Encephalopathy-causing mutations in Gβ1 (GNB1) alter regulation of neuronal GIRK channels

Abstract

Summary Mutations in the GNB1 gene, encoding the Gβ 1 subunit of heterotrimeric G proteins, cause GNB1 Encephalopathy. Patients experience seizures, pointing to abnormal activity of ion channels or neurotransmitter receptors. We studied three Gβ 1 mutations (K78R, I80N and I80T) using computational and functional approaches. In heterologous expression models, these mutations did not alter the coupling between G protein-coupled receptors to G i/o , or the Gβγ regulation of the neuronal voltage-gated Ca 2+ channel Ca V 2.2. However, the mutations profoundly affected the Gβγ regulation of the G protein-gated inwardly rectifying potassium channels (GIRK, or Kir3). Changes were observed in Gβ 1 protein expression levels, Gβγ binding to cytosolic segments of GIRK subunits, and in Gβγ function, and included gain-of-function for K78R or loss-of-function for I80T/N, which were GIRK subunit-specific. Our findings offer new insights into subunit-dependent gating of GIRKs by Gβγ, and indicate diverse etiology of GNB1 Encephalopathy cases, bearing a potential for personalized treatment.

Epilepsy in a mouse model of GNB1 encephalopathy arises from altered potassium (GIRK) channel signaling and is alleviated by a GIRK inhibitor

“…In line with the increased GABA B -GIRK signaling in Gnb 1 K78R/+ neurons, we show in Xenopus oocytes that G β 1K78R exerts a GoF toward GIRK1/2 and GIRK2 activation at physiologically relevant mild G β RNA doses, owing to higher surface expression of G β K78R compared with G β WT , in a wide range of G βγ expression levels ( Figure 6 ). The gain-of-expression of G β K78R was confirmed in our follow-up work, which also demonstrated that the K78R mutation does not affect the coupling of Gα i/o βγ to GPCRs and the G βγ regulation of voltage-gated Ca 2+ channels, Ca V 2.2 (Reddy et al, 2021 ), supporting the hypothesis that a defective G βγ -GIRK signaling plays an important role in the pathological neuronal function in K78R. However, in the case of GIRK1/2, there was a significant LoF toward GIRK1/2 at high G βγ doses, despite robust surface expression of G β K78R .…”

“…Overall, these results suggest that K78R is a gain-of-expression mutation, and the apparent GoF is due exclusively to higher surface expression compared to G β WT γ. Further investigation of this phenomenon showed suppression of GIRK1/2 channel expression by high doses of G β K78R γ RNA and a reduction in channel open probability (Reddy et al, 2021 ), explaining the attenuated GIRK1/2 currents at high expression levels of G β K78R γ.…”

“…However, in the case of GIRK1/2, there was a significant LoF toward GIRK1/2 at high G βγ doses, despite robust surface expression of G β K78R . This LoF was most probably due to a decrease in GIRK1/2 protein expression and open probability caused by overexpressed G βγ (Reddy et al, 2021 ). Mild levels of exogenously expressed G βγ mimic physiological conditions better than the high levels usually attained by saturating expression of a protein in heterologous cell models (Falkenburger et al, 2010 ; Yakubovich et al, 2015 ).…”

“…The DNAs encoding the proteins expressed in the oocytes, and RNA preparation, were as described (Reddy et al, 2021 ). GIRK1 (rat), GIRK2A (mouse), G β 1 (bovine), and Gγ 2 (bovine) cDNAs used in this study were cloned into high-expression oocyte vectors pGEM-HE or pGEM-HJ (Rishal et al, 2005 ).…”

“…In a recent study, inspired by the results of the present article published on a preprint server (Colombo et al, 2019 ), we investigated the effects of three GNB1 mutations seen in patients with GNB1 encephalopathy—K78R, I80N, and I80T—in heterologous expression systems. We demonstrated that these mutations did not alter GPCR-G i/o protein coupling but had a significant effect on G βγ regulation of GIRK channels (Reddy et al, 2021 ).…”