V. Franca Sacchi

Bacillus thuringiensis toxin inhibits K+‐gradient‐dependent amino acid transport across the brush border membrane of Pieris brassicae midgut cells

The luminal membrane of larval midgut cells is the site of action of insecticidal delta‐endotoxin from Bacillus thuringiensis. At concentrations that correspond to normal effective doses in vivo, the toxin inhibits the uptake of amino acids by brush border membrane vesicles prepared from midguts of Pieris brassicae larvae. The toxin does not interact with the K+‐amino acid symport but rather increases the K+ permeability of the membrane. The toxin does not increase the permeability of lepidopteran midgut brush border membrane to either Na+ or H+ nor does it increase the K+ permeability of brush border membrane vesicles prepared from mammalian small intestine,

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).

The Glial Glutamate Transporter 1 (GLT1) Is Expressed by Pancreatic β-Cells and Prevents Glutamate-induced β-Cell Death

Glutamate is the major excitatory neurotransmitter of the central nervous system (CNS) and may induce cytotoxicity through persistent activation of glutamate receptors and oxidative stress. Its extracellular concentration is maintained at physiological concentrations by high affinity glutamate transporters of the solute carrier 1 family (SLC1). Glutamate is also present in islet of Langerhans where it is secreted by the α-cells and acts as a signaling molecule to modulate hormone secretion. Whether glutamate plays a role in islet cell viability is presently unknown. We demonstrate that chronic exposure to glutamate exerts a cytotoxic effect in clonal β-cell lines and human islet β-cells but not in α-cells. In human islets, glutamate-induced β-cell cytotoxicity was associated with increased oxidative stress and led to apoptosis and autophagy. We also provide evidence that the key regulator of extracellular islet glutamate concentration is the glial glutamate transporter 1 (GLT1). GLT1 localizes to the plasma membrane of β-cells, modulates hormone secretion, and prevents glutamate-induced cytotoxicity as shown by the fact that its down-regulation induced β-cell death, whereas GLT1 up-regulation promoted β-cell survival. In conclusion, the present study identifies GLT1 as a new player in glutamate homeostasis and signaling in the islet of Langerhans and demonstrates that β-cells critically depend on its activity to control extracellular glutamate levels and cellular integrity.

Electrogenic K+-Basic Amino-Acid Cotransport in the Midgut of Lepidopteran Larvae

SummaryExperiments performed on isolated midgut demonstrate that the model proposed for the absorption of neutral amino acids in the K+-transporting intestinal epithelium of lepidopteran larvae applies also to the transport of the basic amino acids histidine and lysine. The characteristics of these K+-basic amino-acid cotransports have been studied in brush-border membrane vesicles. Histidine and lysine are transported by different transport agencies, which share, to a different degree, a high sensitivity to transmembrane electrical potential difference. Kinetic analysis showed thatKm for histidine and lysine increased 10-fold and three-fold, respectively, whereasVmax was only slightly modified when the electrical potential difference was abolished. The relationship between potassium concentration and histidine uptake indicates a cooperative binding of more than one potassium to the transporter. Countertransport experiments with glutamine as elicitor show that histidine and glutamine are transported through the same system.

Interaction between na+ and the k+-dependent amino-acid-transport in midgut brush-border membrane-vesicles from philosamia-cynthia larvae

Abstract Both sodium and potassium can drive leucine uptake into brush-border membrane vesicles from Philosamia cynthia midgut, but the effects of these cations are not additive. 2 mM sodium reduces leucine uptake at saturating potassium concentrations, which indicates that these cations interact with the same transporter. The mixed type inhibition of sodium was explained in terms of different kinetic parameters of the co-transporter when this cation binds to the transport protein instead potassium. At 0.2 mM leucine, the affinity of sodium for the transporter was about 18 times that of potassium, whereas leucine Vmax was 2.5 times higher with potassium. Kinetic experiments performed to characterize Na+-dependent leucine uptake showed that the leucine kinetics at different sodium concentrations did not follow Michaelis-Menten kinetics and that the effect of sodium was mainly to increase the affinity of the amino acid for the co-transporter. Na+-activation curves, at fixed leucine concentrations, showed that the Vmax increased with leucine concentration. Since both cations are present in the midgut lumen of P. cynthia larva (200 mM potassium and 1 mM sodium), the interaction between sodium and the K+-dependent co-transporter was observed by measuring leucine uptake as a function of potassium concentration at different fixed sodium concentrations. In accordance with the kinetic parameters that characterize the co-transport in the presence of either Na+ or K+, sodium reduces leucine uptake at high potassium concentrations and increases leucine uptake at low potassium concentrations. Assuming that the translocation is the rate limiting step of the process, we present a model for two alternative drivers (Na+ and K+) and for leucine translocation in the absence of any cation. The derived velocity equation adequately describes the experiments reported here and previously. The physiological meaning of this transport mechanism, probably evolved from a more primitive Na+-dependent co-transport, is discussed and its role in ensuring sodium intake, in an epithelium which contains no conventional sodium pump, is suggested.

Simultaneous measurements of ionic currents and leucine uptake at the amino acid cotransporter KAAT1 expressed in Xenopus laevis oocytes

The transport properties of the intestinal amino acid cotransporter KAAT1, heterologously expressed in Xenopus oocytes, were studied using simultaneous voltage-clamp and tritiated leucine uptake measurements. While addition of 1 mM leucine to oocytes kept at -80 mV in presence of Na(+) or K(+) caused an increase in holding current, in presence of Li(+) the current was reduced. Uptake measurements in voltage-clamp conditions showed that a comparable accumulation of amino acid occurred in all three ionic conditions and irrespective of the direction and amount of the current change. The ratio of moles of transferred charge to moles of transported amino acid ranges from 1.45 for K(+) to 3.52 for Li(+). A hypothetical interpretation involving the coexistence of two populations of transporters, one operating in the uncoupled mode and the other in the substrate transport mode is discussed.

Inhibition of the lepidopteran amino acid co-transporter KAAT1 by phenylglyoxal: role of arginine 76.

Phenylglyoxal (PGO), an arginine‐modifying reagent, is an irreversible inhibitor of KAAT1‐mediated leucine transport, expressed in Xenopus oocytes. The PGO effect was dose‐dependent and 5 mm PGO determined a Vmax reduction to 24% of the control, consistent with the covalent binding to transporter arginine residues not located in the leucine binding site. The use of labelled [14C]PGO confirmed that the inhibitor binds KAAT1. The protein membrane domain contains seven arginine residues one of which, arginine 76, is conserved in the family of GABA transporters. Using site‐directed mutagenesis we showed that only arginine 76 is crucial for KAAT1 activity and is involved in PGO binding.

Neutral amino acid absorption in the midgut of lepidopteran larvae

Publisher Summary Several studies and systematic analysis suggests a great variability in the ionic composition of the hemolymph in different orders of insects. Lepidoptera represent an extreme case in which the [Na+]/[K +] ratio is

Age-related modifications of leucine uptake in brush-border membrane vesicles from rat jejunum.

Leucine uptake in brush-border membrane vesicles purified from rat jejunum is sodium-dependent, sensitive to the membrane electrical potential difference and enhanced by the intravesicular presence of potassium. This last effect is not mediated by the genesis of an electrical potential difference, since potassium activation and electrical potential effects are additive. Sodium-dependent leucine Vmax (1568 +/- 91 pmol/mg per 3 s, is higher in young rats than in adult and old animals. The diffusion component of leucine transport decreases with increasing age. Preloading the vesicles with 100 mM KCl increases leucine Vmax 200% in young animals, 100% in adult and 44% in old animals. The potassium activation is a saturation function of the cation concentration. Leucine uptake in brush border membrane from old animals is less sensitive to the electrical potential difference than in membranes from adult and young animals.

Leucine transport in Xenopus laevis oocytes: Functional and morphological analysis of different defolliculation procedures

L-leucine uptake in stage V Xenopus laevis oocytes was affected by the specific methods used to remove the follicle cells. In the presence of 100 mM NaCl, L-leucine uptake was reduced by 67.5% +/- 5.7 when defolliculation was performed enzymatically by collagenase treatment, whereas the reduction was 30.5% +/- 6.4 after mechanical defolliculation. The Na(+)-dependent uptake of 0.1 mM L-leucine was 18.6 +/- 4.6 pmol oocyte-1 40 min-1 in folliculated oocytes and 5.6 +/- 1.9 in collagenase defolliculated oocytes (means +/- SE). L-leucine uptake was not affected by the removal of the follicular layer if defolliculation occurred after the transport period; radiolabeled L-leucine is therefore not taken up into a compartment that is removed by the defolliculation process. The different L-leucine uptake rates observed in folliculated and defolliculated oocytes were not due to non-specific L-leucine binding to membranes. L-leucine kinetics showed that the L-leucine Vmax and Km values were lower in oocytes deprived of the follicular layer than in control oocytes enveloped in intact follicular layers. The Vmax and Km values of Na(+)-dependent L-leucine transport, calculated from data obtained the day after defolliculation by collagenase treatment, were: 16 +/- 1.5 pmol oocyte-1 40 min-1 and 57 +/- 21 mumol (mean +/- SD). The Na(+)-activation curve of 0.1 mM L-leucine was hyperbolic in folliculated oocytes and sigmoidal in defolliculated oocytes. The morphological analysis performed in parallel with the transport experiments showed that after defolliculation, the fibers forming the vitelline membrane tended to be arranged in a more regular orthogonal array, and the number of oocyte microvilli was reduced after collagenase treatment. Mechanical defolliculation did not appreciably affect the oocyte microvilli, however this procedure did not completely remove all follicle cells. The damage to collagenase treated oocytes was reversible, and the functional and structural features of most oocytes improved upon subsequent in vitro incubation. The recovery process seemed to involve protein synthesis in view of the increased value of L-leucine Vmax, and microscopic observation showing recovery of the microvillar apparatus.