Loss of expression and function of Gβγ by GNB1 encephalopathy-associated L95P mutation of the Gβ₁ subunit

Background: G-proteins areindispensable regulators of cellular signaling, with G-protein-gated inwardly rectifying potassium channels (GIRK) as key effectors. GNB1 encephalopathy (GNB1E) is a congenital neurological syndrome resulting from mutations in the GNB1 gene, encoding the Gβ₁ subunit of G-proteins trimer (Gαβγ). GNB1E manifests as a global developmental delay, accompanied by tonus disturbances, ataxia, and epilepsy. Methods: We utilized the Xenopus laevis oocyte heterologous expression system to investigate the impact of the L95P mutation in Gβ₁ (Gβ₁-L95P) on the activation of neuronal GIRK channels GIRK2 and GIRK1/2. Mutant and wild-type (WT) Gβ₁ RNAs were co-injected with RNAs encoding the Gγ₂ and GIRK channel subunits. The expression levels of both Gβ₁ and the channel proteins, as well as the channel activity, were systematically monitored. Additionally, rigid-body docking was used to model the GIRK1/2–Gβγ complex, evaluating L95P’s effect on channel–Gβγ interaction, Gβγ stability, and Gβγ–effector affinity. Results:. Gβ₁-L95P exhibited reduced protein expression compared to WT. Even after RNA adjustments to restore comparable membrane localization, the mutant failed to effectively activate GIRK2 and GIRK1/2. Structural analysis revealed that L95 was not consistent in the Gβγ–effector interface. Thermodynamic calculations suggested that the mutation primarily destabilized Gβ₁ and Gβ₁–effector complex. Conclusion: Gβ₁-L95P leads to both reduced protein expression and impaired function in the GIRK–Gβγ interaction system. The later effect can be attributed to the changes associated with protein misfolding.