Loss of expression and function of Gβγ by GNB1 Encephalopathy-associated L95P mutation of Gβ 1 subunit

Haritha Polagari Reddy^1,2*Tal Keren-Raifman¹Galit Tabak¹Nathan Dascal^1,3Daniel Yakubovich^4,5*

• ¹School of Medicine, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
• ²Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Capital Region of Denmark, Denmark
• ³Sagol School of Neuroscience, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
• ⁴Ariel University, Ariel, Israel
• ⁵Laniado Hospital, Neonatology Department, Netanya, Israel

Background: G-proteins are indispensable 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.We utilized the Xenopus laevis oocyte heterologous expression system to investigate the impact of the L95P mutation in Gβ 1 (Gβ 1 -L95P) on activation of neuronal GIRK channels GIRK2 and GIRK1/2. Mutant and wild-type (WT) Gβ 1 RNAs were co-injected with RNAs encoding the Gγ 2 and GIRK channel subunits. The expression levels of both Gβ 1 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.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 consistently in the Gβγ-effector interface. Thermodynamic calculations suggested that the mutation primarily destabilized Gβ 1 and Gβ 1 -effector complex.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.