Transport Metrics for Gluin Mutants
| Turnover rate (s−1) | H+ pumping (nmol/µg protein) | Vr, Cl− (mV) | Vr, pH (mV) | |
| Coupled transporters | ||||
| E (WT) | 2,200 ± 200 (3) | 23 ± 3 (7) | 38.0 ± 0.7 (4) | −58.0 ± 0.4 (4) |
| D | 1,700 ± 90 (4) | 22 ± 4 (6) | 37.8 ± 0.3 (4) | −57.0 ± 0.6 (4) |
| H | 1,700 ± 50 (4) | 22 ± 2 (8) | 40 ± 1 (11) | −51± 1 (11) |
| K | 700 ± 30 (4) | 7.5 ± 1 (7) | 44 ± 2 (6) | −34 ± 1 (6) |
| R | 300 ± 30 (4) | 3.8 ± 0.5 (6) | 44 ± 1 (3) | −18 ± 1 (3) |
| Uncoupled transporters | ||||
| S | 500 ± 30 (4) | 2.9 ± 0.5 (5) | 51.7 ± 0.8 (10) | |
| G | 330 ± 20 (4) | 2.5 ± 0.03 (3) | ||
| T | 160 ± 30 (4) | 1.7 ± 0.1 (3) | ||
| Q | 700 ± 40 (4) | 1.5 ± 0.2 (3) | ||
| A | 190 ± 10 (4) | 1.5 ± 0.1 (5) | 53.7 ± 0.9 (5) | 0.5 ± 0.2 (5) |
| C | 70 ± 3 (4) | 1.3 ± 0.03 (3) | 51.0 ± 1.4 (5) | 0.3 ± 0.7 (5) |
| F | 210 ± 10 (4) | 1.1 ± 0.03 (3) | ||
| Y | 140 ± 10 (3) | 0.8 ± 0.2 (4) | ||
| L | 70 ± 3 (4) | 0.4 ± 0.05 (4) | ||
| I | 50 ± 2 (4) | 0.3 ± 0.03 (3) | ||
| V | 40 ± 3 (4) | 0.3 ± 0.1 (4) | 54.0 ± 2.1 (3) | 0.0 ± 0.0 (3) |
| Non-natural side chains (see Fig. 5) | ||||
| E203C/Cys-less | 0.3 ± 0.1 (4) | |||
| Cys-1 | 5.7 ± 0.2 (3) | |||
| Cys-2 | 1.2 ± 0.1 (4) | |||
| Cys-3 | 3.3 ± 0.13 (3) | |||
| Cys-4 | 0.3 ± 0.1 (4) |
| Turnover rate (s−1) | H+ pumping (nmol/µg protein) | Vr, Cl− (mV) | Vr, pH (mV) | |
| Coupled transporters | ||||
| E (WT) | 2,200 ± 200 (3) | 23 ± 3 (7) | 38.0 ± 0.7 (4) | −58.0 ± 0.4 (4) |
| D | 1,700 ± 90 (4) | 22 ± 4 (6) | 37.8 ± 0.3 (4) | −57.0 ± 0.6 (4) |
| H | 1,700 ± 50 (4) | 22 ± 2 (8) | 40 ± 1 (11) | −51± 1 (11) |
| K | 700 ± 30 (4) | 7.5 ± 1 (7) | 44 ± 2 (6) | −34 ± 1 (6) |
| R | 300 ± 30 (4) | 3.8 ± 0.5 (6) | 44 ± 1 (3) | −18 ± 1 (3) |
| Uncoupled transporters | ||||
| S | 500 ± 30 (4) | 2.9 ± 0.5 (5) | 51.7 ± 0.8 (10) | |
| G | 330 ± 20 (4) | 2.5 ± 0.03 (3) | ||
| T | 160 ± 30 (4) | 1.7 ± 0.1 (3) | ||
| Q | 700 ± 40 (4) | 1.5 ± 0.2 (3) | ||
| A | 190 ± 10 (4) | 1.5 ± 0.1 (5) | 53.7 ± 0.9 (5) | 0.5 ± 0.2 (5) |
| C | 70 ± 3 (4) | 1.3 ± 0.03 (3) | 51.0 ± 1.4 (5) | 0.3 ± 0.7 (5) |
| F | 210 ± 10 (4) | 1.1 ± 0.03 (3) | ||
| Y | 140 ± 10 (3) | 0.8 ± 0.2 (4) | ||
| L | 70 ± 3 (4) | 0.4 ± 0.05 (4) | ||
| I | 50 ± 2 (4) | 0.3 ± 0.03 (3) | ||
| V | 40 ± 3 (4) | 0.3 ± 0.1 (4) | 54.0 ± 2.1 (3) | 0.0 ± 0.0 (3) |
| Non-natural side chains (see Fig. 5) | ||||
| E203C/Cys-less | 0.3 ± 0.1 (4) | |||
| Cys-1 | 5.7 ± 0.2 (3) | |||
| Cys-2 | 1.2 ± 0.1 (4) | |||
| Cys-3 | 3.3 ± 0.13 (3) | |||
| Cys-4 | 0.3 ± 0.1 (4) |
Three types of functional assays were quantified as described in Materials and methods on CLC-ec1 transporters with Gluin substituted by the indicated residues. Columns report: (1) Cl− turnover rate, (2) amplitude of H+ uptake after 30 s, (3) reversal potential in 10-fold Cl− gradient, and (4) reversal potential in 3-unit pH gradient. Values represent mean ± SE of the indicated number of separate reconstitutions, in some cases with separate protein preparations. For comparison, values for the uncoupled Gluex mutant E148A are (Accardi and Miller, 2004; Jayaram et al., 2008): turnover rate: 460 ± 10 s−1 ; Vr (H+): 0 ± 3 mV.