Elena Bossi

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).

Exogenous Protein Expression in Xenopus Oocytes

The oocytes of the South African clawed frog Xenopus laevis have been widely used as a reliable system for the expression and characterization of different types of proteins, including ion channels and membrane receptors. The large size and resilience of these oocytes make them easy to handle and to microinject with different molecules such as natural mRNAs, cRNAs, and antibodies. A variety of methods can then be used to monitor the expression of the proteins encoded by the microinjected mRNA/cRNA, and to perform a functional characterization of the heterologous polypeptides. In this chapter, after describing the equipment required to maintain X. laevis in the laboratory and to set up a microinjection system, we provide detailed procedures for oocyte isolation, micropipet and cRNA preparation, and oocyte microinjection. A method for the labeling of oocyte-synthesized proteins and for the immunological detection of the heterologous polypeptides is also described.

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.

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.

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.

Triggering of Suicidal Erythrocyte Death by Pazopanib

Background/Aims: The multi-targeted kinase inhibitor pazopanib, a drug employed for the treatment of a wide variety of malignancies, has previously been shown to trigger apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Mechanisms involved in the triggering of eryptosis include Ca2+ entry, oxidative stress and ceramide. The present study explored, whether pazopanib induces eryptosis and, if so, whether it is effective by Ca2+ entry, oxidative stress and/or ceramide. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, reactive oxygen species (ROS) formation from DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to pazopanib significantly increased the percentage of annexin-V-binding (≥ 25 µg/ml) and of shrunken erythrocytes (≥ 50 µg/ml). Pazopanib treatment further resulted in significant hemolysis (≥ 25 µg/ml). The effect of pazopanib on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. Pazopanib significantly increased DCF fluorescence (50 µg/ml) and ceramide abundance (50 µg/ml). Conclusions: Pazopanib triggers eryptosis, an effect involving Ca2+ entry, oxidative stress and ceramide.

Role of anion-cation interactions on the pre-steady-state currents of the rat Na(+)-Cl(-)-dependent GABA cotransporter rGAT1.

The effects of sodium and chloride on the properties of the sodium‐dependent component of the ‘pre‐steady‐state’ currents of rGAT1, a GABA cotransporter of the Na+‐Cl−‐dependent family, were studied using heterologous oocyte expression and voltage clamp. Reductions in either extracellular sodium or chloride shifted the charge‐voltage (Q‐V) and time constant‐voltage (τ‐V) characteristics of the process towards more negative potentials. The shift induced by sodium (TMA+, tetramethylammonium substitution) was stronger than that induced by chloride (acetate substitution), and the shift of τ was accompanied by a decrease in its maximum value. Increasing extracellular Ca2+ did not produce significant shifts in Q‐V and τ‐V curves. The negative shift of the Q‐V curve upon chloride reduction and the decrease in the value of the relaxation time constant, τ, when either sodium or chloride were lowered, contrasted with the prediction of the Hill‐Boltzmann interpretation of the process. Analysis of the unidirectional rate constants under different conditions revealed that both sodium and chloride shifted the outward rate more than the inward rate; furthermore, the shifts induced by sodium were larger than those induced by chloride. These observations are qualitatively compatible with the existence of a selective vestibule at the mouth of the transporters, acting similarly to a Donnan system.

Properties of the Ca2+-activated Cl− current of Xenopus oocytes

Abstract. The properties of the Ca2+-activated Cl− current of Xenopus oocytes have been investigated by voltage-clamp and injections of D-3-deoxy-3-fluoro-myo-inositol 1,4,5-trisphosphate (3-F-InsP3). Following 3-F-InsP3 injection, a transient phase of Ca2+-activated Cl− current occurred, caused by Ca2+ release from internal stores; subsequently, a secondary, long-lasting, current was recorded, signaling Ca2+ influx from the exterior (ICRAC). Changes in external Cl− during the sustained phase produced the expected shifts in reversal potential (Erev), while the conductance varied opposite to the predictions of simple electrodiffusional theory. Application of depolarizing pulses soon (10 s) after 3-F-InsP3 injection elicited membrane currents exhibiting a single exponential rise. During the sustained subsequent phase, the current elicited by depolarizations showed an early peak followed by a prominent decline. During the sustained phase, removal of calcium from the external solution, or its substitution with Ba2+, abolished voltage- and time-dependent components of the depolarization-induced current. Slope conductance analysis of the inactivating records revealed, in addition to the decline of the Ca2+-activated Cl− current, the presence of a second, inwardly directed current. This could be identified as a slowly inducible Na+ current already described in Xenopus oocytes.