Antonio Peres

Ion binding and permeation through the lepidopteran amino acid transporter KAAT1 expressed in Xenopus oocytes

1 The transient and steady‐state currents induced by voltage jumps in Xenopus oocytes expressing the lepidopteran amino acid co‐transporter KAAT1 have been investigated by two‐electrode voltage clamp. 2 KAAT1‐expressing oocytes exhibited membrane currents larger than controls even in the absence of amino acid substrate (uncoupled current). The selectivity order of this uncoupled current was Li+ > Na+≈ Rb+≈ K+ > Cs+; in contrast, the permeability order in non‐injected oocytes was Rb+ > K+ > Cs+ > Na+ > Li+. 3 KAAT1‐expressing oocytes gave rise to ‘pre‐steady‐state currents’ in the absence of amino acid. The characteristics of the charge movement differed according to the bathing ion: the curves in K+ were strongly shifted (> 100 mV) towards more negative potentials compared with those in Na+, while in tetramethylammonium (TMA+) no charge movement was detected. 4 The charge‐voltage (Q–V) relationship in Na+ could be fitted by a Boltzmann equation having V½ of −69 ± 1 mV and slope factor of 26 ± 1 mV; lowering the Na+ concentrations shifted the Q–V relationship to more negative potentials; the curves could be described by a generalized Hill equation with a coefficient of 1.6, suggesting two binding sites. The maximal movable charge (Qmax) in Na+, 3 days after injection, was in the range 2.5–10 nC. 5 Addition of the transported substrate leucine increased the steady‐state carrier current, the increase being larger in high K+ compared with high Na+ solution; in these conditions the charge movement disappeared. 6 Applying Eyring rate theory, the energy profile of the transporter in the absence of organic substrate included a very high external energy barrier (25.8 RT units) followed by a rather deep well (1.8 RT units).

Temperature effects on the presteady-state and transport-associated currents of GABA cotransporter rGAT1.

The effects of temperature on the γ‐aminobutyric acid (GABA) uptake and on the presteady‐state and transport‐associated currents of the GABA cotransporter, rat γ‐aminobutyric acid transporter 1 (rGAT1), have been studied using heterologous oocyte expression and voltage‐clamp. Increasing temperature from 15 to 30°C increased GABA uptake, diminished the maximal value of the relaxation time constant of the presteady‐state currents and increased the amplitude of the current associated with the transport of GABA. The curve of the presteady‐state charge versus voltage was shifted toward negative potentials by increasing the temperature, while the maximal amount of charge (Q max) remained constant; the τ versus V curve was also negatively shifted by increasing temperatures. Analysis of the outward (α) and inward (β) rate constants as functions of temperature showed that they are affected differently, with a Q 10=3.4 for α and Q 10=1.5 for β. The different temperature coefficients of the rate constants account for the observed shifts. These observations are consistent with a charge moving mechanism based on a conformational change of the protein; the weaker temperature sensitivity of the inward rate constant suggests a rate‐limiting diffusional component on this process.

Ca-mediated and independent effects of arachidonic acid on gap junctions and Ca-independent effects of oleic acid and halothane

In Novikoff hepatoma cell pairs studied by double perforated patch clamp (DPPC), brief (20 s) exposure to 20 microM arachidonic acid (AA) induced a rapid and reversible uncoupling. In pairs studied by double whole-cell clamp (DWCC), uncoupling was completely prevented by effective buffering of Cai2+ with BAPTA. Similarly, AA (20 s) had no effect on coupling in cells perfused with solutions containing no added Ca2+ (SES-no-Ca) and studied by DPPC, suggesting that Ca2+ influx plays an important role. Parallel experiments monitoring [Ca2+]i with fura-2 showed that [Ca2+]i increases with AA to 0.7-1.5 microM in normal [Ca2+]o, and to approximately 400 nM in SES-no-Ca solutions. The rate of [Ca2+]i increase matched that of Gj decrease, but [Ca2+]i recovery was faster. In cells studied by DWCC with 2 mM BAPTA in the pipette solution and superfused with SES-no-Ca, long exposure (1 min) to 20 microM AA caused a slow and virtually irreversible uncoupling. This result suggests that AA has a dual mechanism of uncoupling: one dominant, fast, reversible, and Ca(2+)-dependent, the other slow, poorly reversible, and Ca(2+)-independent. In contrast, uncoupling by oleic acid (OA) or halothane was insensitive to internal buffering with BAPTA, suggesting a Ca(2+)-independent mechanism only.

The effective membrane capacity of Xenopus eggs: its relations with membrane conductance and cortical granule exocytosis

The effective membrane capacity (ceff) of theXenopus egg has been measured integrating the membrane current transients in response to small voltage-clamp pulses. Before activationceff has a value of 1.34±SE. 0.13 μF/cm2 (apparent surface area, 13 eggs from 3 females) and is essentially constant over the voltage range between −30 and +60 mV. During artificial activation of the eggs by pricking or by addition of Ca2+ ionophore A23187,ceff increases by about 60% in 2–3 min and then slowly decreases returning to near the initial value in 15–20 min. Electron microscopic observations of the egg surface at different times reveal that the capacity time course parallels the changes in plasma membrane area due to cortical granule exocytosis and to a later reduction of microvillar extension. Simultaneous measurements of capacity and conductance show that the capacity changes are slower and delayed in comparison with the transient development of the chloride conductance responsible for the activation potential. In CO2-treated eggs the cortical granule exocytosis is prevented and, correspondingly, the transient capacity increase is strongly reduced or absent, but the development of the chloride conductance remains normal. This technique gives a method to electrophysiologically monitor the cortical granule exocytosis; moreover our results show that the exocytotic process can be blocked without affecting the membrane conductance changes.

Mutation K448E in the external loop 5 of rat GABA transporter rGAT1 induces pH sensitivity and alters substrate interactions

1 The effect of the mutation K448E in the rat GABA transporter rGAT1 was studied using heterologous expression in Xenopus oocytes and voltage clamp. 2 At neutral pH, the transport‐associated current vs. voltage (I–V) relationship of the mutated transporter was different from wild‐type, and the pre‐steady‐state currents were shifted towards more positive potentials. The mutated transporter showed an increased apparent affinity for Na+ (e.g. 62 vs. 152 mm at −60 mV), while the opposite was true for GABA (e.g. 20 vs. 13 μm at −60 mV). 3 In both isoforms changes in [Na+]o shifted the voltage dependence of the pre‐steady‐state and of the transport‐associated currents by similar amounts. 4 In the K448E form, the moved charge and the relaxation time constant were shifted by increasing pH towards positive potentials. The transport‐associated current of the mutated transporter was strongly reduced by alkalinization, while acidification slightly decreased and distorted the shape of the I–V curve. Accordingly, uptake of [3H]GABA was strongly reduced in K448E at pH 9.0. The GABA apparent affinity of the mutated transporter was reduced by alkalinization, while acidification had the opposite result. 5 These observations suggest that protonation of negatively charged residues may regulate the Na+ concentration in the proximity of the transporter. Calculation of the unidirectional rate constants for charge movement shows that, in the K448E form, the inward rate constant is increased at alkaline pH, while the outward rate constant does not change, in agreement with an effect due to mass action law. 6 A possible explanation for the complex effect of pH on the transport‐associated current may be found by combining changes in local [Na+]o with a direct action of pH on GABA concentration or affinity. Our results support the idea that the extracellular loop 5 may participate to form a vestibule to which sodium ions must have access before proceeding to the steps involving charge movement.

The relation between charge movement and transport-associated currents in the rat GABA cotransporter rGAT1.

Most cotransporters characteristically display two main kinds of electrical activity: in the absence of organic substrate, transient presteady‐state currents (Ipre) are generated by charge relocation during voltage steps; in the presence of substrate, sustained, transport‐associated currents (Itr) are recorded. Quantitative comparison of these two currents, in Xenopus oocytes expressing the neural GABA cotransporter rGAT1, revealed several unforeseen consistencies between Ipre and Itr, in terms of magnitude and kinetic parameters. The decay rate constant (r) of Ipre and the quantity of charge displaced to an inner position in the transporter (Qin(0)) depended on voltage and ionic conditions. Saturating GABA concentrations, applied under the same conditions, suppressed Ipre (i.e. Qin(∞) = 0) and produced a transport‐associated current with amplitude Itr=Qin(0)r. At non‐saturating levels of GABA, changes of Itr were compensated by corresponding variations in Qin, such that Ipre and Itr complemented each other, according to the relation: Itr= (Qin(0) ‐ Qin) r. Complementarity of magnitude, superimposable kinetic properties and equal dependence on voltage and [Na+]o point to the uniqueness of the charge carrier for both processes, suggesting that transport and charge migration arise from the same molecular mechanism. The observed experimental relations were correctly predicted by a simple three‐state kinetic model, in which GABA binding takes place after charge binding and inward migration have occurred. The model also predicts the observed voltage dependence of the apparent affinity of the transporter for GABA, and suggests a voltage‐independent GABA binding rate with a value around 0.64 μm−1 s−1.

Voltage-dependent channels permeable to K+ and Na+ in the membrane ofAcer pseudoplatanus vacuoles

SummaryThe patch-clamp technique in whole-cell configuration was used to study the electrical properties of the tonoplast in isolated vacuoles fromAcer pseudoplatanus cultured cells. In symmetrical KCl or K2 malate solutions, voltage- and time-dependent inward currents were elicited by hyperpolarizing the tonoplast (inside negative), while in the positive range of potential the conductance was very small. The specific conductance of the tonoplast at −100 mV, in 100mm symmetrical KCl was about 160 μS/cm2. The reversal potentials (Erev) of the current, measured in symmetrical or asymmetrical ion concentrations (cation, anion or both) were very close to the values of the K+ equilibrium potential. Experiments performed in symmetrical or asymmetrical NaCl indicate that Na+ too can flow through the channels. NeitherErev nor amplitude and kinetics of the current changed by replacing NaCl with KCl in the external solution. These results indicate the presence of hyperpolarization-activated channels in tonoplasts, which are permeable to K+ as well as to Na+. Anions such as Cl− or malate seem to contribute little to the channel current.

InsP3- and Ca2+-induced Ca2+ release in single mouse oocytes

To better understand the mechanism of intracellular Ca2+ mobilization, mouse oocytes were micro‐injected with ‘caged’‐inositol‐1,4,5 trisphosphate (caged‐InsP3) together with the Ca2+ indicator Fluo‐3 ??? and monitor Ca2+ redistribution. Photo‐released InsP3 olicits [Ca2+], changes exhibiting several kinetics phases and threshold ??? oscillations were induced after a single InsP3 pulse. Autoregenerative Ca2+ transients could also be induced by injections of Ca2+ itself, demonstrating unequivocally the presence of a Ca2+‐induced Ca2+‐release mechanism in these cells.

Modulation of the Inward Rectifier Potassium Channel IRK1 by the Ras Signaling Pathway

In this study, we investigated the role of Ras and the mitogen-activated protein kinase (MAPK) pathway in the modulation of the inward rectifier potassium channel IRK1. We show that although expression of IRK1 in HEK 293 cells leads to the appearance of a potassium current with strong inward rectifying properties, coexpression of the constitutively active form of Ras (Ras-L61) results in a significant reduction of the mean current density without altering the biophysical properties of the channel. The inhibitory effect of Ras-L61 is not due to a decreased expression of IRK1 since Northern analysis indicates that IRK1 mRNA level is not affected by Ras-L61 co-expression. Moreover, the inhibition can be relieved by treatment with the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor PD98059. Confocal microscopy analysis of cells transfected with the fusion construct green fluorescent protein-IRK1 shows that the channel is mainly localized at the plasma membrane. Coexpression of Ras-L61 delocalizes fluorescence to the cytoplasm, whereas treatment with PD98059 partially restores the membrane localization. In conclusion, our data indicate that the Ras-MAPK pathway modulates IRK1 current by affecting the subcellular localization of the channel. This suggests a role for Ras signaling in regulating the intracellular trafficking of this channel.

Role of the conserved glutamine 291 in the rat γ-aminobutyric acid transporter rGAT-1

Abstract.We investigated the role of the Q291 glutamine residue in the functioning of the rat γ-aminobutyric acid (GABA) transporter GAT-1. Q291 mutants cannot transport GABA or give rise to transient, leak and transport-coupled currents even though they are targeted to the plasma membrane. Coexpression experiments of wild-type and Q291 mutants suggest that GAT-1 is a functional monomer though it requires oligomeric assembly for membrane insertion. We determined the accessibility of Q291 by investigating the impact of impermeant sulfhydryl reagents on cysteine residues engineered in close proximity to Q291. The effect of these reagents indicates that Q291 faces the external aqueous milieu. The introduction of a steric hindrance close to Q291 by means of [2-(trimethylammonium)ethyl] methanethiosulfonate bromide modification of C74A/T290C altered the affinity of the mutant for cations. Taken together, these results suggest that this irreplaceable residue is involved in the interaction with sodium or in maintaining the cation accessibility to the transporter.