TABLE 1.
Substrate kinetic parameters for select TM1 cysteine mutants
COS-1 cells transiently transfected as described under “Experimental Procedures.” Uptake measured at substrate concentrations ranging from 0–200 μm for TCA, or 0–200 mm for NaCl (measured in triplicate). Kinetic data was analyzed using Graphpad Prism software, with constants determined via nonlinear regression, and one-way ANOVA for significance.
| Na+ kinetics |
TCA kinetics |
|||
|---|---|---|---|---|
| Jmax | KNa | Jmax | KTCA | |
| pmol/min/mg protein | mm | pmol/min/mg protein | μm | |
| C270A | 380.3 ± 16.8 | 8.9 ± 1.6 | 892.8 ± 57.7 | 9.7 ± 2.4 |
| N27Cab | 934 ± 8.2 | 34.0 ± 8.3e | 312.5 ± 28.7 | 21.5 ± 6.2 |
| N28C | 347.7 ± 25.1 | 5.5 ± 2.0 | 1469.0 ± 55.6 | 17.7 ± 2.2 |
| L30Cabc | 198.1 ± 8.2 | 19.5 ± 2.6e | 1047.0 ± 183.5 | 59.8 ± 25.0e |
| L34Cacd | 178.4 ± 6.9 | 7.5 ± 1.2 | 592.7 ± 23.3 | 11.6 ± 1.7 |
| S35Cac | 270.3 ± 9.4 | 11.3 ± 1.5 | 389.5 ± 35.6 | 5.7 ± 2.6 |
| T36Cacd | 336.5 ± 16.7 | 9.9 ± 1.9 | 801.8 ± 37.1 | 8.6 ± 1.6 |
| L38Cbcd | 716.6 ± 54.7 | 24.1 ± 5.6e | 1507.0 ± 32.2e | 10.6 ± 0.9 |
| T39Cbc | 406.9 ± 17.5 | 16.4 ± 2.4e | 553.5 ± 70.0 | 6.3 ± 3.6 |
| M46Cab | 164.2 ± 8.4 | 1.3 ± 0.8e | 239.2 ± 25.2 | 8.4 ± 3.3 |
| F47C | 297.6 ± 13.0 | 12.8 ± 2.0 | 453.8 ± 64.6 | 6.5 ± 4.0 |
a Decreased uptake due to mutation.
b Sodium sensitive.
c MTSET inhibition.
d Substrate protection.
e Parameters significantly different from the C270A control are indicated, *p ≤ 0.05.