Concentration Jumps Research Articles

Calcium concentration jumps reveal dynamic ion selectivity of calcium-activated chloride currents in mouse olfactory sensory neurons and TMEM16b-transfected HEK 293T cells

Ca(2+)-activated Cl(-) channels play relevant roles in several physiological processes, including olfactory transduction, but their molecular identity is still unclear. Recent evidence suggests that members of the transmembrane 16 (TMEM16, also named anoctamin) family form Ca(2+)-activated Cl(-) channels in several cell types. In vertebrate olfactory transduction, TMEM16b/anoctamin2 has been proposed as the major molecular component of Ca(2+)-activated Cl(-) channels. However, a comparison of the functional properties in the whole-cell configuration between the native and the candidate channel has not yet been performed. In this study, we have used the whole-cell voltage-clamp technique to measure functional properties of the native channel in mouse isolated olfactory sensory neurons and compare them with those of mouse TMEM16b/anoctamin2 expressed in HEK 293T cells. We directly activated channels by rapid and reproducible intracellular Ca(2+) concentration jumps obtained from photorelease of caged Ca(2+) and determined extracellular blocking properties and anion selectivity of the channels. We found that the Cl(-) channel blockers niflumic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and DIDS applied at the extracellular side of the membrane caused a similar inhibition of the two currents. Anion selectivity measured exchanging external ions and revealed that, in both types of currents, the reversal potential for some anions was time dependent. Furthermore, we confirmed by immunohistochemistry that TMEM16b/anoctamin2 largely co-localized with adenylyl cyclase III at the surface of the olfactory epithelium. Therefore, we conclude that the measured electrophysiological properties in the whole-cell configuration are largely similar, and further indicate that TMEM16b/anoctamin2 is likely to be a major subunit of the native olfactory Ca(2+)-activated Cl(-) current.

Water and biomolecules—physical chemistry of life

This book contains contributions from research associates of the Japanese Ministry of Education, Science, Culture, Sports and Technology on the research project “Water in Biomolecules” as well as several invited speakers from the final workshop of the project in the beginning of 2008. In 15 chapters and 307 pages, the book covers the physical chemistry of biological processes that involve the joint action of biomolecules and water, primarily in protein folding, dynamics, and function. By necessity, stemming from the proceedings of a single research project, it is a rather uneven and rugged overview of the field of synergy and interaction between biomolecules and water. Not all contributions in this volume are directly about water, but they are never far from it either. Nevertheless, the book contains some very interesting and well-written contributions to the physical chemistry of life processes that I can recommend to researchers working in this field. Some chapters also present extremely well-written overviews that would be interesting to read also for non-specialists. The first two chapters, viz. “Mapping Protein Folding Landscapes by NMR Relaxation” (P.E. Wright, D.J. Felitsky, K. Sugase, and H.J. Dyson) and “Experimental and Simulation Studies of the Folding/Unfolding of Goat α-Lactalbumin” (K. Kuwajima, T. Oroguchi, T. Nakamura, M. Ikeguchi, and A. Kidera) deal roughly with—nuclear magnetic resonance (NMR) in the former and circular dichroism (CD), fluorescence, and heteronuclear single quantum coherence spectroscopy in the latter—determination of the main features of the dynamics of the protein-folding process. Apomyoglobin is the subject of the first chapter, with specific attention being paid to the sites of local hydrophobic collapse, secondary structure formation, and transient long-range interactions via formation of transient hydrophobic contacts. Furthermore, an NMR relaxation study of coupled folding and binding of a disordered protein (pKID adsorption onto CBP) also shows that the folding–binding proceeds via the transient formation of hydrophobic contacts. The second chapter is centered on the folding and unfolding properties of authentic and recombinant forms of α-lactalbumin induced by GdnHCl concentration jumps and studied experimentally as well

Electrophysiological characterization of ATPases in native synaptic vesicles and synaptic plasma membranes

Vesicular V-ATPase (V-type H+-ATPase) and the plasma membrane-bound Na+/K+-ATPase are essential for the cycling of neurotransmitters at the synapse, but direct functional studies on their action in native surroundings are limited due to the poor accessibility via standard electrophysiological equipment. We performed SSM (solid supported membrane)-based electrophysiological analyses of synaptic vesicles and plasma membranes prepared from rat brains by sucrose-gradient fractionation. Acidification experiments revealed V-ATPase activity in fractions containing the vesicles but not in the plasma membrane fractions. For the SSM-based electrical measurements, the ATPases were activated by ATP concentration jumps. In vesicles, ATP-induced currents were inhibited by the V-ATPase-specific inhibitor BafA1 (bafilomycin A1) and by DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate). In plasma membranes, the currents were inhibited by the Na+/K+-ATPase inhibitor digitoxigenin. The distribution of the V-ATPase- and Na+/K+-ATPase-specific currents correlated with the distribution of vesicles and plasma membranes in the sucrose gradient. V-ATPase-specific currents depended on ATP with a K0.5 of 51+/-7 microM and were inhibited by ADP in a negatively co-operative manner with an IC50 of 1.2+/-0.6 microM. Activation of V-ATPase had stimulating effects on the chloride conductance in the vesicles. Low micromolar concentrations of DIDS fully inhibited the V-ATPase activity, whereas the chloride conductance was only partially affected. In contrast, NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid] inhibited the chloride conductance but not the V-ATPase. The results presented describe electrical characteristics of synaptic V-ATPase and Na+/K+-ATPase in their native surroundings, and demonstrate the feasibility of the method for electrophysiological studies of transport proteins in native intracellular compartments and plasma membranes.

Shock formation and breaking in granular avalanches

In this paper, we explore properties of shock wave solutions of the Gray-Thornton model for particle size segregation in granular avalanches.The model equation is a nonlinear scalar conservation law expressing conservation of mass under shear for the concentration of small particles in a bidisperse mixture. Shock waves are weak solutions of the partial differential equation across which the concentration jumps. We give precise criteria on smooth initial conditions under which a shock wave forms in the interior of the avalanche in finite time. Shocks typically lose stability as they are sheared by the flow, giving way to a complex structure in which a two-dimensional rarefaction wave interacts dynamically with a pair of shocks. The rarefaction represents a mixing zone, in which small and large particles are mixed as they are transported up and down (respectively) through the zone. The mixing zone expands and twice changes its detailed structure before reaching the boundary.

Inhibitory Effect of Pb2+ on the Transport Cycle of the Na+,K+-ATPase

The effect of Pb(2+) on the transport cycle of the Na(+),K(+)-ATPase was characterized in detail at a molecular level by combining electrical and biochemical measurements. Electrical measurements were performed by adsorbing purified membrane fragments containing Na(+),K(+)-ATPase on a solid-supported membrane. Upon adsorption, the Na(+),K(+)-ATPase was activated by carrying out concentration jumps of different activating substrates, for example, Na(+) and ATP. Charge movements following Na(+),K(+)-ATPase activation were measured in the presence of various Pb(2+) concentrations to investigate the effect of Pb(2+) on different ion translocating steps of the pump cycle. These charge measurements were then compared to biochemical measurements of ATPase activity in the presence of increasing Pb(2+) concentration. Our results indicate that Pb(2+) inhibits cycling of the enzyme, but it does not affect cytoplasmic Na(+) binding and release of Na(+) ions at the extracellular side at concentrations below 10 muM. To explain the inhibitory effect of Pb(2+) on the Na(+),K(+)-ATPase, we propose that Pb(2+) may interfere with the hydrolytic cleavage of the phosphorylated intermediate E(2)P, which occurs in the K(+)-related branch of the pump cycle.

Tricks of Perspective: Insights and Limitations to the Study of Macroscopic Currents for the Analysis of nAChR Activation and Desensitization

Activation, inactivation, and desensitization are key features of ion channel behavior. We endeavor to understand these processes at the level of the single molecules and extrapolate from such microscopic models the behavior of ion channels in contexts of cellular physiology and therapeutics. In the case of ligand-gated ion channels, such as nicotinic acetylcholine receptors (nAChRs), it is also important to consider the nature of the dynamic changes in the chemical stimulus required for activation. The amplitude and time course of the agonist pulse provided to nAChR at a fast synapse will be vastly different from those of the ACh stimulus presented to presynaptic receptors in the brain and neither of these physiological processes will resemble the stimuli presented by nicotine self-administration or with systemic delivery of a therapeutic agent. Likewise, specific experimental protocols will provide unique stimulus profiles, which will impact the relationship between the macroscopic data and the underlying molecular processes. In this work, ion channel simulations intended to model heteromeric neuronal nAChR are conducted under varying conditions of agonist presentation, and the impact of a key microscopic process, desensitization, is studied on the macroscopic responses. With instantaneous jumps in agonist concentrations, the microscopic desensitization rate impacts essentially all aspects of the macroscopic responses, rise rates, decay rates, and both peak and steady-state currents. In contrast, with an agonist pulse like that used in Xenopus oocyte experiments, microscopic desensitization rates have a profound impact on peak current amplitude and very little effect on the kinetics of the macroscopic responses.

Electrogenic Ion Pumps Investigated on a Solid Supported Membrane: Comparison of Current and Voltage Measurements

Current and voltage measurements were performed on Na,K-ATPase and sarcoplasmic reticulum (SR) Ca-ATPase. Measurements of current transients under short-circuit conditions and of voltage transients under open-circuit conditions were carried out by employing a solid supported membrane (SSM). Purified membrane fragments containing Na,K-ATPase or native SR vesicles were adsorbed on a SSM and were activated by performing substrate concentration jumps. Current and voltage transients were recorded in the external circuit. They are related to pump activity and can be attributed to electrogenic events in the reaction cycles of the two enzymes. While current transients of very small amplitude are difficult to detect, the corresponding voltage transients can be measured with higher accuracy because of a much more favorable signal-to-noise ratio. Therefore, voltage measurements are preferable for the investigation of slow processes generating low current signals, e.g., for the analysis of low turnover transporters.

Cell-Free Electrophysiology of Native Inner Mitochondrial Membranes

Transporters, pumps and channels of inner mitochondrial membranes are involved in all central mitochondrial functions such as regulation of ATP synthesis, thermogenesis, control of cellular calcium, regulation of apoptosis, and in balancing production of cellular reactive oxygen species. However, functional investigation of mitochondrial transport proteins is tedious. Typically, the functional studies rely on the reconstitution of recombinant proteins in proteoliposomes. This work describes the use of the SURFE2R technology for electrical measurements of transporters in native mitochondrial membranes. The inner mitochondrial membranes were isolated from pig heart and adsorbed on gold electrodes according to standard protocols. These biosensors were measured with the standard SURFE2R equipment. Three transport proteins were characterized: the ADP/ATP exchanger (ANT), the F-type ATPase (complex V), and the Cytochrome c Oxidase (COX, complex IV). The transport was activated through substrate concentration jumps (ATP or ADP in case of ANT and F-type ATPase) or, in case of COX, via concentration jumps with reduced cytochrome c. In case of activation via ATP-concentration jumps, both, the ANT and F-type ATPase activity could be detected independently on the same sensor by applying ANT- or ATPase-selective solutions. The specificity of the currents was verified by ATPase- and ANT-specific inhibitors. As expected, the ATP-induced activity of ANT was increased in presence of ADP gradients across the membranes. The ANT-signals depended on the applied ATP-concentration. The cytochrome c-induced currents were inhibited by COX-specific inhibitor cyanide and depended on the redox state of the protein. In summary, the presented results demonstrate a novel, easy and reliable approach for functional studies of mitochondrial transporters and pumps in their native surroundings, and can potentially help to understand the complex molecular mechanisms of different mitochondrial functions. Additionally, the technology platform can also be used to perform pharmacological screens for mitochondria-targeted drugs.

Electrophysiological Investigation of the Lactose Permease from Escherichia coli on a Solid-Supported Membrane

Electrogenic events associated with the activity of the wild-type lactose permease (LacY) of Escherichia coli were investigated by using proteoliposomes containing purified LacY adsorbed onto a solid-supported membrane. Activation of the proteoliposomes with concentration jumps of different substrates generated transient currents. Analysis of the transient currents at different lipid to protein ratios and different pH values show that the currents represent stationary turnover of LacY. Furthermore, selective inactivation of the substrate binding by alkylation of C148 with N-ethyl maleimide (NEM) suppressed the transient currents, indicating that the transients correspond to the electrogenic activity of LacY.Mutant E325A LacY was used to investigate possible electrogenic steps in the transport cycle unrelated with proton translocation. In addition, electrogenic steps taking place before proton translocation were investigated with C154G LacY, which binds sugar as well as the wild-type but catalyzes very little transport activity. Both mutants show electrogenic activity after activation with different substrates. Therefore, either substrate binding or a conformational change following substrate binding is responsible for these electrical transients. As exchange (but not efflux) is almost voltage independent in wild-type LacY, it is concluded that there are least two electrogenic steps in the transport cycle.

An automatic electrophysiological assay for the neuronal glutamate transporter mEAAC1

A rapid and robust electrophysiological assay based on solid supported membranes (SSM) for the murine neuronal glutamate transporter mEAAC1 is presented. Measurements at different concentrations revealed the EAAC1 specific affinities for l-glutamate ( K m = 24 μM), l-aspartate ( K m = 5 μM) and Na + ( K m = 33 mM) and an inhibition constant K i for dl- threo-β-benzyloxyaspartic acid (TBOA) of 1 μM. Inhibition by 3-hydroxy-4,5,6,6a-tetrahydro-3a H-pyrrolo[3,4- d]isoxazole-6-carboxylic acid (HIP-B) was not purely competitive with an IC 50 of 13 μM. Experiments using SCN − concentration jumps yielded large transient currents in the presence of l-glutamate showing the characteristics of the glutamate-gated anion conductance of EAAC1. Thus, SSM-based electrophysiology allows the analysis of all relevant transport modes of the glutamate transporter on the same sample. K + and Na + gradients could be applied to the transporter. Experiments in the presence and absence of Na + and K + gradients demonstrated that the protein is still able to produce a charge translocation when no internal K + is present. In this case, the signal amplitude is smaller and a lower apparent affinity for l-glutamate of 144 μM is found. Finally the assay was adapted to a commercial fully automatic system for SSM-based electrophysiology and was validated by determining the substrate affinities and inhibition constants as for the laboratory setup. The combination of automatic function and its ability to monitor all transport modes of EAAC1 make this system an universal tool for industrial drug discovery.

Activation of recombinant NR1/NR2C NMDA receptors

The N-methyl-d-aspartate (NMDA) subtype of ionotropic glutamate receptors comprises both NR1 and NR2 subunits, and plays numerous roles in both physiological and pathophysiological processes in the central nervous system (CNS). NR2C-containing NMDA receptors are most abundant in cerebellum, thalamus and olfactory bulb, and are also expressed in oligodendrocytes and hippocampal interneurons. We have used patch clamp recording to explore the activation properties of recombinant NR1/NR2C receptors expressed in HEK293 cells. NR1/NR2C receptors activated by a maximally effective concentration of glutamate and glycine had two main conductance levels of 45 pS and 28 pS when the extracellular Ca(2+) concentration was 0.5 mm and the holding potential was -80 mV. The occurrence of the lower subconductance state was reduced in the absence of extracellular Ca(2+). The distribution of closed durations recorded from patches with a high probability of containing only one active channel were best fitted by five exponential functions; the apparent open duration histogram could be fitted by two exponential functions (n = 10 patches). The apparent mean open time of NR1/NR2C receptors was brief (0.52 +/- 0.04 ms), suggesting that the stability of the open state of the NR1/NR2C receptors is lower than other NR2-containing receptors. NR1/NR2C open probability was exceptionally low, being 0.011 +/- 0.002 in patches containing a single active receptor (n = 8). Fast agonist concentration jumps were performed on outside out patches with multiple NR1/NR2C channels, which activated with a 10-90% rise time of 3.9 +/- 0.4 ms, faster than other NR2-containing receptors. The deactivation time constant after a brief (5-8 ms) application of a maximally effective concentration of agonists was 319 +/- 34 ms. The majority of the patches also showed a modest level of desensitization that could be described by either a single or a double exponential time course with the fastest time constant between 15 and 47 ms. Conceptual models of activation were fitted using the maximum interval likelihood (MIL) method to the sequence of open and closed durations recorded from outside-out patches that contained one active NR1/NR2C channel. NR1/NR2C receptor properties including modest desensitization and low open probability could be described by gating schemes similar to those previously proposed for other NMDA receptor subunit combinations.

Thermodynamics of Activation Gating in Olfactory-Type Cyclic Nucleotide-Gated (CNGA2) Channels

Olfactory-type cyclic nucleotide-gated (CNG) ion channels open by the binding of cyclic nucleotides to a binding domain in the C-terminus. Employing the Eyring rate theory, we performed a thermodynamic analysis of the activation gating in homotetrameric CNGA2 channels. Lowering the temperature shifted the concentration-response relationship to lower concentrations, resulting in a decrease of both the enthalpy ΔH and entropy ΔS upon channel opening, suggesting that the order of an open CNGA2 channel plus its environment is higher than that of the closed channel. Activation time courses induced by cGMP concentration jumps were used to study thermodynamics of the transition state. The activation enthalpies ΔH‡ were positive at all cGMP concentrations. In contrast, the activation entropy ΔS‡ was positive at low cGMP concentrations and became then negative at increasing cGMP concentrations. The enthalpic and entropic parts of the activation energies approximately balance each other at all cGMP concentrations, leaving the free enthalpy of activation in the range between 19 and 21kcal/mol. We conclude that channel activation proceeds through different pathways at different cGMP concentrations. Compared to the unliganded channel, low cGMP concentrations generate a transitional state of lower order whereas high cGMP concentrations generate a transitional state of higher order.

Blocking kinetics of memantine on NR1a/2A receptors recorded in inside-out and outside-out patches from Xenopus oocytes

Previous experiments on primary cultures of hippocampal/cortical neurones revealed that the block and unblock of N-Methyl-D-Aspartate (NMDA) receptor channels by memantine showed double exponential kinetics and that the offset kinetics following a voltage-step were much faster than following a concentration jump. There are, however, two major problems when using such cultured primary neurones for these experiments (1) the almost certain expression of heterogeneous NMDA receptor subunits which could underlie double exponential kinetics due to different potencies at receptor subtypes and (2) slow space- and concentration-clamp due to neuronal morphology which could mask even faster kinetics. Therefore, we performed similar experiments with Xenopus oocytes exclusively expressing one NMDA receptor type (NR1a/2A) at high levels which allowed recordings from membrane patches with large currents. The use of inside-out patches for voltage-step and outside-out patches in combination with a piezo driven fast application system largely negated potential space- and concentration-clamp problems. Block and unblock of the NMDA receptor by memantine after both voltage jump and concentration jumps showed triple exponential kinetics. The fast onset kinetics of NMDA receptor channel block following both concentration-clamp and voltage jumps from +70 to -70 mV were similar. In contrast, offset kinetics after a voltage-step from -70 to +70 mV were much faster than following a concentration jump at the holding potential of -70 mV. These results provide further support for the hypothesis that rapid relief of block via strong synaptic membrane depolarisation underlies the good therapeutic profile of memantine.

Currents in Response to Rapid Concentration Jumps of Amphetamine Uncover Novel Aspects of Human Dopamine Transporter Function

Amphetamine (AMPH) is a widely abused psychostimulant that acts as a substrate for the human dopamine transporter (hDAT). Using a piezoelectric rapid application system, we measured AMPH-induced currents mediated by hDAT. Whole-cell patch-clamp recordings in a heterologous expression system reveal that AMPH induces a rapidly activating and subsequently decaying inward current mediated by hDAT. We hypothesize that this transient inward current reflects a conformational change associated with substrate translocation. The AMPH-induced current strictly depends on extracellular Na+. Elevated intracellular Na+ has no effect on the peak AMPH-induced current amplitude but inhibits the steady-state current. In addition, elevated intracellular Na+ causes an overshoot outward current upon washout of AMPH that reflects hDAT locked in a Na+-exchange mode. Furthermore, elevated intracellular Na+ dramatically accelerates the recovery time from desensitization of the AMPH-induced current, revealing a new role for intracellular Na+ in promoting the transition to the hDAT "outward-facing" conformation. Ion substitution suggests that both extracellular and intracellular Cl- facilitate transporter turnover in contrast to the classical model of Cl- as a cotransported ion. We present an alternating-access model of hDAT function that accurately fits the main features of the experimental data. The model predicts that translocation of substrate occurs within milliseconds of substrate binding but that slow reorientation of the empty transporter is the rate-limiting factor for turnover. The model provides a framework for interpreting perturbations of hDAT activity.

Resolving the Fast Kinetics of Cooperative Binding: Ca2+ Buffering by Calretinin

Cooperativity is one of the most important properties of molecular interactions in biological systems. It is the ability to influence ligand binding at one site of a macromolecule by previous ligand binding at another site of the same molecule. As a consequence, the affinity of the macromolecule for the ligand is either decreased (negative cooperativity) or increased (positive cooperativity). Over the last 100 years, O2 binding to hemoglobin has served as the paradigm for cooperative ligand binding and allosteric modulation, and four practical models were developed to quantitatively describe the mechanism: the Hill, the Adair-Klotz, the Monod-Wyman-Changeux, and the Koshland-Némethy-Filmer models. The predictions of these models apply under static conditions when the binding reactions are at equilibrium. However, in a physiological setting, e.g., inside a cell, the timing and dynamics of the binding events are essential. Hence, it is necessary to determine the dynamic properties of cooperative binding to fully understand the physiological implications of cooperativity. To date, the Monod-Wyman-Changeux model was applied to determine the kinetics of cooperative binding to biologically active molecules. In this model, cooperativity is established by postulating two allosteric isoforms with different binding properties. However, these studies were limited to special cases, where transition rates between allosteric isoforms are much slower than the binding rates or where binding and unbinding rates could be measured independently. For all other cases, the complex mathematical description precludes straightforward interpretations. Here, we report on calculating for the first time the fast dynamics of a cooperative binding process, the binding of Ca2+ to calretinin. Calretinin is a Ca2+-binding protein with four cooperative binding sites and one independent binding site. The Ca2+ binding to calretinin was assessed by measuring the decay of free Ca2+ using a fast fluorescent Ca2+ indicator following rapid (<50-μs rise time) Ca2+ concentration jumps induced by uncaging Ca2+ from DM-nitrophen. To unravel the kinetics of cooperative binding, we devised several approaches based on known cooperative binding models, resulting in a novel and relatively simple model. This model revealed unexpected and highly specific nonlinear properties of cellular Ca2+ regulation by calretinin. The association rate of Ca2+ with calretinin speeds up as the free Ca2+ concentration increases from cytoplasmic resting conditions (∼100 nM) to approximately 1 μM. As a consequence, the Ca2+ buffering speed of calretinin highly depends on the prevailing Ca2+ concentration prior to a perturbation. In addition to providing a novel mode of action of cellular Ca2+ buffering, our model extends the analysis of cooperativity beyond the static steady-state condition, providing a powerful tool for the investigation of the dynamics and functional significance of cooperative binding in general.

Electrogenic steps of the SR Ca-ATPase enzymatic cycle and the effect of curcumin

Sarcoplasmic reticulum (SR) vesicles were adsorbed on an octadecanethiol/phosphatidylcholine mixed bilayer anchored to a gold electrode, and the Ca-ATPase contained in the vesicles was activated by ATP concentration jumps in the presence of calcium ions. The resulting capacitive current transients are compared with those calculated on the basis of the enzymatic cycle of the calcium pump. This comparison provides information on the kinetics of the E 2–E 1 conformational change and on its pH dependence. The alteration in the current transients following ATP concentration jumps in the presence of curcumin is examined. In particular, curcumin decreases the rate of slippage of the Ca-ATPase, and at concentrations above 10 μM reduces calcium transport by this pump.

Time resolved kinetics of the guinea pig Na–Ca exchanger (NCX1) expressed in Xenopus oocytes: voltage and Ca2+ dependence of pre-steady-state current investigated by photolytic Ca2+concentration jumps

Kinetic properties of the Na-Ca exchanger (guinea pig NCX1) expressed in Xenopus oocytes were investigated by patch clamp techniques and photolytic Ca(2+) concentration jumps. Current measured in oocyte membranes expressing NCX1 is almost indistinguishable from current measured in patches derived from cardiac myocytes. In the Ca-Ca exchange mode, a transient inward current is observed, whereas in the Na-Ca exchange mode, current either rises to a plateau, or at higher Ca(2+) concentration jumps, an initial transient is followed by a plateau. No comparable current was observed in membrane patches not expressing NCX1, indicating that photolytic Ca(2+) concentrations jumps activate Na-Ca exchange current. Electrical currents generated by NCX1 expressed in Xenopus oocytes are about four times larger than those obtained from cardiac myocyte membranes enabling current recording with smaller concentration jumps and/or higher time resolution. The apparent affinity for Ca(2+) of nonstationary exchange currents (0.1 mM) is much lower than that of stationary currents (6 muM). Measurement of the Ca(2+) dependence of the rising phase provides direct evidence that the association rate constant for Ca(2+) is about 5 x 10(8) M(-1) s(-1) and voltage independent. In both transport modes, the transient current decays with a voltage independent but Ca(2+)-dependent rate constant, which is about 9,000 s(-1) at saturating Ca(2+) concentrations. The voltage independence of this relaxation is maintained for Ca(2+) concentrations far below saturation. In the Ca-Ca exchange mode, the amount of charge translocated after a concentration jump is independent of the magnitude of the jump but voltage dependent, increasing at negative voltages. The slope of the charge-voltage relation is independent of the Ca(2+) concentration. Major conclusions are: (1) Photolytic Ca(2+) concentration jumps generate current related to NCX1. (2) The dissociation constant for Ca(2+) at the cytoplasmic transport binding site is about 0.1 mM. (3) The association rate constant of Ca(2+) at the cytoplasmic transport sites is high (5 x 10(-8) M(-1)s(-1)) and voltage independent. (4) The minimal five-state model (voltage independent binding reactions, one voltage independent conformational transition and one very fast voltage dependent conformational transition) used before to describe Ca(2+) translocation at saturating Ca(2+) concentrations is valid for Ca(2+) concentrations far below saturation.

Cooperation of the Conserved Aspartate 439 and Bound Amino Acid Substrate Is Important for High-Affinity Na+ Binding to the Glutamate Transporter EAAC1

The neuronal glutamate transporter EAAC1 contains several conserved acidic amino acids in its transmembrane domain, which are possibly important in catalyzing transport and/or binding of co/countertransported cations. Here, we have studied the effects of neutralization by site-directed mutagenesis of three of these amino acid side chains, glutamate 373, aspartate 439, and aspartate 454, on the functional properties of the transporter. Transport was analyzed by whole-cell current recording from EAAC1-expressing mammalian cells after applying jumps in voltage, substrate, or cation concentration. Neutralization mutations in positions 373 and 454, although eliminating steady-state glutamate transport, have little effect on the kinetics and thermodynamics of Na+ and glutamate binding, suggesting that these two positions do not constitute the sites of Na+ and glutamate association with EAAC1. In contrast, the D439N mutation resulted in an approximately 10-fold decrease of apparent affinity of the glutamate-bound transporter form for Na+, and an ∼2,000-fold reduction in the rate of Na+ binding, whereas the kinetics and thermodynamics of Na+ binding to the glutamate-free transporter were almost unchanged compared to EAAC1WT. Furthermore, the D439N mutation converted l-glutamate, THA, and PDC, which are activating substrates for the wild-type anion conductance, but not l-aspartate, into transient inhibitors of the EAAC1D439 anion conductance. Activation of the anion conductance by l-glutamate was biphasic, allowing us to directly analyze binding of two of the three cotransported Na+ ions as a function of time and [Na+]. The data can be explained with a model in which the D439N mutation results in a dramatic slowing of Na+ binding and a reduced affinity of the substrate-bound EAAC1 for Na+. We propose that the bound substrate controls the rate and the extent of Na+ interaction with the transporter, depending on the amino acid side chain in position 439.

Evaporation and condensation of a binary mixture of vapors

AbstractHalf‐space problem of evaporation and condensation of a binary mixture of vapors is investigated on the basis of the linearized Boltzmann equation for hard‐sphere molecules with the complete condensation condition. The problem is analyzed numerically by a finite‐difference method, in which the complicated collision integrals are computed by the extension of the method proposed by Y. Sone, T. Ohwada, and K. Aoki [Phys. Fluids A 1, 363 (1989)] to the case of a gas mixture. As a result, the behavior of the mixture is clarified not only at the level of the macroscopic quantities but also at the level of the velocity distribution function. In addition, accurate formulas of the temperature, pressure, and concentration jumps caused by the evaporation and condensation are constructed for arbitrary values of the concentration of the background reference state by the use of the Chebyshev polynomial approximation. (© 2006 WILEY‐VCH Verlag GmbH &amp; Co. KGaA, Weinheim)

The Inner Interhelix Loop 4–5 of the Melibiose Permease from Escherichia coli Takes Part in Conformational Changes after Sugar Binding

Cytoplasmic loop 4-5 of the melibiose permease from Escherichia coli is essential for the process of Na+-sugar translocation (Abdel-Dayem, M., Basquin, C., Pourcher, T., Cordat, E., and Leblanc, G. (2003) J. Biol. Chem. 278, 1518-1524). In the present report, we analyze functional consequences of mutating each of the three acidic amino acids in this loop into cysteines. Among the mutants, only the E142C substitution impairs selectively Na+-sugar translocation. Because R141C has a similar defect, we investigated these two mutants in more detail. Liposomes containing purified mutated melibiose permease were adsorbed onto a solid supported lipid membrane, and transient electrical currents resulting from different substrate concentration jumps were recorded. The currents evoked by a melibiose concentration jump in the presence of Na+, previously assigned to an electrogenic conformational transition (Meyer-Lipp, K., Ganea, C., Pourcher, T., Leblanc, G., and Fendler, K. (2004) Biochemistry 43, 12606-12613), were much smaller for the two mutants than the corresponding signals in cysteineless MelB. Furthermore, in R141C the stimulating effect of melibiose on Na+ affinity was lost. Finally, whereas tryptophan fluorescence spectroscopy revealed impaired conformational changes upon melibiose binding in the mutants, fluorescence resonance energy transfer measurements indicated that the mutants still show cooperative modification of their sugar binding sites by Na+. These data suggest that: 1) loop 4-5 contributes to the coordinated interactions between the ion and sugar binding sites; 2) it participates in an electrogenic conformational transition after melibiose binding that is essential for the subsequent obligatory coupled translocation of substrates. A two-step mechanism for substrate translocation in the melibiose permease is suggested.