and calculations of pK(a)s in protein interiors is clarified, pointing out that approaches that are applied to study the aquaporin barrier should be validated by pK(a)s calculations. Such calculations also help to clarify the crucial role of solvation energies in establishing the barrier in aquaporins.     

The mechanism of proton exclusion in aquaporin channels

The mechanism of proton exclusion in aquaporin channels is elucidated through free energy calculations of the pathway of proton transport. The second generation multistate empirical valence bond (MS-EVB2) model was applied to simulate the interaction of an excess proton with the channel environment. Jarzynski's equality was employed for rapid convergence of the free energy profile. A barrier sufficiently high to block proton transport is located near the channel center at the NPA motif-a site involved in bi-orientational ordering of the embedded water-wire in absence of the excess proton. A second and lower barrier is observed at the selectivity filter near the periplasmic outlet where the channel is narrowest. This secondary barrier may be essential in filtering other large solutes and cations.     

Charge delocalization in proton channels, I: the aquaporin channels and proton blockage

The explicit contribution to the free energy barrier and proton conductance from the delocalized nature of the excess proton is examined in aquaporin channels using an accurate all-atom molecular dynamics computer simulation model. In particular, the channel permeation free energy profiles are calculated and compared for both a delocalized (fully Grotthuss shuttling) proton and a classical (nonshuttling) hydronium ion along two aquaporin channels, Aqp1 and GlpF. To elucidate the effects of the bipolar field thought to arise from two alpha-helical macrodipoles on proton blockage, free energy profiles were also calculated for computational mutants of the two channels where the bipolar field was eliminated by artificially discharging the backbone atoms. Comparison of the free energy profiles between the proton and hydronium cases indicates that the magnitude of the free energy barrier and position of the barrier peak for the fully delocalized and shuttling proton are somewhat different from the case of the (localized) classical hydronium. The proton conductance through the two aquaporin channels is also estimated using Poisson-Nernst-Planck theory for both the Grotthuss shuttling excess proton and the classical hydronium cation.     

On the stimulation by insulin of tryptophan transport across the blood-brain barrier

Following previous studies showing that in vivo insulin administration increases brain tryptophan levels, we have tested the effect of insulin on tryptophan uptake by isolated bovine brain capillaries, which represent the in vitro equivalent of the blood-brain barrier. In the presence of insulin and Na+ ions, the uptake of 14C-labelled tryptophan was significantly increased with respect to controls, this increase being essentially due to a higher affinity of the transport system for the amino acid, while the Vmax was not affected. Insulin increased also, to a similar extent, the uptake of alpha-methylaminoisobutyrate in the presence of Na+ ions, while the uptake of beta-aminobicyclo(2.2.1)heptane carboxylic acid was not affected. Addition of phloretine, or of anti-insulin antibodies, as well as omission of Na+ ions from the buffer abolished the effect of insulin. Insulin appears therefore to increase specifically the substrate affinity of the A-system for neutral amino acid transport, without exerting any influence on the L-system. The absence of the A-system from the luminal side of the microvessels, and the high insulin concentrations needed, raise however some problems as to the physiological significance of this effect.     

Knockdown of aquaporin 3 is involved in intestinal barrier integrity impairment

AQP3 is a water/glycerol transporter expressed at the basolateral membrane of colonic epithelial cells. Although AQPs are expressed in the gastrointestinal tract, their effect on intestinal barrier has not been clear. Here, we showed that knockdown of AQP3 caused a dramatic, dose-dependent increase in E. coli C25 translocation, with the reduction of TEER and increasing LY permeability. Western blots revealed that expression of Claudin-1 and Occludin were significantly decreased in the AQP3 knockdown group, demonstrating that this treatment enhances paracellular permeability via an opening of the tight junction complex. These data not only describe the correlation between transcellular and paracellular pathways in human intestines, but also show that targeted knockdown of AQP3 might impair the intestinal barrier integrity.     

On the origin of lipid asymmetry: the flip side of ion transport

Membrane lipid asymmetry influences a multitude of cellular functions, including membrane vesiculation, cell division, and lifespan. Most cells retain the bulk of aminophospholipids to the cytosolic membrane leaflet by means of ATP-fuelled flippases or translocases. Converging lines of evidence indicate that members of the P(4) subfamily of P-type ATPases catalyze aminophospholipid transport and create lipid asymmetry in late secretory and endocytic compartments. Yet P-type ATPases usually pump small cations or metal ions. Atomic structures revealed important aspects of the transport mechanism, and sequence homology indicates that this mechanism is conserved throughout the family. Consequently, understanding how P(4) ATPases acquired the ability to translocate phospholipids instead of simple ions has become a major focus of interest.     

Water transport in AQP0 aquaporin: molecular dynamics studies

A double lipid bilayer structure containing opposing tetramers of AQP0 aquaporin, in contact through extracellular face loop regions, was recently modeled using an intermediate-resolution map obtained by electron crystallographic methods. The pores of these water channels were found to be critically narrow in three regions and subsequently interpreted to be those of a closed state of the channel. The subsequent determination of a high-resolution AQP0 tetramer structure by X-ray crystallographic methods yielded a pore model featuring two of the three constrictions as noted in the EM work and water molecules within the channel pore. The extracellular-side constriction region of this AQP0 structure was significantly larger than that of the EM-based model and similar to that of the highly water permeable AQP1. The X-ray-based study of AQP0 however could not ascertain if the water molecules found in the pore were the result of water entering from one or both ends of the channel, nor whether water could freely pass through all constriction points. Additionally, this X-ray-based structure could not provide an answer to the question of whether the double lipid bilayer configuration of AQP0 could functionally maintain a water impermeable state of the channel. To address these questions we conducted molecular dynamics simulations to compare the time-dependent behavior of the AQP0 and AQP1 channels within lipid bilayers. The simulations demonstrate that AQP0, in single or double lipid bilayers, is not closed to water transport and that thermal motions of critical side-chains are sufficient to facilitate the movement of water past any of its constriction regions. These motional requirements do however lead to significant free energy barriers and help explain physiological observations that found water permeability in AQP0 to be substantially lower than in the AQP1 pore.     

On the mechanism of proton transport by the neuronal excitatory amino acid carrier 1

Uptake of glutamate from the synaptic cleft is mediated by high affinity transporters and is driven by Na(+), K(+), and H(+) concentration gradients across the membrane. Here, we characterize the molecular mechanism of the intracellular pH change associated with glutamate transport by combining current recordings from excitatory amino acid carrier 1 (EAAC1)-expressing HEK293 cells with a rapid kinetic technique with a 100-micros time resolution. Under conditions of steady state transport, the affinity of EAAC1 for glutamate in both the forward and reverse modes is strongly dependent on the pH on the cis-side of the membrane, whereas the currents at saturating glutamate concentrations are hardly affected by the pH. Consistent with this, the kinetics of the pre-steady state currents, measured after saturating glutamate concentration jumps, are not a function of the pH. In addition, we determined the deuterium isotope effect on EAAC1 kinetics, which is in agreement with proton cotransport but not OH(-) countertransport. The results can be quantitatively explained with an ordered binding model that includes a rapid proton binding step to the empty transporter followed by glutamate binding and translocation of the proton-glutamate-transporter complex. The apparent pK of the extracellular proton binding site is approximately 8. This value is shifted to approximately 6.5 when the substrate binding site is exposed to the cytoplasm.     

Annexin A4 reduces water and proton permeability of model membranes but does not alter aquaporin 2-mediated water transport in isolated endosomes

Annexin A4 (Anx4) belongs to a ubiquitous family of Ca2+-dependent membrane-binding proteins thought to be involved in membrane trafficking and membrane organization within cells. Anx4 localizes to the apical region in epithelia; however, its physiological role is unclear. We show that Anx4 exhibited binding to liposomes (phosphatidylcholine:phosphatidylserine, 1:1) in the presence of Ca2+ and binding was reversible with EDTA. Anx4 binding resulted in liposome aggregation and a reduction in membrane water permeability of 29% (P < 0.001) at 25 degrees C. These effects were not seen in the presence of Ca2+ or Anx4 alone and were reversible with EDTA. Measurements of membrane fluidity made by monitoring fluorescence anisotropy of 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-HPC) demonstrated that Anx4 binding rigidified the outer leaflet of the bilayer (P < 0.001), thus providing a molecular explanation for the inhibition of water flux. To determine whether Anx4 would produce similar effects on physiological membranes we constructed liposomes which recapitulated the lipid composition of the inner leaflet of the MDCK apical membrane. These membranes exhibited reductions to water permeability upon Anx4 binding (19.5% at 25 degrees C, 31% at 37 degrees C; P < 0.01 and P < 0.001, respectively) and to proton permeability (15% at 25 degrees C, 19.5% at 37 degrees C; P < 0.05). Since our in vitro experiments indicated an effect on membrane permeability, we examined localization of Anx4 in the kidney collecting duct, a region of the nephron responsible for concentrating urine through water reabsorbtion. Anx4 was shown to colocalize apically with aquaporin 2 (AQP2) in collecting duct epithelia. To test for the existence of a functional interaction between Anx4 and AQP2 we isolated AQP2-containing endosomes and exposed them to Anx4/Ca2+. Water flux rates were unchanged, indicating Anx4 does not directly regulate AQP2. We conclude that Anx4 can alter the physical properties of membranes by associating with them and regulate passive membrane permeability to water and protons. These properties represent important new functions for Anx4.     

Transient transport across the blood-retina barrier

A mathematical model of the transport of fluorescein across the blood-retina barrier in the transient state and the subsequent diffusion of fluorescein in the vitreous body is presented. The function of the barrier is lumped in a single parameter—the permeability. The sensitivity of this parameter due to changes in the other parameters of the model is given. This establishes the foundation for the quantitative assessment of the barrier function through vitreous fluorophotometry.     

Hypoxanthine transport through the blood-brain barrier

The unidirectional influx of hypoxanthine across cerebral capillaries, the anatomical locus of the blood=brain barrier, was measured with an in situ rat brain perfusion technique employing [³H]hypoxanthine. Hypoxanthine was transported across the blood-brain barrier by a saturable system with a one-half saturation concentration of approximately 0.4 mM. The permeability-surface area product was 3×10^–4 sec^–1 with a hypoxanthine concentration of 0.02 M in the perfusate. Adenine (4 mM) and uracil and theophylline (both 10 mM), but not inosine (10 mM) or leucine (1 mM), inhibited hypoxanthine transfer through the blood-brain barrier. Thus, hypoxanthine is transported through the blood-brain barrier by a high-capacity, saturable transport system with a half-saturation concentration about 100 times the plasma hypoxanthine concentration. Although involved in the transport hypoxanthine from blood into brain, this system is not powerful enough to transfer important quantities of hypoxanthine from blood into brain.     

Myo-inositol transport through the blood-brain barrier

The unidirectional transport of [³H]myo-inositol across cerebral capillaries, the anatomical locus of the blood-brain barrier, was measured using an in situ rat brain perfusion technique. Myo-inositol was transported across the blood-brain barrier by a low capacity, saturable system with a one-half saturation concentration of 0.1 mM. The permeability surface-area product was 6.2×10^–5S^–1 with a myo-inositol concentration of 0.02 mM in the perfusate. The myo-inositol stereoisomer scyllo-inositol but not (+)-chiro-inositol (both 1 mM) inhibited myo-inositol transfer through the blood-brain barrier. These observations provide evidence that myo-inositol is transferred through the blood-brain barrier by simple diffusion and a stereospecific, saturable transport system.     

Niacinamide transport through the blood-brain barrier

The unidirectional influx of niacinamide across cerebral capillaries, the anatomical locus of the blood-brain barrier, was measured with an in situ rat brain perfusion technique employing [¹⁴C]niacinamide. Niacinamide was transported rapidly across the blood-brain barrier by a system that was not saturable with 10 mM niacinamide in the perfusate. However, with periods of perfusion longer than 30 seconds, there was substantial backflow of [¹⁴C]niacinamide into the perfusate. Niacinamide (1.7 M) transport through the blood-brain barrier was not significantly inhibited by 3-acetylpyridine. Thus, niacinamide is transported rapidly and bidirectionally through the blood-brain barrier by a high capacity transport system. Although involved in the transfer of niacinamide between blood and brain, this transport system does not play an important regulatory role in the synthesis of NMN, NAD, and NADP from niacinamide in brain.     

Electrogenic proton transport in epithelial membranes

Summary Certain polar epithelial cells have strong transport capacities for protons and can be examinedin vitro as part of an intact epithelial preparation. Recent studies in the isolated turtle bladder and other tight urinary epithelia indicate that the apical membranes of the carbonic anhydrase-containing cell population of these tissues contain an electrogenic proton pump which has the characteristics of a proton-translocating ATPase. The translocation of protons is tightly coupled to the energy of ATP hydrolysis. Since the pump translocates protons without coupling to the movement of other ions, it may be regarded as an ideal electrogenic pump. The apparent simplicity of the functional properties has led to extensive studies of the characteristics of this pump and of the cellular organization of the secondary acid-base flows in the turtle bladder. Over a rather wide range of electrochemical potential gradients for protons ( ) across the epithelium, the rate of H⁺ transport is nearly linear with . The formalisms of equivalent circuit analysis and nonequilibrium thermodynamics have been useful in describing the behavior of the pump, but these approaches have obvious limitations. We have attempted to overcome some of these limitations by developing a more detailed set of assumptions about each of the transport steps across the pump complex and to formulate a working model for proton transport in the turtle bladder that can account for several otherwise unexplained experimental results. The model suggests that the real pump is neither a simple electromotive force nor a constant current source. Depending on the conditions, it may behave as one or the other.     

Upregulation of the transport system for TNFalpha at the blood-brain barrier

The transport system for the cytokine tumor necrosis factor-alpha (TNFalpha) at the blood-brain barrier (BBB) enables an enhanced yet saturable entry of TNFalpha from blood to the CNS. This review focuses on the selective upregulation of the transport system for TNFalpha at the BBB that is specific for type of pathology, region, and time. The upregulation is reflected by increased CNS tissue uptake of radiolabeled TNFalpha after iv injection in mice and by inhibition of this increase with excess non-radiolabeled TNFalpha. (1) Spinal cord injury (SCI): upregulation of TNFalpha uptake after thoracic transection is seen in the delayed phase of BBB disruption at the lumbar spinal cord. Thoracic SCI by compression, however, has a longer lasting impact on TNFalpha transport that involves thoracic and lumbar spinal cord, in contrast to the upregulation confined to the lumbar region in lumbar SCI by compression. Regardless, the uptake of TNFalpha by spinal cord does not parallel BBB disruption as measured by the leakage of radiolabeled albumin. (2) Experimental autoimmune encephalomyelitis (EAE): the increase in the differential permeability to TNFalpha is seen in all CNS regions (brain and cervical, thoracic, and lumbar spinal cord) and has a distinct time course and reversibility. Exogenous TNFalpha has biphasic effects in modulating functional scores. The BBB, a dynamically regulated barrier, is actively involved in disease processes.     

Azide reduces the hydrophobic barrier of the bacteriorhodopsin proton channel

The sensitivity of a nitroxide spin label to the polarity of its environment has been used to estimate the hydrophobic barrier of the proton channel of the transmembrane proton pump bacteriorhodopsin. By means of site-specific mutagenesis, single cysteine residues were introduced at 10 positions located at the protein surface, in the protein interior, and along the proton pathway. After reaction with a methanethiosulfonate spin label, the principle values of the hyperfine tensor A and the g-tensor were determined from electron paramagnetic resonance spectra measured at 170 K. The shape of the hydrophobic barrier of the proton channel is characterized in terms of a polarity index, DeltaA, determined from the variation of the hyperfine coupling constant Azz. The maximum of the hydrophobic barrier is found to be close to the retinal chromophore in the proton uptake pathway. The effect of the asymmetric distribution of charged and polar residues in the proton release and uptake pathways is clearly reflected in the behavior of the hydrophobic barrier. The presence of azide reduces the barrier height of both the cytoplasmic and extracellular channels. This finding supports the view of azide and other weakly acidic anions as catalysts for the formation of hydrogen-bonded networks in proton pathways of proteins.     

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 E₁ 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.     

Inhibitors of proton pumping: effect on passive proton transport

Reported inhibitors of the Characean plasmalemma proton pump were tested for their ability to inhibit the passive H⁺ conductance which develops in Chara corallina Klein ex Willd. at high pH. Diethylstilbestrol inhibits the proton pump and the passive H⁺ conductance with about the same time course, at concentrations that have no effect on cytoplasmic streaming. N-Ethylmaleimide, a sulfhydryl reagent which is small and relatively nonpolar, also inhibits both pumping and passive conductance of H⁺. However, it also inhibits cytoplasmic streaming with about the same time course, and therefore could not be considered a specific ATPase inhibitor. p-Chloromercuribenzene sulfonate (PCMBS), a sulfhydryl reagent which is large and charged and hence less able to penetrate the membrane, does not inhibit pumping or conductance at low concentration. At high concentration, PCMBS sometimes inhibits pumping without affecting H⁺ conductance, but since streaming is also inhibited, the effect on the pump cannot be said to be specific. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, a water soluble carbodiimide, weakly inhibits both pump and conductance, apparently specifically.