Article Figures & Data
Figures
- Fig. 1.
Transport cycle and membrane topology of glutamate transporters. Model of the glutamate translocation cycle (Kanner and Bendahan, 1982; Kavanaugh et al., 1997). The order of binding of the three sodium ions, glutamate, and the proton is not indicated, although it seems that at least one of the sodium ions binds before glutamate, and this is followed by the binding of one or two additional sodium ions (Watzke et al., 2001). After glutamate and the coions bind to the transporter (T) from the external medium (going clockwise), they are translocated and released into the inside of the cell. These steps represent half-cycle I. Then, potassium binds from the intracellular side and, after translocation to the outside, is released there. After completion of half-cycle II, a new translocation cycle can commence. The steps in this scheme are reversible, and therefore, elevated levels of extracellular potassium can cause the transporter to become inward-facing (going counterclockwise). If the interaction with potassium is abolished by mutation, half-cycle II is not operative, but the transporters can still exchange labeled glutamate (or aspartate), added to the outside with internal glutamate by reversible translocation via half-cycle I (A). The membrane topology shown is based on the high-resolution structure of GltPh (Yernool et al., 2004). TMs are indicated by Arabic numerals, and the two reentrant loops by HP1 and HP2. The gray regions, including the linkers between the structural elements (thick gray lines) are those analyzed in the accessibility studies reported here. HP1a and HP1b are the parts of HP1 closest to TMs 6 and 7, respectively. The residue numbers mark the boundaries of the regions analyzed. Some of the amino acid residues, critical for transport, are indicated together with the functional aspect affected upon mutation. Residues Asn396 and Asp398 play multiple roles in transport (Rosental et al., 2006; Tao et al., 2006). Glu404 is also believed to be important for the interaction with the cotransported proton (Grewer et al., 2003) (B).
- Fig. 2.
d-[3H]aspartate transport activity of single cysteine mutants in HP1. Transport of d-[3H]aspartate by the single cysteine mutants in HP1a (A) and HP1b, including the linker between HP1a and b (B), was measured as described under Materials and Methods, and the activity of the mutants is expressed as a percentage of activity of CL-GLT-1.
- Fig. 3.
Inhibition of d-[3H]aspartate transport in single cysteine mutants in HP1 by NEM. Inhibition of the transport activity of the single cysteine mutants after treatment by NEM in the presence of either sodium (□) or potassium (
) was performed as described under Materials and Methods and is given as a percentage of remaining activity of the same mutant, treated under the same conditions but without NEM. The concentration of NEM used was 0.5 mM, except for I350C (1 mM), A353C and T366C (0.15 mM), A364C (0.04 mM), G365C (0.2 mM), and L374C (0.25 mM). No significant inhibition by NEM in the presence of either sodium or potassium was observed with the following active mutants: F344C, S345C, F347C, A348C, F351C, Q352C, W354C, I355C, T356C, L358C, V369C, F371C, and R372C (A). In the case of S373C, the preincubation was done with the concentrations of NEM indicated on the abscissa (B). - Fig. 4.
Transport activity and inhibition by NEM in single cysteine mutants in TMs 7 and 8. The mutants were from the “cytoplasmic” parts of TMs 7 (TM 7a), including the linker between HP1b and TM 7a (positions 376–395) and 8 (positions 498–496), as indicated in Fig. 1B. Transport of d-[3H]aspartate by the single cysteine mutants was measured as described under Materials and Methods, and the activity of the mutants is expressed as a percentage of activity of CL-GLT-1 as in Fig. 2 (A). Inhibition of transport by NEM was done as described in the legend to Fig. 3. The concentration of NEM used was 0.5 mM except for L378C, V388C, A393C, T394C, I395C, and V492C (all at 0.3 mM), T385C (0.075 mM), L389C (0.2 mM), V391C (0.4 mM), A489C (1 mM), and G490C (0.15 mM) (B). No significant inhibition by NEM, in the presence of either sodium or potassium, was observed with the following active mutants: D376C, N377C, G379C, K382C, F387C, and H494C.
- Fig. 5.
Transport activity and inhibition by NEM in single cysteine mutants in TM 6. The mutants were from the “cytoplasmic” parts of TM 6, as indicated in Fig. 1B. The conditions were the same as described in the legends to Figs. 2 and 3. The concentration of NEM used was 0.5 mM for all single cysteine mutants.
- Fig. 6.
Effect of kainate on the inhibition of transport of single cysteine mutants by NEM. Transport by the indicated mutants was measured after pretreatment of the cells with the concentrations of NEM specified in the legends to Figs. 3 and 4 in sodium-containing medium in the presence and absence of 0.2 mM kainate.
- Fig. 7.
Effect of NEM and MTSET on transport by A364C and S440C. HeLa cells expressing A364C (A) and S440C (B) were pretreated in the presence of sodium or potassium with NEM and MTSET at the concentrations indicated. Then, transport of d-[3H]aspartate was measured.
- Fig. 8.
Inhibition of A364C/S440C and A412C/V427C by CuPh. HeLa cells expressing the double mutants A364C/S440C (A) and A412C/V427C (B) were pretreated with the indicated concentrations of CuPh in the presence of sodium or potassium as described under Materials and Methods. Then, transport of d-[3H]aspartate was measured.
