Stefano Giovannardi

THE FRESHWATER CYANOBACTERIUM PLANKTOTHRIX SP. FP1: MOLECULAR IDENTIFICATION AND DETECTION OF PARALYTIC SHELLFISH POISONING TOXINS

A filamentous cyanobacterium, belonging to the Order of Oscillatoriales, was found to be responsible for a toxic algal bloom in Lake Varese, Italy, during the summer of 1997. Morphological characters, as well as near complete 16S rRNA gene sequencing, revealed that the dominant species of the bloom was most closely related to the genus Planktothrix. In addition, genetic analysis of the phycocyanin operon of Planktothrix sp. FP1 revealed a novel primary structure, previously undescribed within the cyanobacteria, which was used as a genetic marker for rapid detection and identification of this toxic strain. The occurrence of saxitoxin (STX), a principal toxin in paralytic shellfish poisoning (PSP), was confirmed in the natural bloom sample by both pre‐column and post‐column derivatization high‐performance liquid chromatography (HPLC) analyses, and eventually by liquid chromatography/mass spectrometry (LC/MS). The toxicity of this field sample was also revealed by electrophysiological assays in which the extract inhibited 90% of the voltage‐dependent Na+ current in human neuroblastoma cells at the STX concentration of 80 nM. The cultured strain showed a lower physiologic activity than the bloom sample (67% blockage of Na+ current at a toxin concentration of 200 nM), and STX was detected only by pre‐column HPLC, indicating the presence of a compound structurally close to STX. Chemical and molecular genetic analyses performed here add Planktothrix sp. FP1 to the growing list of diverse cyanobacterial species capable of synthesizing STX and its related compounds.

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).

Evidence for the interaction of d-amino acid oxidase with pLG72 in a glial cell line

Accumulating genetic evidence indicates that the primate-specific gene locus G72/G30 is related to schizophrenia: it encodes for the protein pLG72, whose function is still the subject of controversy. We recently demonstrated that pLG72 negatively affects the activity of human d-amino acid oxidase (hDAAO, also related to schizophrenia susceptibility), which in neurons and (predominantly) in glia is expected to catabolize the neuromodulator d-serine. The d-serine regulation mechanism relying on hDAAO-pLG72 interaction does not match with the subcellular localizations proposed for hDAAO (peroxisomes) and pLG72 (mitochondria). By using glioblastoma U87 cells transfected with plasmids encoding for hDAAO and/or pLG72 we provide convergent lines of evidence that newly synthesized hDAAO, transitorily present in cytosol before being delivered to the peroxisomes, colocalizes and interacts with pLG72 which we propose to be exposed on the external membrane of mitochondria. We also report that newly synthesized cytosolic hDAAO is catalytically active, and therefore pLG72 binding-and ensuing hDAAO inactivation-plays a protective role against d-serine depletion.

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 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.

Toxic cyanobacterial blooms in Lake Varese (Italy): A multidisciplinary approach

During the summer of 1997, a toxic cyanobacterial bloom was observed in Lake Varese. The cyanobacterial community is recognized morphologically as being dominated by species belonging to the genera Aphanizomenon, Oscillatoria, Anabaena, and Microcystis. HPLC analyses were performed on phytoplankton samples collected on the lake from July to September 1997. A HPLC peak that had the same retention time as saxitoxin was detected in the sample collected on August 19. In addition, extracts from shellfish and fish tissues showed the same peak. In electrophysiological experiments, extracts of cyanobacteria showed an almost complete inhibition of Na+ channels, the typical biological effect of saxitoxin. The genomic region corresponding to the phycocyanin intergenic spacer of cyanobacteria from the last summer bloom has been characterized by means of a PCR amplification technique. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 127–134, 1999

Functionally independent subunits in the oligomeric structure of the GABA cotransporter rGAT1.

Abstract.We have combined structural and functional approaches to investigate the role of oligomerization in the operation of the GABA transporter rGAT1. Xenopus laevis oocytes were induced to express, either separately or simultaneously, the wild-type form of rGAT1 and a mutated (Y140W) form, unable to translocate GABA and to generate transport currents, although its intramembrane charge movement properties are only slightly affected. These characteristics, together with the insensitivity of Y140W to the blocking action of SKF89976A, were used to study the possible functional interaction of the two forms in an heteromeric structure. The electrophysiological data from oocytes coexpressing wild-type and Y140W rGAT1 were consistent with a completely independent activity of the two forms. Oligomerization was also studied by fluorescence resonance energy transfer (FRET) in tsA201 cells expressing the transporters fused with cyan and yellow fluorescent proteins (ECFP and EYFP). All combinations tested (WT-ECFP/WTEYFP, Y140W-ECFP/Y140W-EYFP and WT-ECFP/ Y140W-EYFP) were able to give rise to FRET, confirming the formation of homo- as well as heterooligomers. We conclude that, although rGAT1 undergoes structural oligomerization, each monomer operates independently.