Insect Biochemistry and Molecular Biology 30 2000 369–376 www.elsevier.comlocateibmb
Substrate-stereoselectivity of a high-affinity glutamate transporter cloned from the CNS of the cockroach Diploptera punctata
C. Donly
a,
, J. Jevnikar
a
, H. McLean
b
, S. Caveney
b
a
Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford St., London, Ontario, Canada N5V 4T3
b
Department of Zoology, University of Western Ontario, London, Ontario, Canada N6A 5B7 Received 30 August 1999; received in revised form 17 December 1999; accepted 21 December 1999
Abstract
A cDNA encoding a Na
+
-dependent glutamate transporter has been cloned from the brain of the cockroach Diploptera punctata. The cDNA encodes a transporter protein of 481 amino acids, designated DipEAAT1, which when expressed in baculovirus infected
insect cells, resulted in a 40–50 fold increase in [
3
H]L-glutamate uptake. DipEAAT1 mRNA is expressed in the brain, as is the RNA encoding TrnEAAT1, a related transporter recently isolated from the caterpillar Trichoplusia ni. The affinity of these transporters for
L-glutamate and several structural analogues was compared. Both have a high affinity for L-glutamate, their presumed primary substrate, but quite different affinities for D-aspartate. TrnEAAT1 was found to be similar to other glutamate transporters in that
its ability to transport [
3
H]L-glutamate into cells was inhibited strongly by D- and L- isomers of aspartate and its analogues. DipEAAT1, by contrast, was inhibited weakly by all D- isomers tested. The affinity of DipEAAT1 for [
3
H]D-aspartate was found to be an order of magnitude lower than that of TrnEAAT1, revealing an unusual stereoselectivity for aspartate substrates by the
cockroach transporter. The activity of DipEAAT1 was also unaffected by the presence of Zn
++
in the bathing solution, despite the presence of a putative Zn
++
-binding motif conferring Zn
++
-sensitivity on some mammalian glutamate transporters.
2000 Elsevier Science Ltd. All rights reserved.
Keywords: Glutamate transporter; Diploptera punctata; Cockroach; Cloning; Substrate-stereoselectivity
1. Introduction
D-aspartate is a high-affinity transport substrate used to characterize the Na
+
-dependent uptake systems for the excitatory amino acid L-glutamate in the mammalian
brain Balcar and Johnston, 1972; Robinson et al., 1993 and non-neuronal tissues Schneider et al., 1980; Balcar,
1992. D-aspartate competes equally with its naturally occurring L-isomer in blocking L-glutamate uptake in
rat brain slices and synaptosomal preparations. D-gluta- mate, on the other hand, does not inhibit L-glutamate
uptake by neuronal tissues Balcar and Johnston, 1972; Robinson et al., 1993. Gazzola et al. 1981 commented
on this stereoselective anomaly in a study of Na
+
-depen- dent dicarboxylic amino acid transport in human skin
Corresponding author. Tel.: +
1-519-457-1470; fax: +
1-519-457- 3997.
E-mail address: donlycem.agr.ca C. Donly.
0965-174800 - see front matter
2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 0 0 0 0 0 0 4 - 7
fibroblasts. As in the mammalian CNS, this transport system was competitively inhibited by both L- and D-
isomers of aspartate, but was selectively stereospecific and not blocked by the D-isomers of glutamate and cys-
teate. In insect tissues, too, D-aspartate but not D- glutamate competes strongly with L-glutamate for both
high-affinity Bermudez et al., 1988; McLean and Cav- eney, 1993; Caveney et al., 1996 and low-affinity Na
+
- dependent uptake Kingan and Hishinuma, 1987.
D-aspartate has been used recently in the functional analysis of several excitatory amino acid transporters
EAATs cloned from mammalian rat, Klockner et al., 1994; rabbit, Kanai and Hediger, 1992; human, reviewed
in Vandenberg, 1998 and insect tissues caterpillar, Donly et al., 1997; fruitfly, Seal et al., 1998. [
3
H]D- aspartate has been used in the in vitro characterization
of three human Na
+
-dependent glutamate transporters EAATs 1–3, Arriza et al., 1994. Unlike [
3
H]L-gluta- mate, [
3
H]D-aspartate is not metabolized after its accumulation by mammalian cells, a property that facili-
tates the kinetic studies of transporters.
370 C. Donly et al. Insect Biochemistry and Molecular Biology 30 2000 369–376
In insects, a glutamate transporter cDNA isolated and cloned from the head of the caterpillar Trichoplusia ni,
TrnEAAT1, was also shown to accept both L- and D- enantiomers of aspartate as transport substrates Donly
et al., 1997, while a glutamate transporter isolated from Drosophila
embryos, dEAAT1, discriminates only weakly between these two substrates Seal et al., 1998.
The situation, however, is quite different for a trans- porter cDNA isolated from the brain of the cockroach,
Diploptera punctata . We report here that functional
characterization of
this cockroach
transporter, DipEAAT1, in a baculovirus-cell expression system
revealed that this transporter is strongly stereoselective. [
3
H]D-aspartate is not a high affinity transport substrate for this transporter, nor are D-aspartate or threo-3-hyd-
roxy-D-aspartate potent blockers of L-glutamate uptake.
Fig. 1. Amino acid sequence comparison of glutamate transporters cloned from insects. Residues identical in all 3 sequences are shaded in black,
while positions having 2 out of 3 matches are shaded grey. Bars are drawn over regions of sequence hydrophobicity that may correspond to transmembrane structures. Two histidine residues in the DipEAAT1 sequence analogous to a putative Zn
++
binding site are boxed. The sequence of TrnEAAT1 is from Donly et al. 1997 and the sequence of DrmEAAT1 is from Seal et al. 1998. The sequence of the DipEAAT1 cDNA
reported here has been deposited in the GenBankEMBL database under the accession AF208521.
2. Methods
