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Functional characterization of a glutamate/aspartate transporter from the mosquito Aedes aegypti

Anita Umesh^*,1,2, Bruce N. Cohen³, Linda S. Ross² and Sarjeet S. Gill^1,2,

¹ Environmental Toxicology Graduate Program
² Department of Cell Biology and Neuroscience, University of California, Riverside, Riverside, CA 92521, USA
³ Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA

View larger version (79K): [in a new window]   Fig. 1. Amino acid sequence analysis of AeaEAAT. (A) Amino acid (aa) sequence alignment of AeaEAAT and other insect glutamate transporters cloned and characterized to date. The deduced aa sequence of AeaEAAT gave a 481-aa residue protein with six clear N-terminal regions of hydrophobicity (overlined), possibly correlating to transmembrane domains, having one putative N-linked glycosylation site (asterisk). (B) Sequence distances, derived from ClustalW based alignment, of the insect and human EAAT superfamily, including the ASC transporters. Of the functionally characterized insect EAATs, AeaEAAT has most identity to DipEAAT (55.3%), and amongst the human counterparts has highest identity to hEAAT3 (47%). Included are putative EAATs from the completed genome of Anopheles gambiae (GenBank accession numbers: AAB01008807; AAB01008797, AAB01008964), the first of which has 74.9% identity to AeaEAAT. AeaEAAT, Aedes aegypti; AmEAAT, Apis mellifera; DipEAAT, Diploptera punctata; dEAAT1 and dEAAT2, Drosophila melanogaster; TrnEAAT, Trichoplusia ni; hEAAT1-5, human; hASCT, human alanine serine cysteine transporter.

View larger version (59K): [in a new window]   Fig. 2. In vitro transcription/translation of AeaEAAT and western analysis. In vitro transcription and translation of the AeaEAAT cDNA produces a translation product that migrates at approximately 40 kDa on an 8% SDS-PAGE gel (lane 1). AeaEAAT is expressed in both the head of the adult Ae. aegypti (lane 2), as well as in Xenopus laevis oocytes injected with the cRNA encoding AeaEAAT as a protein (lane 4) with mobility of 55 kDa. Membranes prepared from the whole head of adult Ae. aegypti and from Xenopus laevis oocytes expressing AeaEAAT were separated by 8% SDS-PAGE, transferred to Immobilon P (Millipore), and subjected to western analysis, using the affinity-purified -AeaEAT antibody. Preincubation of the primary antibody with the antigenic peptide abolished any signal. Lanes 2 and 5, adult Ae. aegypti head membranes; lanes 3 and 6, Xenopus laevis oocytes injected with cRNA of a Drosophila sodium channel; lanes 4 and 7, X. laevis oocytes injected with cRNA of pBSAMVBC10. Note that lanes 5–7 are probed with the primary antibody upon preadsorption with the antigenic peptide. The positions of molecular mass (kDa) proteins are shown.

View larger version (17K): [in a new window]   Fig. 3. Ion dependence of substrate transport. HeLa cells were transfected either with pBSAMVBC10 carrying the open reading frame of AeaEAAT or with no DNA (–, control), and were assayed for the transport of its substrates, L-glutamate, L-aspartate and D-aspartate, 24 h post infection/transfection. Both A and B are the result of one representative experiment, which was repeated three times and shown to give the same result qualitatively each time. (A) Sodium dependence of substrate transport was assessed by substituting equimolar choline chloride for NaCl in the transport assay buffer (striped bars). (B) Anion dependence of substrate transport was determined by replacing chloride ions in the transport assay buffer either with equimolar concentrations of acetate (gray bars) or gluconate (hatched bars).

View larger version (27K): [in a new window]   Fig. 4. Amino acid inhibition of D-aspartate transport. The ability of the 20 amino acids (each at 5 mmol l-1) to compete the transport of D-aspartate (as a mixture of 1 µmol l-1 cold and 30 nmol l-1 3H-D-aspartate) was examined. Values are a mean of triplicate experiments, plotted as a percentage of D-aspartate transport (fmol cell-1 h-1) in the absence of any competing amino acid. Asterisks indicate significant inhibition at the 99% confidence interval. Note that the results for tyrosine (Y) and tryptophan (W) also appeared to give significant inhibition, but are not indicated by asterisks due to the acidic conditions required for dissolving these amino acids at 5 mmol l-1, which skewed the results. Amino acids are indicated by the single letter code. –, control (no competing amino acid).

View larger version (18K): [in a new window]   Fig. 5. Substrate-induced currents. (A) Inward currents induced by 100 µmol l-1 D-aspartate onto Xenopus oocytes expressing AeaEAAT for the duration indicated by the black bar. A holding potential of -60 mV was used. Similar inward currents were produced by application of L-Glu, L-Asp and L-Cys (data not shown). (B) Reversal of current upon exposing AeaEAAT-expressing oocytes to a sodium-free buffer containing a high potassium concentration (98 mmol l-1), at a holding potential of -60 mV. This represents the transport of endogenous excitatory amino acids out of the oocyte, and was not observed in water-injected oocytes (data not shown). (C) AeaEAAT-mediated substrate-activated currents. These current–voltage relationships are the difference between substrate-activated currents and currents in the buffer alone, obtained by off-line subtraction of the steady state portion of voltage-jump curves. Reversal potentials for the four amino acids (L-Glu, L-and D-Asp and L-Cys) applied at 100 µmol l-1 each are approximately +37 mV. (D) Sodium dependence of substrate-activated currents. Replacement of extracellular sodium with equimolar choline completely abolishes the D-aspartate (100 µmol l-1)-activated current.

View larger version (24K): [in a new window]   Fig. 6. Effect of CL- on substrate-induced transporter-mediated currents. (A) D-Aspartate (100 µmol l-1) activated AeaEAAT currents in ND96 (squares) and various chloride-substituted buffers: gluconate (circles), nitrate (triangles) and thiocyanate (inverted triangles). (B) As A, but with currents in the thiocyanate buffer omitted, so as to expand the y-axis. (C) Substrate-induced current-voltage relationships upon dialysis of intracellular CL- from AeaEAAT-expressing oocytes, by incubation in CL--free saline substituted by gluconate at least 24 h prior to recordings. In the absence of intracellular CL-, the presence of extracellular CL- (squares) causes outward current. This outward current is absent when extracellular CL- is replaced by gluconate (circles). In all cases, currents were recorded from potentials between -60 and +50 mV in 10 mV intervals. (D) Radiolabeled ligand transport by oocytes under voltage clamp (-60 mV), in ND96 buffer, gluconate-substituted buffer (Gluconate), and dialysed oocytes in gluconate-substituted buffer (Dialysed).

View larger version (47K): [in a new window]   Fig. 7. Western analysis of adult Ae. aegypti. Membrane and cytosolic fractions separated from head (lanes 1, 2, 7, 8), thorax (lanes 3, 4, 9, 10) and abdomen (Abd., lanes 5, 6, 11, 12) of adult Ae. aegypti were probed with affinity-purified -AeaEAAT antibody (lanes 1–6) or with the -AeaEAAT antibody that had been preadsorbed with the antigenic peptide (10 µmol l-1) (lanes 7–12). Odd-numbered lanes correspond to membrane fractions, and even-numbered lanes to cytosolic fractions. The -AeaEAAT specific band migrates at a molecular mass of 50–52 kDa (arrowheads in lanes 1, 3, 5). Arrowheads in lanes 7, 9 and 11 indicate the missing band upon peptide preadsorption. The positions of molecular mass (kDa) proteins are shown.

View larger version (120K): [in a new window]   Fig. 8. Immunohistochemistry of Ae. aegypti adult thorax, embedded in paraffin and sectioned. (A) Samples treated with -AeaEAAT antibody preadsorbed with the antigenic peptide (50 µmol l-1); (B) samples treated with -AeaEAAT antibody, showing specific staining in the neuropile of the thoracic ganglia. (C) Staining in the thoracic ganglia (boxed areas in A and B) shown at a higher magnification. Arrowheads indicate examples of stained cell bodies. Scale bars, 100 µm.

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