Table 2.

Characteristics of Glyt1b background constructs

Glyt1b	KM (µM)	Vmax (pmol/min/mg)	Cell surface expression (% of WT)	Rate of binding inactivation by MTSEA, M−1 ⋅ sec−1	Rate of binding inactivation by MTSET, M−1 · sec−1	Rate of transport inactivation by MTSET, M−1 · sec−1
WT	35.2 ± 2.1	1560 ± 93	100	3.29 ± 0.09	1.79 ± 0.07	0.61 ± 0.03
C62A	36.4 ± 2.6	1466 ± 81	89.3 ± 5.3	0.40 ± 0.01	0.12 ± 0.01	0.02 ± 0.01
Y60C-C62A	41 ± 4	715 ± 50**	65.3 ± 4.7*	nd	nd	6.52 ± 0.42
S244C-C62A	35.2 ± 2.8	645 ± 18**	50.7 ± 5.9*	32.10 ± 1.49	20.8 ± 0.70	nd

Transport rates were measured over a range (0.05 to 200 µM) of glycine concentrations, and surface expression was determined by biotinylation as described in Materials and Methods. K_M and V_max values represent the means ± SEM of three experiments for kinetic analysis and four experiments for biotinylation. Sensitivity to MTSEA and MTSET modification was determined from the concentration of methanethiosulfonate reagents and the half-time for inactivation of CHIBA-3007 binding (S244C-C62A) or glycine transport (Y60C-C62A) as described in Materials and Methods. Asterisks indicate significant differences from WT (*P < 0.01, **P < 0.005) using Student’s t test. nd, not determined.