Table 4.
Rates of MTS inhibition
| Residue | Reagent | κ, M−1·s−1 (MTSEA Equivalent) |
|---|---|---|
| N78C | MTSEA (3 μM) | 1,000 |
| H83C | MTSEA (1 μM) | 4,000 |
| L87C | MTSEA (2 μM) | 10,000 |
| F101C | MTSET (40 μM) | 500 |
| Y290C | MTSET (25 μM) | 800 |
| S392C | MeMTS* | na |
| S393C | MeMTS* | na |
| F453C | MTSET (2 μM) | 2,000 |
| Q457C | MTSEA (0.3 μM) | 14,000 |
Labeling conditions and estimated methanethiosulfonate (MTS) reaction rates: for each reagent we determined the concentration that inhibited hSGLT1 Na+/glucose currents by 50% in 2 min in 100 mM NaCl buffer. Because 2-aminoethyl methanethiosulfonate (MTSEA) is inherently more reactive than 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET), we normalized the MTSET reaction rate to that of MTSEA, e.g., the concentration for ∼50% inhibition of H83C was determined for MTSEA (1 μM) and MTSET (5 μM) in Na buffer for 2 min: κ = 0.5/([MTS]·t). Since the rate was 5-fold higher for MTSEA than MTSET, the rates for MTSET were multiplied by 5 to normalize the results.
Neither MTSEA nor MTSET inhibited S392C or S393C, but 100 μM methyl methanethiosulfonate (MeMTS) inhibited 50%. Low functional expression of E102C, and L452C precluded estimation of rates, and neither MTSEA nor MTSES inhibited W291C. Note that tetramethylrhodamine-6-maleimide(TMR6M) partially inhibited E102C and L452C and TMR5M partially inhibited W291C and L452C.