Wild-Type and Mutant GABA Concentration–Response Relationships
| E Mutants . | . | . | . | K Mutants . | . | . | . | Neutral Mutants . | . | . | . | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Mutant . | EC50 . | Hill slope . | n . | Mutant . | EC50 . | Hill slope . | n . | Mutant . | EC50 . | Hill slope . | n . | |||||||||
| ρ1 | 0.8 ± 0 | 3.4 ± 0.1 | 8 | |||||||||||||||||
| V280E (−12′) | 4.7 ± 0.5 | 1.1 ± 0.1 | 5 | |||||||||||||||||
| S281E (−11′) | 1.0 ± 0.1 | 1.6 ± 0.1 | 5 | |||||||||||||||||
| F282E (−10′) | NFE | 30 | ||||||||||||||||||
| W283E (−9′) | NFE | 30 | ||||||||||||||||||
| I284E (−8′) | 2.8 ± 0.2 | 2.0 ± 0.1 | 5 | |||||||||||||||||
| D285E (−7′) | 1.0 ± 0.1 | 2.3 ± 0.3 | 5 | D285N | 1.8 ± 0.4 | 1.8 ± 0.2 | 6 | |||||||||||||
| R286E (−6′) | 0.3 ± 0.0 | 2.5 ± 0.3 | 5 | R286M | 0.6 ± 0.1 | 2.9 ± 0.1 | 6 | |||||||||||||
| R287E (−5′) | 1.0 ± 0.1 | 3.0 ± 0.3 | 4 | R287K | 24.0 ± 2.6 | 2.4 ± 0.8 | 5 | R286Q/R287Q | 0.4 ± 0.03 | 2.8 ± 0.5 | 6 | |||||||||
| A288D (−4′) | 1.5 ± 0.2 | 2.4 ± 0.2 | 5 | A288K | 2.3 ± 0.4 | 2.3 ± 0.3 | 5 | A288G | 2.6 ± 0.3 | 1.4 ± 0.1 | 5 | |||||||||
| V289E (−3′) | 1.3 ± 0.1 | 2.1 ± 0.1 | 5 | V289K | 4.4 ± 0.5 | 1.1 ± 0.1 | 5 | V289A | 4.8 ± 0.3 | 2.1 ± 0.1 | 5 | |||||||||
| P290E (−2′) | 4.1 ± 0.3 | 1.8 ± 0.2 | 6 | P290K | 19.4 ± 1.9 | 1.4 ± 0.3 | 5 | P290A | 2.8 ± 0.1 | 1.1 ± 0.1 | 6 | |||||||||
| A291E (−1′) | 2.0 ± 0.1 | 1.9 ± 0.1 | 5 | A291K | 2.0 ± 0.1 | 1.9 ± 0.1 | 5 | A291G | 1.6 ± 0.2 | 2.7 ± 0.2 | 5 | |||||||||
| R292E (0′) | 1.3 ± 0.1 | 1.4 ± 0.2 | 5 | R292K | 11.9 ± 1.3 | 0.7 ± 0.1 | 5 | R292M | 1.8 ± 0.1 | 1.0 ± 0.1 | 6 | |||||||||
| V293E (1′) | 3.9 ± 0.3 | 1.4 ± 0.1 | 5 | V293K | 0.6 ± 0.1 | 1.3 ± 0.1 | 5 | V293A | 2.3 ± 0.2 | 2.1 ± 0.2 | 5 | |||||||||
| P294E (2′) | 3.3 ± 0.2 | 1.2 ± 0.1 | 5 | P294K | 0.07 ± 0.01 | 1.0 ± 0.1 | 5 | P294A | 2.4 ± 0.6 | 2.0 ± 0.3 | 5 | |||||||||
| L295E (3′) | 1.6 ± 0.2 | 1.9 ± 0.4 | 5 | L295A | 1.6 ± 0.2 | 1.6 ± 0.2 | 5 | |||||||||||||
| G296E (4′) | 1.4 ± 0.1 | 2.1 ± 0.2 | 5 | G296K | NFE | 30 | G296A | 2.4 ± 0.1 | 1.6 ± 0.2 | 5 | ||||||||||
| I297E (5′) | 4.7 ± 0.2 | 1.0 ± 0.1 | 5 | I297K | NFE | 30 | I297A | 1.5 ± 0.1 | 2.1 ± 0.1 | 5 | ||||||||||
| T298E (6′) | NFE | 30 | T298K | NFE | 30 | T298A | 1.4 ± 0.2 | 1.9 ± 0.2 | 5 | |||||||||||
| T299E (7′) | 1.2 ± 0.1 | 1.8 ± 0.2 | 5 | T299K | NFE | 30 | T299A | 2.2 ± 0.2 | 1.7 ± 0.1 | 5 | ||||||||||
| V300E (8′) | NFE | 30 | ||||||||||||||||||
| L301E (9′) | 2.1 ± 0.4 | 1.5 ± 0.4 | 5 | L301K | HL | 30 | ||||||||||||||
| T302E (10′) | HL | 30 | T302K | HL | 30 | |||||||||||||||
| M303E (11′) | 0.7 ± 0.1 | 2.4 ± 0.2 | 5 | |||||||||||||||||
| S304E (12′) | 1.2 ± 0.1 | 2.3 ± 0.2 | 5 | S304A | 0.9 ± 0.1 | 2.7 ± 0.3 | 5 | |||||||||||||
| T305E (13′) | 0.06 ± 0.008 | 2.7 ± 0.3 | 5 | T305A | 0.2 ± 0.05 | 2.7 ± 0.3 | 5 | |||||||||||||
| I306E (14′) | NFE | I306A | 0.3 ± 0.1 | 2.3 ± 0.2 | 5 | |||||||||||||||
| I307E (15′) | NFE | I307A | 0.1 ± 0.01 | 1.4 ± 0.2 | 5 | |||||||||||||||
| T308E (16′) | 0.7 ± 0.1 | 3.1 ± 0.1 | 5 | |||||||||||||||||
| G309E (17′) | 0.02 ± 0.003 | 1.46 ± 0.1 | 5 | |||||||||||||||||
| V310E (18′) | 0.06 ± 0.01 | 1.84 ± 0.1 | 5 | V310A | 1.9 ± 0.1 | 2.3 ± 0.1 | 5 | |||||||||||||
| N311E (19′) | 0.6 ± 0.05 | 2.3 ± 0.3 | 5 | N311A | 0.7 ± 0.1 | 2.8 ± 0.3 | 5 | |||||||||||||
| A312E (20′) | 0.2 ± 0.01 | 3.1 ± 0.3 | 5 | D285N/R286Q/ R287Q/R292M | 1.6 ± 0.2 | 2.0 ± 0.2 | 6 | |||||||||||||
| E Mutants . | . | . | . | K Mutants . | . | . | . | Neutral Mutants . | . | . | . | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Mutant . | EC50 . | Hill slope . | n . | Mutant . | EC50 . | Hill slope . | n . | Mutant . | EC50 . | Hill slope . | n . | |||||||||
| ρ1 | 0.8 ± 0 | 3.4 ± 0.1 | 8 | |||||||||||||||||
| V280E (−12′) | 4.7 ± 0.5 | 1.1 ± 0.1 | 5 | |||||||||||||||||
| S281E (−11′) | 1.0 ± 0.1 | 1.6 ± 0.1 | 5 | |||||||||||||||||
| F282E (−10′) | NFE | 30 | ||||||||||||||||||
| W283E (−9′) | NFE | 30 | ||||||||||||||||||
| I284E (−8′) | 2.8 ± 0.2 | 2.0 ± 0.1 | 5 | |||||||||||||||||
| D285E (−7′) | 1.0 ± 0.1 | 2.3 ± 0.3 | 5 | D285N | 1.8 ± 0.4 | 1.8 ± 0.2 | 6 | |||||||||||||
| R286E (−6′) | 0.3 ± 0.0 | 2.5 ± 0.3 | 5 | R286M | 0.6 ± 0.1 | 2.9 ± 0.1 | 6 | |||||||||||||
| R287E (−5′) | 1.0 ± 0.1 | 3.0 ± 0.3 | 4 | R287K | 24.0 ± 2.6 | 2.4 ± 0.8 | 5 | R286Q/R287Q | 0.4 ± 0.03 | 2.8 ± 0.5 | 6 | |||||||||
| A288D (−4′) | 1.5 ± 0.2 | 2.4 ± 0.2 | 5 | A288K | 2.3 ± 0.4 | 2.3 ± 0.3 | 5 | A288G | 2.6 ± 0.3 | 1.4 ± 0.1 | 5 | |||||||||
| V289E (−3′) | 1.3 ± 0.1 | 2.1 ± 0.1 | 5 | V289K | 4.4 ± 0.5 | 1.1 ± 0.1 | 5 | V289A | 4.8 ± 0.3 | 2.1 ± 0.1 | 5 | |||||||||
| P290E (−2′) | 4.1 ± 0.3 | 1.8 ± 0.2 | 6 | P290K | 19.4 ± 1.9 | 1.4 ± 0.3 | 5 | P290A | 2.8 ± 0.1 | 1.1 ± 0.1 | 6 | |||||||||
| A291E (−1′) | 2.0 ± 0.1 | 1.9 ± 0.1 | 5 | A291K | 2.0 ± 0.1 | 1.9 ± 0.1 | 5 | A291G | 1.6 ± 0.2 | 2.7 ± 0.2 | 5 | |||||||||
| R292E (0′) | 1.3 ± 0.1 | 1.4 ± 0.2 | 5 | R292K | 11.9 ± 1.3 | 0.7 ± 0.1 | 5 | R292M | 1.8 ± 0.1 | 1.0 ± 0.1 | 6 | |||||||||
| V293E (1′) | 3.9 ± 0.3 | 1.4 ± 0.1 | 5 | V293K | 0.6 ± 0.1 | 1.3 ± 0.1 | 5 | V293A | 2.3 ± 0.2 | 2.1 ± 0.2 | 5 | |||||||||
| P294E (2′) | 3.3 ± 0.2 | 1.2 ± 0.1 | 5 | P294K | 0.07 ± 0.01 | 1.0 ± 0.1 | 5 | P294A | 2.4 ± 0.6 | 2.0 ± 0.3 | 5 | |||||||||
| L295E (3′) | 1.6 ± 0.2 | 1.9 ± 0.4 | 5 | L295A | 1.6 ± 0.2 | 1.6 ± 0.2 | 5 | |||||||||||||
| G296E (4′) | 1.4 ± 0.1 | 2.1 ± 0.2 | 5 | G296K | NFE | 30 | G296A | 2.4 ± 0.1 | 1.6 ± 0.2 | 5 | ||||||||||
| I297E (5′) | 4.7 ± 0.2 | 1.0 ± 0.1 | 5 | I297K | NFE | 30 | I297A | 1.5 ± 0.1 | 2.1 ± 0.1 | 5 | ||||||||||
| T298E (6′) | NFE | 30 | T298K | NFE | 30 | T298A | 1.4 ± 0.2 | 1.9 ± 0.2 | 5 | |||||||||||
| T299E (7′) | 1.2 ± 0.1 | 1.8 ± 0.2 | 5 | T299K | NFE | 30 | T299A | 2.2 ± 0.2 | 1.7 ± 0.1 | 5 | ||||||||||
| V300E (8′) | NFE | 30 | ||||||||||||||||||
| L301E (9′) | 2.1 ± 0.4 | 1.5 ± 0.4 | 5 | L301K | HL | 30 | ||||||||||||||
| T302E (10′) | HL | 30 | T302K | HL | 30 | |||||||||||||||
| M303E (11′) | 0.7 ± 0.1 | 2.4 ± 0.2 | 5 | |||||||||||||||||
| S304E (12′) | 1.2 ± 0.1 | 2.3 ± 0.2 | 5 | S304A | 0.9 ± 0.1 | 2.7 ± 0.3 | 5 | |||||||||||||
| T305E (13′) | 0.06 ± 0.008 | 2.7 ± 0.3 | 5 | T305A | 0.2 ± 0.05 | 2.7 ± 0.3 | 5 | |||||||||||||
| I306E (14′) | NFE | I306A | 0.3 ± 0.1 | 2.3 ± 0.2 | 5 | |||||||||||||||
| I307E (15′) | NFE | I307A | 0.1 ± 0.01 | 1.4 ± 0.2 | 5 | |||||||||||||||
| T308E (16′) | 0.7 ± 0.1 | 3.1 ± 0.1 | 5 | |||||||||||||||||
| G309E (17′) | 0.02 ± 0.003 | 1.46 ± 0.1 | 5 | |||||||||||||||||
| V310E (18′) | 0.06 ± 0.01 | 1.84 ± 0.1 | 5 | V310A | 1.9 ± 0.1 | 2.3 ± 0.1 | 5 | |||||||||||||
| N311E (19′) | 0.6 ± 0.05 | 2.3 ± 0.3 | 5 | N311A | 0.7 ± 0.1 | 2.8 ± 0.3 | 5 | |||||||||||||
| A312E (20′) | 0.2 ± 0.01 | 3.1 ± 0.3 | 5 | D285N/R286Q/ R287Q/R292M | 1.6 ± 0.2 | 2.0 ± 0.2 | 6 | |||||||||||||
Values are mean ± SEM with n equal to the number of oocytes tested. Note that most charge neutralization or glutamate substitutions had little effect on the agonist dose–response curve, while some lysine mutations resulted in modest increases in the GABA EC50. Constructs that failed to exhibit currents upon application of GABA are indicated by NFE (no functional expression). Constructs that exhibited small (<100 nA) currents with a concomitant high background (>1 μA) are labeled HL (high leak). The glutamate substitution mutant A288E was nonfunctional; results from an aspartate substitution mutant (A288D) are reported for this position.