Activity‐restoring mutations in the histamine <scp> H ₃ </scp> receptor increase constitutive activity and reduce structural stability

The histamine H₃ receptor (H₃R) is a class A G protein-coupled receptor (GPCR) that regulates neurotransmitter release in the central nervous system. Although structures of H₃R in both inactive and active states have been elucidated, the functional roles of specific residues remain unclear. We previously identified four point mutations-L73^2.43M, F193^ECL2S, S359^6.36Y, and C415^7.56R-that restore signaling in yeast, where wild-type H₃R is otherwise inactive. Here, we show that these mutations also enhance constitutive (ligand-independent) activity. All six possible double mutants exhibited markedly increased basal activity, indicating that these mutations act cooperatively to shift the conformational equilibrium toward the active state. In mammalian cells, all single mutations also increased constitutive activity compared with the wild-type receptor. Additional mutagenesis at and around the four sites revealed that activity restoration cannot be explained solely by steric clashes introduced by residue substitution. Radioligand binding assays showed minimal changes in histamine affinity, implying that the mutations affect receptor function through conformational modulation. FSEC and FSEC-TS analyses further demonstrated that increased constitutive activity correlates with reduced structural stability. When the same mutations were introduced into the histamine H₁ receptor (H₁R), only the C471^7.56R mutant enhanced signaling, suggesting that the functional consequences of these mutations are receptor dependent. Collectively, our findings reveal a close relationship between structural destabilization and constitutive activation in H₃R, while also underscoring the complexity of GPCR activation.