FIGURE 9.

Chloride dependence of glycine-induced steady-state currents and glycine transport by wild-type GlyTs and mutant transporters. (A,B) Glycine-induced currents: the membrane voltage of oocytes expressing the indicated transporters was stepped from a holding potential of –60 mV to voltages between –150 to 30 mV in –20 mV increments. Currents in 100 mM NaCl recording solution (A) or in chloride-free solution (described in material and methods) (B) were subtracted from those in the same medium supplemented with 1 mM Gly. At each potential glycine-induced currents were averaged and normalized to those at –150 mV. (C,D) COS7 cells expressing the indicated transporters were assayed for glycine transport in the presence of increasing extracellular NaCl concentrations (isotonic substitution by sodium gluconate). Control glycine transport by wild-type GlyT2 and GlyT1 was 3.3 ± 0.3 and 2.6 ± 0.3 nmol glycine/mg protein/10 min, respectively. Representative experiments are shown repeated at least in three separated experiments performed in quadruplicate. Experimental data were fitted to the Michaelis–Menten equation.