Simple Carrier Kinetics in Complex Membrane Transporters

The four-state simple carrier model (SCM) has been employed to describe facilitative transport of ligands across biological membranes. Two basic mechanisms have been invoked to account for carrier-mediated ligand translocation: (i) binding to a mobile carrier, and (ii) displacement determined by conformational changes of an integral protein. While translatory carriers may be accurately represented by a four-state diagram, it is unlikely that the transport process mediated by a complex membrane protein can be strictly described by the elementary SCM. The purpose of this article is to test whether facilitative transporters with a more complex kinetic design than the SCM can exhibit macroscopic kinetic properties indistinguishable from it. For this, I studied a ``general carrier model' (GCM), and evaluated whether the relevant kinetic parameters are subject to the same basic restrictions as in the SCM. The fundamental finding is that there is a general kinetic design embodied with SCM-like properties, that can be shared by many transporters. In particular, the classical SCM is shown here to represent a particular case of the GCM. A main conclusion of this work is therefore that the finding of a macroscopic SCM-like kinetic behavior for a particular process of facilitative transport does not represent a sufficient argument in favor of a particular type of mechanism, like the typical one involving a two-conformational single-site carrier. Received: 9 February 1998/Revised: 19 June 1998     

A General Model for the Dynamics of the Cell Volume

The conservation of the cell volume within values compatible with the overall cell functions represents an ubiquitous property, shared by cells comprising the whole biological world. Water transport across membranes constitutes the main process associated to the dynamics of the cell volume, its chronic and acute regulations therefore represent crucial aspects of cell homeostasis. In spite of the biological diversity, the dynamics of the cell volume exhibits common basic features in the diverse types of cells. The purpose of this study is to show that there is a general model capable to describe the basic aspects of the dynamics of the cell volume. It is demonstrated here that the steady states of this model represent asymptotically stable configurations. As illustrations, several cases of non-polarized (i.e., symmetrical) and polarized (e.g., epithelial) cells performing water transport are shown here to represent particular cases of the general model. From a biological perspective, the existence of a general model for the dynamics of the cell volume reveals that, in spite of physiological and morphological peculiarities, there is a basic common design of the membrane transport processes. In view of its stability properties, this basic design may represent an ancestral property that has proven to be successful regarding the overall homeostatic properties of cells.     

Modeling Facilitative Sugar Transporters: Transitions Between Single and Double Ligand Occupancy of Multiconformational Channel Models Explain Anomalous Kinetics

The four-state simple carrier model (SCM) is employed to describe ligand translocation by diverse passive membrane transporters. However, its application to systems like facilitative sugar transporters (GLUTs) is controversial: unidirectional fluxes under zero-trans and equilibrium-exchange experimental conditions fit a SCM, but flux data from infinite-cis and infinite-trans experiments appear not to fit the same SCM. More complex kinetic models have been proposed to explain this ``anomalous' behavior of GLUTs, but none of them accounts for all the experimental findings. We propose an alternative model in which GLUTs are channels subject to conformational transitions, and further assume that the results from zero-trans and equilibrium-exchange experiments as well as trans-effects corresponds to a single-occupancy channel regime, whereas the results from the infinite-cis and infinite-trans experiments correspond to a regime including higher channel occupancies. We test the plausibility of this hypothesis by studying a kinetic model of a two-site channel with two conformational states. In each state, the channel can bind the ligand from only one of the compartments. Under single-occupancy, for conditions corresponding to zero-trans and equilibrium-exchange experiments, the model behaves as a SCM capable of exhibiting trans-stimulations. For a regime including higher degrees of occupancy and infinite-cis and infinite-trans conditions, the same channel model can exhibit a behavior qualitatively similar to a SCM, albeit with kinetic parameters different from those for the single-occupancy regime. Numerical results obtained with our model are consistent with available experimental data on facilitative glucose transport across erythrocyte membranes. Hence, if GLUTs are multiconformational channels, their particular kinetic properties can result from transitions between single and double channel occupancies. Received: 12 April 1995/Revised: 28 August 1995     

Identification and Functional Characterization of a Novel Mitochondrial Carrier for Citrate and Oxoglutarate in Saccharomyces cerevisiae

Mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and coenzymes across the mitochondrial membrane. The function of only a few of the 35 Saccharomyces cerevisiae mitochondrial carriers still remains to be uncovered. In this study, we have functionally defined and characterized the S. cerevisiae mitochondrial carrier Yhm2p. The YHM2 gene was overexpressed in S. cerevisiae, and its product was purified and reconstituted into liposomes. Its transport properties, kinetic parameters, and targeting to mitochondria show that Yhm2p is a mitochondrial transporter for citrate and oxoglutarate. Reconstituted Yhm2p also transported oxaloacetate, succinate, and fumarate to a lesser extent, but virtually not malate and isocitrate. Yhm2p catalyzed only a counter-exchange transport that was saturable and inhibited by sulfhydryl-blocking reagents but not by 1,2,3-benzenetricarboxylate (a powerful inhibitor of the citrate/malate carrier). The physiological role of Yhm2p is to increase the NADPH reducing power in the cytosol (required for biosynthetic and antioxidant reactions) and probably to act as a key component of the citrate-oxoglutarate NADPH redox shuttle between mitochondria and cytosol. This protein function is based on observations documenting a decrease in the NADPH/NADP⁺ and GSH/GSSG ratios in the cytosol of ΔYHM2 cells as well as an increase in the NADPH/NADP⁺ ratio in their mitochondria compared with wild-type cells. Our proposal is also supported by the growth defect displayed by the ΔYHM2 strain and more so by the ΔYHM2ΔZWF1 strain upon H₂O₂ exposure, implying that Yhm2p has an antioxidant function.     

Resolving Linear and Non-Linear Interactive Kinetic Mechanisms for Ions in Membrane Channels

Thallous ion in gramicidin channels displays the anomalous molefraction effect and other behavior that suggests its permeationmechanism might be more complicated than the mechanisms for sodiumor potassium ion permeation. The permeation is modeled by eithermultistate first order kinetics where the number of states and therate constants are modified to fit the data or an ion displacementmechanism that requires higher order rate terms. Although the twoclasses of mechanism are difficult to distinguish usingcurrent-voltage data, the two classes give different responses toa modulated transmembrane potential with frequency comparable tothe rate constants for intrachannel ion transitions. Themultistate first order kinetics give currents only at themodulation frequency. Information is transmitted in the phase andamplitude of the observed current. The non-linear iondisplacement mechanism produces harmonic frequencies. A detailedspectral analysis then distinguishes the two classes of mechanismand provides a range of frequency and phase data that permitsdetermination of the appropriate rate constants.     

RT—PCR测定大鼠妊娠早期子宫孕激素受体基因的表达

孕激素在妊娠的建立和维持过程中起着非常重要的作用,但目前为止未见有关大鼠子宫 激素受体（ＰＲ）基因在该过程中表达情况的系统报道。本和反转偶联聚合酶链式反应（ＲＴ－ＰＣＲ）方法测定了大鼠动情周期和妊娠早期子宫孕激素受体基因的转录,结果表明：１．动情周期中,子吕ＰＲｍＲＮＡ水平在动情期最高,在动情后期最低,动情期水平约为动情后期的２倍。２．妊娠开始后,子宫ＰＲｍＲＮＡ水平迅速上地ｄ３－ｄ４（着床前）达     

A New Role for PTEN in Regulating Transient Receptor Potential Canonical Channel 6-mediated Ca2+ Entry,Endothelial Permeability,and Angiogenesis

Phosphatase and tensin homologue (PTEN) is a dual lipid-protein phosphatase that catalyzes the conversion of phosphoinositol 3,4,5-triphosphate to phosphoinositol 4,5-bisphosphate and thereby inhibits PI3K-Akt-dependent cell proliferation, migration, and tumor vascularization. We have uncovered a previously unrecognized role for PTEN in regulating Ca²⁺ entry through transient receptor potential canonical channel 6 (TRPC6) that does not require PTEN phosphatase activity. We show that PTEN tail-domain residues 394–403 permit PTEN to associate with TRPC6. The inflammatory mediator thrombin promotes this association. Deletion of PTEN residues 394–403 prevents TRPC6 cell surface expression and Ca²⁺ entry. However, PTEN mutant, C124S, which lacks phosphatase activity, did not alter TRPC6 activity. Thrombin failed to increase endothelial monolayer permeability in the endothelial cells, transducing the Δ394–403 PTEN mutant. Paradoxically, we also show that thrombin failed to induce endothelial cell migration and tube formation in cells transducing the Δ394–403 PTEN mutant. Our results demonstrate that PTEN, through residues 394–403, serves as a scaffold for TRPC6, enabling cell surface expression of the channel. Ca²⁺ entry through TRPC6 induces an increase in endothelial permeability and directly promotes angiogenesis. Thus, PTEN is indicated to play a role beyond suppressing PI3K signaling.     

Membrane transport models with fast and slow reactions: General analytical solution for a single relaxation

Summary Membrane transport models are usually expressed on the basis of chemical kinetics. The states of a transporter are related by rate constants, and the time-dependent changes of these states are given by linear differential equations of first order. To calculate the time-dependent transport equation, it is necessary to solve a system of differential equations which does not have a general analytical solution if there are more than five states. Since transport measurements in a complex system rarely provide all the time constants because some of them are too rapid, it is more appropriate to obtain approximate analytical solutions, assuming that there are fast and slow reaction steps. The states of the fast steps are related by equilibrium constants, thus permitting the elimination of their differential equations and leaving only those for the slow steps. With a system having only two slow steps, a single differential equation is obtained and the state equations have a single relaxation. Initial conditions for the slow reactions are determined after the perturbation which redistribute the states related by fast reactions. Current and zero-trans uptake equations are calculated. Curve fitting programs can be used to implement the general procedure and obtain the model parameters.     

Functional and structural dynamics of NhaA,a prototype for Na and H antiporters,which are responsible for Na and H homeostasis in cells

The crystal structure of down-regulated NhaA crystallized at acidic pH 4 [21] has provided the first structural insights into the antiport mechanism and pH regulation of a Na⁺/H⁺ antiporter [22]. On the basis of the NhaA crystal structure [21] and experimental data (reviewed in [2,22,38] we have suggested that NhaA is organized into two functional regions: (i) a cluster of amino acids responsible for pH regulation (ii) a catalytic region at the middle of the TM IV/XI assembly, with its unique antiparallel unfolded regions that cross each other forming a delicate electrostatic balance in the middle of the membrane. This unique structure contributes to the cation binding site and allows the rapid conformational changes expected for NhaA. Extended chains interrupting helices appear now a common feature for ion binding in transporters. However the NhaA fold is unique and shared by ASBTNM [30] and NapA [29]. Computation [13], electrophysiology [69] combined with biochemistry [33,47] have provided intriguing models for the mechanism of NhaA. However, the conformational changes and the residues involved have not yet been fully identified. Another issue which is still enigma is how energy is transduced “in this ‘nano-machine.’” We expect that an integrative approach will reveal the residues that are crucial for NhaA activity and regulation, as well as elucidate the pHand ligand-induced conformational changes and their dynamics. Ultimately, integrative results will shed light on the mechanism of activity and pH regulation of NhaA, a prototype of the CPA2 family of transporters. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

A Probit Latent Class Model with General Correlation Structures for Evaluating Accuracy of Diagnostic Tests

Summary Traditional latent class modeling has been widely applied to assess the accuracy of dichotomous diagnostic tests. These models, however, assume that the tests are independent conditional on the true disease status, which is rarely valid in practice. Alternative models using probit analysis have been proposed to incorporate dependence among tests, but these models consider restricted correlation structures. In this article, we propose a probit latent class model that allows a general correlation structure. When combined with some helpful diagnostics, this model provides a more flexible framework from which to evaluate the correlation structure and model fit. Our model encompasses several other PLC models but uses a parameter‐expanded Monte Carlo EM algorithm to obtain the maximum‐likelihood estimates. The parameter‐expanded EM algorithm was designed to accelerate the convergence rate of the EM algorithm by expanding the complete‐data model to include a larger set of parameters and it ensures a simple solution in fitting the PLC model. We demonstrate our estimation and model selection methods using a simulation study and two published medical studies.     

General method for the derivation and numerical solution of epithelial transport models

Summary A general method is presented for the formulation and numerical evaluation of mathematical models describing epithelial transport. The method is based on the principles of conservation of mass, and maintenance of electroneutrality within the cells and bathing solutions. It is therefore independent of the specific membrane transport mechanisms, and can be used to evaluate different models describing arbitrary transport processes (including passive, active and cotransport processes). Detailed numerical methods are presented that allow computation of steady-state and transient responses under open-circuit, current-clamp and voltage-clamp conditions, using a general-purpose laboratory minicomputer. To evaluate the utility of this approach, a specific model is presented that is consistent with the Koefoed-Johnson and Ussing hypothesis of sodium transport in tight epithelia (Acta Physiol. Scand. 42:298–308, 1958). This model considers passive transport of an arbitrary number of permeant solutes, active transport of sodium and potassium, and osmotically induced water transport across the apical and basolateral membranes. Results of the model are compared to published experimental measurements in rabbit urinary bladder epithelium.     

TPC2 Is a Novel NAADP-sensitive Ca2+ Release Channel, Operating as a Dual Sensor of Luminal pH and Ca2+

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a molecule capable of initiating the release of intracellular Ca²⁺ required for many essential cellular processes. Recent evidence links two-pore channels (TPCs) with NAADP-induced release of Ca²⁺ from lysosome-like acidic organelles; however, there has been no direct demonstration that TPCs can act as NAADP-sensitive Ca²⁺ release channels. Controversial evidence also proposes ryanodine receptors as the primary target of NAADP. We show that TPC2, the major lysosomal targeted isoform, is a cation channel with selectivity for Ca²⁺ that will enable it to act as a Ca²⁺ release channel in the cellular environment. NAADP opens TPC2 channels in a concentration-dependent manner, binding to high affinity activation and low affinity inhibition sites. At the core of this process is the luminal environment of the channel. The sensitivity of TPC2 to NAADP is steeply dependent on the luminal [Ca²⁺] allowing extremely low levels of NAADP to open the channel. In parallel, luminal pH controls NAADP affinity for TPC2 by switching from reversible activation of TPC2 at low pH to irreversible activation at neutral pH. Further evidence earmarking TPCs as the likely pathway for NAADP-induced intracellular Ca²⁺ release is obtained from the use of Ned-19, the selective blocker of cellular NAADP-induced Ca²⁺ release. Ned-19 antagonizes NAADP-activation of TPC2 in a non-competitive manner at 1 μm but potentiates NAADP activation at nanomolar concentrations. This single-channel study provides a long awaited molecular basis for the peculiar mechanistic features of NAADP signaling and a framework for understanding how NAADP can mediate key physiological events.     

A mechanism for nitrate transport and reduction

It is proposed that a tetrahedron-shaped, transmembrane nitrate reductase tetramer functions as a carrier for nitrate transport. Reduction and transport are thereby brought about by the same enzyme complex. An ATPase is visualized to be closely associated with the nitrate reductase tetramer. The tetramer is apparently oriented such that one monomer is exposed to the outside of the plasmalemma while the other three are exposed to the cytoplasmic side. This orientation yields a reaction mechanism where the transport and reduction of one nitrate ion is accompanied by the transport of two additional nitrate ions (i.e. a 3 : 1 transport-reduction ratio). The proportion of transported nitrate that is reduced is apparently modulated by thiol reversible ADP inhibition of reduction. This inhibition, however, is probably the result of adenylate binding at sites on the proposed nitrate-activated ATPase to which nitrate reductase is tightly coupled. An analogous system consisting of a nitrate reductase dimer that spans a unit membrane plus an ATPase is proposed to be responsible for nitrate transport and reduction in some algae and chloroplasts.     

A simple method for the isolation of basolateral plasma membrane vesicles from rat kidney cortex Enzyme activities and some properties of glucose transport

A procedure for preparing basolateral membrane vesicles from rat renal cortex was developed by differential centrifugation and Percoll density gradient centrifugation, and the uptake of d-[³H]glucose into these vesicles was studied by a rapid filtration technique. (Na⁺ + K⁺)-ATPase, the marker enzyme for basolateral membranes, was enriched 22-fold compared with that found in the homogenate. The rate of d-glucose uptake was almost unaffected by Na⁺ gradient (no overshoot).     

A ditopic receptor for cation binding and facilitated transport through a supported liquid membrane

The binding properties of an artificial receptor towards a series of cations including Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ba²⁺, Fe²⁺ and Al³⁺ in acetonitrile are described. The receptor comprises a photo-responsive pyrene unit connected via a short spacer to a 2,2′:6′,2″-terpyridine metal ion binding site. Interaction of cations with the receptor was monitored by changes in absorption profile and the association constants calculated for 1:1 and 1:2 cation:ligand binding fall within the range log β = 3-12. The receptor is highly fluorescent and quenching of the emission is observed upon cation binding. The potassium picrate transport properties of the membrane-bound receptor are also described. This receptor when immobilised in a polymer support, which separates two aqueous solutions, has been shown to transport potassium ions in the dark with a flux rate of 1.5 × 10⁸ mol/s m². In contrast, when the membrane-bound receptor is selectively illuminated with light (λ > 400 nm), the flux increases to 2.0 × 10⁸ mol/s m². The transport efficiency depends on the nature of the trap used in the receiver phase.     

Glycosylation of a Vesicular Monoamine Transporter: A Mutation in a Conserved Proline Residue Affects the Activity, Glycosylation, and Localization of the Transporter

Abstract: The role of N -glycosylation in the expression, ligand recognition, activity, and intracellular localization of a rat vesicular monoamine transporter (rVMAT1) was investigated. The glycosylation inhibitor tunicamycin induced a dose-dependent decrease in the rVMAT1-mediated uptake of [³H]serotonin. Part of this effect was due to a general toxic effect of the drug. Therefore, to assess the contribution of each of the glycosylation sites to the transporter activity, the three putative N -glycosylation sites were mutated individually, in combination, and in toto ("triple" mutant). Mutation of each glycosylation site caused a minor and additive decrease in activity, up to the triple mutant, which retained at least 50% of the wild-type activity. No significant differences were found either in the time dependence of uptake or the apparent affinity for ligands of the triple mutant compared with the wild-type protein. It is interesting that in contrast to plasma-membrane neurotransmitter transporters, the unglycosylated form of rVMAT1 distributed in the cell as the wild-type protein. Pro⁴³ is a highly conserved residue located at the beginning of the large loop in which all the potential glycosylation sites are found. A Pro⁴³Leu mutant transporter was inactive. It is remarkable that despite the presence of glycosylation sites, the mutant transporter was not glycosylated. Moreover, the distribution pattern of the Pro⁴³Leu mutant clearly differed from that of the wild type. In contrast, a Pro⁴³Gly mutant displayed an activity practically identical to the wild-type protein. As this replacement generated a protein with wild-type characteristics, we suggest that the conformation conferred by the amino acid at this position is essential for activity.     

In glycine and GABA(A) channels, different subunits contribute asymmetrically to channel conductance via residues in the extracellular domain

Single-channel conductance in Cys-loop channels is controlled by the nature of the amino acids in the narrowest parts of the ion conduction pathway, namely the second transmembrane domain (M2) and the intracellular helix. In cationic channels, such as Torpedo ACh nicotinic receptors, conductance is increased by negatively charged residues exposed to the extracellular vestibule. We now show that positively charged residues at the same loop 5 position boost also the conductance of anionic Cys-loop channels, such as glycine (α1 and α1β) and GABA(A) (α1β2γ2) receptors. Charge reversal mutations here produce a greater decrease on outward conductance, but their effect strongly depends on which subunit carries the mutation. In the glycine α1β receptor, replacing Lys with Glu in α1 reduces single-channel conductance by 41%, but has no effect in the β subunit. By expressing concatameric receptors with constrained stoichiometry, we show that this asymmetry is not explained by the subunit copy number. A similar pattern is observed in the α1β2γ2 GABA(A) receptor, where only mutations in α1 or β2 decreased conductance (to different extents). In both glycine and GABA receptors, the effect of mutations in different subunits does not sum linearly: mutations that had no detectable effect in isolation did enhance the effect of mutations carried by other subunits. As in the nicotinic receptor, charged residues in the extracellular vestibule of anionic Cys-loop channels influence elementary conductance. The size of this effect strongly depends on the direction of the ion flow and, unexpectedly, on the nature of the subunit that carries the residue.     

Formulation of a coupled mechanism between solute diffusion,phosphatase-kinase reactions and membrane potentials for the primary active transport of phosphorylated substrates through biological membranes

Coupled interrelations occurring between a phosphatase/kinase reaction sequence acting in unstirred layers and on both sides of a charged biomembrane pore structure are presented as a plausible kinetic model for the primary active transport of phosphorylated molecules. Simulations conducted at the cell level and with credible numerical values demonstrate that the enzymes positions strongly regulate the membrane permeability for the transported substrate. Depending on both the enzymes positions (more or less far from the membrane) and the membrane charges, the membrane may appear either impervious, either permeable or able to actively transport a phosphorylated substrate. Globally all happens as if, in function of the enzymes positions, a permanent pore may be regulated, changing from a more closed to a more open conformation.     

A tomato ER-type Ca-ATPase, LCA1, has a low thapsigargin-sensitivity and can transport manganese

Recombinant Ca²⁺-ATPase from tomato (i.e. LCA1 for Lycopersicon esculentum [Since the identification and naming of LCA1, the scientific name for the tomato has been changed to Solanum lycopersicum.] Ca-ATPase) was heterologously expressed in yeast for structure-function characterization. We investigate the differences between plant and animal Ca pumps utilizing comparisons between chicken and rabbit SERCA-type pumps with Arabidopsis (ECA1) and tomato plant (LCA1) Ca²⁺-ATPases. Enzyme function was confirmed by the ability of each Ca²⁺-ATPase to rescue K616 growth on EGTA-containing agar and directly via in vitro ATP hydrolysis. We found LCA1 to be ∼300-fold less sensitive to thapsigargin than animal SERCAs, whereas ECA1 was thapsigargin-resistant. LCA1 showed typical pharmacological sensitivities to cyclopiazonic acid, vanadate, and eosin, consistent with it being a P_IIA-type Ca²⁺-ATPase. Possible amino acid changes responsible for the reduced plant thapsigargin-sensitivity are discussed. We found that LCA1 also complemented K616 yeast growth in the presence of Mn²⁺, consistent with moving Mn²⁺ into the secretory pathway and functionally compensating for the lack of secretory pathway Ca-ATPases (SPCAs) in plants.