| Titrant . | CNBD (titrad) . | Kd ± SEM (μM) . | P value (compared to WT) . | n . |
|---|---|---|---|---|
| cAMP | WT | 1.9 ± 0.1 | — | 3 |
| R650E | 4 ± 0.1 | 3.2E-04 | 3 | |
| K665E | 6.3 ± 0.3 | 1.2E-06 | 3 | |
| N547D/A548C | 1.8 ± 0.1 | 0.98 | 3 | |
| TRIP8bnano WT | WT | 1.3 ± 0.1 | — | 3 |
| TRIP8bnano D252N | WT | 3.5 ± 0.5 | 0.1 | 3 |
| TRIP8bnano WT | N547D/A548C | 9.3 ± 0.9 | 4.8E-5 | 3 |
| TRIP8bnano D252N | N547D/A548C | 3.6 ± 0.6 | 0.09 | 3 |
| Titrant . | CNBD (titrad) . | Kd ± SEM (μM) . | P value (compared to WT) . | n . |
|---|---|---|---|---|
| cAMP | WT | 1.9 ± 0.1 | — | 3 |
| R650E | 4 ± 0.1 | 3.2E-04 | 3 | |
| K665E | 6.3 ± 0.3 | 1.2E-06 | 3 | |
| N547D/A548C | 1.8 ± 0.1 | 0.98 | 3 | |
| TRIP8bnano WT | WT | 1.3 ± 0.1 | — | 3 |
| TRIP8bnano D252N | WT | 3.5 ± 0.5 | 0.1 | 3 |
| TRIP8bnano WT | N547D/A548C | 9.3 ± 0.9 | 4.8E-5 | 3 |
| TRIP8bnano D252N | N547D/A548C | 3.6 ± 0.6 | 0.09 | 3 |
ITC measurements of cAMP and Strep-tagged TRIP8bnano (WT and mutant) binding to His6-MBP–tagged human HCN2 CNBD (WT and mutants). Data are reported as mean ± SEM. Statistical analysis performed with one-way ANOVA, followed by post-hoc Tukey test.