Table 1.

Kinetic constants for d-aspartate and l-glutamate transport and inhibition by dihydrokainate

	d-Aspartate		l-Glutamate		DHK
	Km	Vmax	Km	Vmax	Ki
WT
Na	2.79  ± 0.71	987  ± 45	5.34  ± 1.3	802  ± 71	21.6  ± 2.4
Li	44.4  ± 4.53	215  ± 21.2	49  ± 1	125  ± 4.2	36  ± 9.4
S440G
Na	2.83  ± 0.55	171  ± 18.9	5.63  ± 0.6	107  ± 7	150  ± 19
Li	3.98  ± 0.25	126  ± 7.1	15  ± 1.7	118  ± 7	216  ± 10.6

Influx of d-[³H]aspartate and l-[³H]glutamate was measured for 1 min in proteoliposomes containing wild-type GLT-1 (WT) or S440G transporters in a final volume of 0.38 ml of 0.15 M NaCl- or LiCl-containing medium. In the NaCl containing medium, 4 μCi of d-[³H]aspartate (10.5 Ci/mmol) or l-[³H]glutamate (60 Ci/mmol) was present in each assay supplemented with 1, 2, 4, 6, and 10 μM unlabeled substrate. In the LiCl containing medium 6 μm, 6 μCi labeled substrate together with 2, 4, 6, 10, and 15 μM of the unlabeled amino acid were used or 10 μCi labeled and 20 and 40 μM unlabeled substrate. The K_i for DHK was determined by performing d-[³H]aspartate transport in the absence or presence of DHK. In sodium-containing medium DHK was present at 80 and 400 μM for wild-type GLT-1 and S440G, respectively. In lithium medium the concentrations were 40 and 200 μM, respectively. In the latter medium 5 μCi of d-[³H]aspartate was present together with 2, 4, 5, 10, 15, and 20 μM unlabeled d-aspartate. In the sodium medium 4 μCi labeled together with 1, 2, 4, 6, and 10 μM unlabeled substrate were used. Transport reactions were terminated after 1 min. K_m and K_i are expressed in μM and V_max is expressed as pmol/mg⋅protein/min. Determination ± SEM as done at least three times.