ATP pharmacology of alanine-substituted mutants
| n | /basal (pA/pF) | EC50 (μm) | pEC50 | nH | Ymax (pA/pF) | (Ymax//30μm) | Δ(ΔG) (kcal/mol) | |
|---|---|---|---|---|---|---|---|---|
| Mock | 4 | 1.96 ± 0.62 | 0 ± 0 | |||||
| WT | 26 | 0.75 ± 0.08 | 5.25 | 5.28 ± 0.07 | 1.76 ± 0.17 | 420 ± 36 | 1.15 ± 0.05 | |
| TM1 | ||||||||
| G30A | 7 | 0.81 ± 0.16 | 3.98 | 5.40 ± 0.06 | 1.64 ± 0.27 | 268 ± 43 | 1.05 ± 0.02 | 0.16 ± 0.08 |
| F31A | 7 | 0.60 ± 0.03 | 4.27 | 5.37 ± 0.03 | 1.88 ± 0.21 | 444 ± 36 | 1.04 ± 0.02 | 0.13 ± 0.04 |
| V32A | 6 | 0.75 ± 0.07 | 11.75 | 4.93 ± 0.07 | 2.00 ± 0.21 | 173 ± 14 | 1.26 ± 0.13 | −0.48 ± 0.10 |
| H33A | 7 | 0.93 ± 0.09 | 1.18 | 5.93 ± 0.07** | 2.21 ± 0.32 | 197 ± 48 | 0.98 ± 0.02 | 0.88 ± 0.10 |
| R34A | 6 | 0.78 ± 0.08 | 13.04 | 4.86 ± 0.04 | 2.00 ± 0.24 | 27 ± 12** | 1.49 ± 0.21 | −0.59 ± 0.06 |
| M35A | 7 | 0.83 ± 0.06 | 7.08 | 5.15 ± 0.09 | 1.45 ± 0.12 | 213 ± 62 | 1.19 ± 0.09 | −0.17 ± 0.12 |
| V36A | 6 | 0.86 ± 0.12 | 5.37 | 5.27 ± 0.06 | 1.64 ± 0.21 | 357 ± 71 | 1.08 ± 0.04 | 0.01 ± 0.08 |
| Q37A | 7 | 0.85 ± 0.15 | 33.11 | 4.48 ± 0.04** | 1.85 ± 0.13 | 203 ± 24 | 1.95 ± 0.21 | −1.08 ± 0.06 |
| L38A | 5 | 0.64 ± 0.03 | 5.50 | 5.26 ± 0.02 | 2.09 ± 0.24 | 298 ± 39 | 1.07 ± 0.04 | 0.03 ± 0.02 |
| L39A | 5 | 0.51 ± 0.04 | 6.03 | 5.22 ± 0.09 | 2.15 ± 0.18 | 311 ± 49 | 1.05 ± 0.04 | 0.08 ± 0.13 |
| I40A | 5 | 1.19 ± 0.09 | 0.96 | 6.02 ± 0.07** | 2.07 ± 0.23 | 209 ± 68 | 1.02 ± 0.02 | 1.01 ± 0.08 |
| L41A | 5 | 0.83 ± 0.09 | 15.14 | 4.82 ± 0.06* | 1.77 ± 0.07 | 218 ± 20 | 1.31 ± 0.08 | −0.63 ± 0.08 |
| L42A | 7 | 0.90 ± 0.05 | 3.89 | 5.41 ± 0.12 | 1.67 ± 0.16 | 384 ± 84 | 1.12 ± 0.07 | 0.17 ± 0.16 |
| Y43A | 9 | 39.54 ± 10.03** | 0.35 | 6.46 ± 0.19** | 1.37 ± 0.62 | 270 ± 52 | 1.01 ± 0.01 | 1.60 ± 0.26 |
| F44A | 9 | 48.99 ± 7.94** | 0.60 | 6.22 ± 0.20** | 2.06 ± 0.30 | 312 ± 17 | 0.99 ± 0.02 | 1.29 ± 0.28 |
| V45A | 6 | 1.02 ± 0.17 | nd | nd | nd | 4 ± 1** | nd | nd |
| W46A | 6 | 0.98 ± 0.14 | 7.76 | 5.11 ± 0.04 | 1.55 ± 0.17 | 399 ± 57 | 1.07 ± 0.05 | −0.22 ± 0.05 |
| Y47A | 5 | 1.23 ± 0.16 | nd | nd | nd | 2 ± 0** | nd | nd |
| V48A | 6 | 1.01 ± 0.06 | 3.98 | 5.40 ± 0.06 | 1.65 ± 0.13 | 218 ± 18 | 1.02 ± 0.01 | 0.16 ± 0.08 |
| F49A | 6 | 1.04 ± 0.11 | 5.88 | 5.23 ± 0.10 | 2.14 ± 0.36 | 272 ± 68 | 1.04 ± 0.02 | −0.07 ± 0.13 |
| I50A | 7 | 1.03 ± 0.03 | 27.54 | 4.56 ± 0.04** | 1.88 ± 0.15 | 225 ± 89 | 1.91 ± 0.19 | −0.98 ± 0.03 |
| V51A | 5 | 1.15 ± 0.14 | nd | nd | nd | 2 ± 0** | nd | nd |
| Q52A | 6 | 0.56 ± 0.14 | 10.72 | 4.97 ± 0.07 | 2.20 ± 0.43 | 275 ± 41 | 1.16 ± 0.02 | −0.42 ± 0.10 |
| K53A | 7 | 0.63 ± 0.09 | 8.91 | 5.05 ± 0.04 | 2.31 ± 0.20 | 206 ± 53 | 1.10 ± 0.04 | −0.32 ± 0.06 |
| S54A | 6 | 0.77 ± 0.12 | 8.32 | 5.08 ± 0.09 | 1.79 ± 0.31 | 206 ± 79 | 1.17 ± 0.07 | 0.27 ± 0.12 |
| TM2 | ||||||||
| I328A | 6 | 0.74 ± 0.11 | 0.62 | 6.21 ± 0.11** | 1.70 ± 0.22 | 272 ± 52 | 0.99 ± 0.02 | 1.26 ± 0.15 |
| P329A | 9 | 1.22 ± 0.11 | 1.70 | 5.77 ± 0.06** | 1.62 ± 0.29 | 416 ± 69 | 1.04 ± 0.02 | 0.70 ± 0.11 |
| T330A | 6 | 0.97 ± 0.11 | 3.55 | 5.45 ± 0.10 | 1.93 ± 0.30 | 286 ± 99 | 1.07 ± 0.05 | 0.23 ± 0.14 |
| I331A | 6 | 0.99 ± 0.10 | 4.57 | 5.34 ± 0.08 | 1.97 ± 0.25 | 305 ± 35 | 1.01 ± 0.02 | 0.08 ± 0.10 |
| I332A | 5 | 0.94 ± 0.09 | 3.55 | 5.45 ± 0.06 | 1.91 ± 0.18 | 245 ± 70 | 1.02 ± 0.02 | 0.23 ± 0.09 |
| N333A | 9 | 1.22 ± 0.06 | 1.07 | 5.97 ± 0.07** | 1.57 ± 0.10 | 339 ± 44 | 1.01 ± 0.01 | 0.94 ± 0.09 |
| L334A | 6 | 0.71 ± 0.07 | 4.37 | 5.36 ± 0.10 | 1.23 ± 0.10 | 363 ± 67 | 1.08 ± 0.05 | 0.11 ± 0.13 |
| T336A | 6 | 0.76 ± 0.08 | 5.62 | 5.25 ± 0.14 | 1.67 ± 0.15 | 222 ± 47 | 1.09 ± 0.05 | −0.05 ± 0.19 |
| L338A | 12 | 1.01 ± 0.08 | 27.54 | 4.56 ± 0.07** | 1.66 ± 0.22 | 283 ± 20 | 2.39 ± 0.30 | −0.98 ± 0.10 |
| T339A | 7 | 1.19 ± 0.10 | 1.55 | 5.81 ± 0.07** | 1.98 ± 0.21 | 370 ± 56 | 1.01 ± 0.01 | 0.72 ± 0.09 |
| S340A | 9 | 0.87 ± 0.06 | 19.50 | 4.71 ± 0.06** | 2.38 ± 0.15 | 211 ± 3 | 1.61 ± 0.14 | −0.78 ± 0.09 |
| I341A | 10 | 1.96 ± 0.13 | 7.59 | 5.12 ± 0.09 | 1.94 ± 0.07 | 229 ± 32 | 1.26 ± 0.13 | −0.22 ± 0.13 |
| G342A | 18 | 1.24 ± 0.19 | 26.30 | 4.59 ± 0.04** | 1.68 ± 0.12 | 289 ± 33 | 2.04 ± 0.15 | −0.95 ± 0.06 |
| V343A | 10 | 1.80 ± 0.09 | 0.66 | 6.18 ± 0.07** | 1.41 ± 0.13 | 301 ± 68 | 1.00 ± 0.01 | 1.23 ± 0.10 |
| G344A | 14 | 1.11 ± 0.17 | 66.07 | 4.18 ± 0.07** | 1.68 ± 0.14 | 251 ± 39 | 2.76 ± 0.26 | −1.50 ± 0.10 |
| S345A | 11 | 0.83 ± 0.16 | 15.49 | 4.81 ± 0.07** | 1.44 ± 0.13 | 236 ± 5 | 1.47 ± 0.09 | −0.64 ± 0.10 |
| F346A | 10 | 0.88 ± 0.12 | 6.46 | 5.19 ± 0.10 | 1.88 ± 0.12 | 397 ± 48 | 1.09 ± 0.04 | −0.12 ± 0.14 |
| L347A | 6 | 1.01 ± 0.03 | 12.02 | 4.92 ± 0.08 | 1.79 ± 0.16 | 217 ± 48 | 1.31 ± 0.09 | −0.49 ± 0.10 |
| C348A | 7 | 1.02 ± 0.09 | 10.72 | 4.97 ± 0.08 | 1.72 ± 0.20 | 457 ± 28 | 1.25 ± 0.15 | −0.43 ± 0.10 |
| D349A | 5 | 1.21 ± 0.14 | nd | nd | nd | 3 ± 1** | nd | nd |
| W350A | 8 | 0.87 ± 0.07 | 8.51 | 5.07 ± 0.09 | 1.81 ± 0.12 | 278 ± 35 | 1.18 ± 0.09 | −0.29 ± 0.12 |
| I351A | 6 | 1.03 ± 0.13 | 9.77 | 5.01 ± 0.06 | 2.21 ± 0.32 | 246 ± 49 | 1.13 ± 0.06 | −0.37 ± 0.08 |
| L352A | 9 | 0.85 ± 0.10 | 8.32 | 5.08 ± 0.05 | 2.11 ± 0.07 | 276 ± 38 | 1.11 ± 0.03 | −0.27 ± 0.07 |
| L353A | 6 | 1.12 ± 0.14 | 9.12 | 5.04 ± 0.08 | 2.03 ± 0.16 | 223 ± 39 | 1.08 ± 0.04 | −0.32 ± 0.11 |
Data from experiments like those shown in Figure 1./basal is the holding current measured in the absence of agonist; all other parameters are defined in Results./basal and Ymax of Y43A and F44A were determined in one set of experiments, and EC50, nH, and Ymax//30 μm were determined in another, also as described in Results. Mock, Cells expressing only GFP; n, the number of times that complete concentration-response curves were generated in single cells; nd, not determined. We did not test for differences in Ymax//30μm or Δ(ΔG). The average cell capacitance was 14.6 ± 0.5 pF (n = 100).
*p < 0.05; **p < 0.01.