Biology subjects

Dascal, N.

Publications and source records attributed to Dascal, N..

10 recordsLinked to original sources

bioRxiv2026

Gβγ dually regulates the M current via increased channel surface expression and PIP2 sensitivity

The M-current, generated by voltage gated KV7.2/7.3 channels, sets the threshold for neuronal action potential and acts as a key brake on repetitive firing. The M-current is tightly regulated by signaling molecules, such as calmodulin and phosphatidylinositol-4,5-bisphosphate (PIP2). Here, we show that coexpression of the ubiquitous subunit dimer of heterotrimeric G-proteins, G{beta}{gamma}, with KV7.2/7.3 in Xenopus laevis oocytes doubles maximal M-current. This regulation requires free, prenylated, membrane-associated G{beta}{gamma} and operates via two distinct mechanisms: 1) increasing plasma membrane (PM) channel density and 2) stabilizing KV7.2/7.3-PIP2 coupling. Conversely, G{beta}{gamma} scavengers reduce basal KV7.2/7.3 current and weaken PIP2 coupling. Proximity ligation assays confirm colocalization of G{beta}{gamma} and KV7.2/7.3 in the PM. Peptide array and AlphaFold modeling identify putative interaction sites on the channel cytoplasmic domain. Finally, the disease-causing G{beta}1 variant I80N abolished G{beta}{gamma}-induced M-current potentiation. Together, these findings establish G{beta}{gamma} as a significant physiological regulator and potential site of vulnerability in neuronal M-current function.

physiology↗

bioRxiv2026

Cross-species analysis of GNB1 I80T encephalopathy: conserved developmental, epileptic and neuronal transcriptome signatures

GNB1 encephalopathy (GNB1E) is a rare neurodevelopmental disorder caused by mutations in GNB1 gene encoding the G protein subunit G{beta}1. Mechanisms linking these variants to neurological dysfunction remain unclear. We investigated the prevalent p.Ile80Thr (I80T) variant using combined clinical, cellular, and in vivo approaches. Longitudinal evaluation of a GNB1E patient revealed developmental delay, progressive peripheral spasticity, and epilepsy with Spike-Wave Activation in Sleep. Heterozygous knock-in Gnb1I80T/+ mice exhibited disease-relevant phenotypes, including impaired early development, mild adult motor and cognitive deficits and epileptiform cortical spike-and-wave discharges. Transcriptomic analysis identified 323 genes concordantly dysregulated in mouse cortex and cortical human neuronal cultures from patient-derived induced pluripotent cells. This gene set was enriched for ion-channel function, epilepsy-associated genes, and Gs/adenylyl cyclase signaling pathway. Our integrated analysis establishes the first cross-species model for GNB1E, suggests common neurological mechanisms and molecular pathways linked to GNB1E, and provides a framework for mechanistic and therapeutic studies. TeaserConserved human/mouse neurological and transcriptomic signatures in GNB1 encephalopathy.

physiology↗

bioRxiv2026

Structural and Functional Characterization of the KCNJ6 G154C Variant Reveals Severe GIRK2 Channel Gain-of-Function and Opportunities for Drug Repurposing

G protein-gated inwardly rectifying potassium (GIRK2) channels regulate neuronal excitability and are implicated in neurodevelopmental disorders. A rare KCNJ6 variant, G154C (hGIRK2G154C), was identified in a patient with mild Keppen-Lubinsky syndrome features, contrasting with severe phenotypes linked to other selectivity filter mutations. Here we combined molecular dynamics simulations and patch-clamp electrophysiology to characterize the hGIRK2G154C mutant, revealing a widened selectivity filter that resulted in loss of potassium selectivity, aberrant sodium permeation, and loss of inward rectification, indicating a severe gain-of-function phenotype. An in silico and electrophysiological drug screen identified FDA-approved compounds, including nefazodone and eletriptan, that potently inhibited GIRK2 and GIRK2G154C through distinct blocking mechanisms. These findings elucidate the structural and functional impact of the G154C mutation and highlight potential pharmacological tools and therapeutic candidates for the treatment of GIRK2 channelopathies.

pharmacology and toxicology↗

bioRxiv2025

Live-cell quantitative monitoring reveals distinct, high-affinity Gβγ regulations of GIRK2 and GIRK1/2 channels

Gi/o protein-coupled receptors (GPCRs) inhibit cardiac and neuronal excitability via G protein-activated K+ channels (GIRK), assembled by combinations of GIRK1 - GIRK4 subunits. GIRKs are activated by direct binding of the G{beta}{gamma} dimer of inhibitory Gi/o proteins. However, key aspects of this textbook signaling pathway remain debated. Recent studies suggested no Gi/o-GIRK pre-coupling and low (>250 {micro}M) G{beta}{gamma}-GIRK interaction affinity, contradicting earlier sub-{micro}M estimates and implying low signaling efficiency. We show that G{gamma} prenylation, which mediates G{beta}{gamma} membrane attachment required for GIRK activation, also contributes to the G{beta}{gamma}-GIRK interaction, explaining the poor affinity obtained with non-prenylated G{beta}{gamma}. Using quantitative protein titration and electrophysiology in live Xenopus oocytes, G{beta}{gamma} affinity for homotetrameric GIRK2 ranged from 4-30 {micro}M. Heterotetrameric GIRK1/2 showed a higher G{beta}{gamma} apparent affinity due to unique G{beta}{gamma}-docking site (anchor) in GIRK1, which enriches G{beta}{gamma} at the channel. Biochemical approaches and molecular dynamic simulations revealed that the G{beta}{gamma} anchor is formed by interacting N-terminal and distal C-terminal domains of the GIRK1 subunits, distinct from the G{beta}{gamma}-binding "activation" site(s) underlying channel opening. Thus, the affinity of G{beta}{gamma}-GIRK interaction is within the expected physiological range, while dynamic pre-coupling of G{beta}{gamma} to GIRK1-containing channels through high-affinity interactions further enhances the GPCR-Gi/o-GIRK signaling efficiency.

physiology↗

bioRxiv2024

Ethosuximide: subunit- and Gβγ-dependent blocker and reporter of allosteric changes in GIRK channels

The antiepileptic drug ethosuximide (ETX) suppresses epileptiform activity in a mouse model of GNB1 syndrome, caused by mutations in G{beta}1 protein, likely through the inhibition of G-protein gated K+ (GIRK) channels. Here we show that ETX is a subunit-selective, allosteric blocker of GIRKs. The potency of ETX block is increased by the G protein subunit dimer G{beta}{gamma}, the physiological activator of GIRKs. Molecular dynamics (MD) simulations and mutagenesis locate the ETX binding site in GIRK2 to a region associated with phosphatidylinositol-4,5-bisphosphate (PIP2) regulation, and suggest that ETX acts by closing the HBC gate and altering channels interaction with PIP2. The apparent affinity of ETX block is highly sensitive to changes in channel gating caused by mutations in G{beta}1 or GIRK subunits. Our findings pose GIRK as a potential therapeutic target for ETX, and ETX as a potent allosteric GIRK blocker and a tool for probing gating-related conformational changes in GIRK.

pharmacology and toxicology↗

bioRxiv2023

Tripartite interactions of PKA catalytic subunit and C-terminal domains of cardiac Ca2+ channel modulate its β-adrenergic regulation

The adrenergic nervous system augments cardiac contraction by increasing the activity of L-type voltage-gated CaV1.2 channels. Dysregulation of this process is linked to severe cardiac dysfunctions. The signaling cascade involves activation of {beta}-adrenergic receptors, elevation of cAMP levels, separation of protein kinase A (PKA) regulatory subunit (PKAR) from catalytic subunit (PKAC), and phosphorylation of the inhibitory protein Rad leading to increased Ca2+ influx. In cardiomyocytes, the core subunit of CaV1.2 (1C) exists in two forms: full-length (FL) or proteolytically processed (truncated), lacking the distal C-terminus (dCT). Specificity and efficiency in the cascade are believed to emanate from unique protein-protein interactions, such as anchoring PKA (via PKAR) to 1C by A-kinase anchoring proteins (AKAPs). However, most AKAPs do not interact with the truncated 1C, and their role in {beta}AR regulation of cardiac CaV1.2 remains unclear. Here we show that PKAC, independently of PKAR or AKAPs, directly interacts with 1C at two domains in 1C-CT: the proximal and distal C-terminal regulatory domains (PCRD and DCRD), which also interact with each other. Furthermore, we find that DCRD competes with PCRD and reduces its interaction with PKAC. The physiological consequences of these complex interactions are incompletely understood; our data suggest that they may fine-tune the {beta}AR regulation of CaV1.2. We propose that the newly discovered interactions take part in governing colocalization of regulatory proteins within the {beta}AR-CaV1.2 multimolecular signaling complexes in cardiomyocytes.

biochemistry↗

bioRxiv2022

Mapping molecular determinants of Cav2.2 inhibition by RGK proteins and homologs in Xenopus oocytes

The CaV1 and CaV2 families of voltage-dependent calcium channels play a crucial role in neurotransmitter release, excitation-contraction and many other cellular processes. Comprised of the membrane pore-forming 1, intracellular {beta} and extracellular 2{delta} subunits, these channels have been targets for pharmacological intervention for decades. Physiological functions of CaV channels are attenuated by either constitutively or transiently bounds proteins in the cellular environment. The RGK (Rad, Gem, Rem, and Rem2) G-protein family potently inhibits CaV1 and CaV2 function in heterologous expression systems. RGK proteins bind to CaV{beta} and inhibit channel localization and activity by forming a ternary complex with CaV1. Here, we evaluated the influence of RGK proteins on CaV2.2 channels heterologously expressed in Xenopus oocytes. Both Gem and Rad showed no nucleotide dependency on its inhibitory function on CaV2.2. The G-domain and C-terminus could inhibit the CaV2.2 channel independently when co-expressed with channel subunits. Our results demonstrated that structural determinants in Gem, crucial for channel inhibition, lie within the 222-296 amino acid region containing both the partial G-domain and C-terminus as determined from chimeric CaV{beta}-Gem constructs. We expanded our mapping efforts and prepared various chimeras of Drosophila melanogaster (Dm) RGK sequences fused to CaV{beta} and showed that 22 residues in RGK2t and RGK3L C-terminal imparted complete CaV2.2 inhibition. Point mutations in the DmRGK C-terminus, conserved in mammalian RGK proteins, abrogated the CaV2.2 inhibition to a significant extent, pointing to a hot region in the extreme C-terminus for inhibition of CaV channels. Since RGK homologs are now recognized as physiological modulators in {beta}-adrenergic regulation of CaV channels, the relevance of this curious G-protein family deserves close examination.

physiology↗

bioRxiv2020

Reconstitution of β-adrenergic regulation of CaV1.2: Rad-dependent and Rad-independent protein kinase A mechanisms.

IntroductionCardiac L-type voltage-gated CaV1.2 channels are crucial in physiological regulation of cardiac excitation-contraction coupling. Adrenergic modulation of CaV1.2 starts with activation of {beta}-adrenergic receptors (AR) and culminates in protein kinase A (PKA) - induced increase of calcium influx through CaV1.2 channels. To date, this cascade has never been fully reconstituted in heterologous systems; even partial reconstitution proved challenging and controversial. A recent study identified Rad, a calcium channel inhibitory protein, as an essential component of the adrenergic signaling cascade. We corroborated this finding, further characterized, and fully reconstituted, the complete {beta}-AR CaV1.2 modulation cascade in a heterologous expression system. ObjectiveOur primary goal was to heterologously reconstitute the complete {beta}-adrenergic cascade, and to investigate the role of Rad and additional molecular determinants in adrenergic regulation of cardiac CaV1.2. Methods and ResultsWe utilized the Xenopus oocyte heterologous expression system. We expressed CaV1.2 channel subunits, without or with Rad and {beta}1-AR or {beta}2-AR. To activate PKA, we injected cyclic AMP (cAMP) into the oocytes, or extracellularly applied isoproterenol (Iso) to stimulate {beta}-AR. Whole-cell Ba2+ currents served as readout. We find and distinguish between two distinct pathways of PKA modulation of CaV1.2: Rad-dependent (~80% of total) and Rad-independent. We separate the two mechanisms by showing distinct requirements for the cytosolic N- and distal C- termini of 1C and for the CaV{beta} subunit. Finally, for the first time, we reconstitute the complete pathway using agonist activation of either {beta}1-AR or {beta}2-AR. The reconstituted system reproduces the known features of {beta}-AR regulation in cardiomyocytes, such as a >2-fold increase in CaV1.2 current, a hyperpolarizing shift in activation curve, and a high constitutive activity of {beta}2-AR. ConclusionsThe adrenergic modulation of CaV1.2 is composed of two distinct pathways, Rad-independent and Rad-dependent. The latter contributes most of the {beta}-AR-induced enhancement of CaV1.2 activity, crucially depends on CaV{beta} subunit, and is differently regulated by {beta}1-AR and {beta}2-AR. The reconstitution of the full {beta}-AR cascade provides the means to address central unresolved issues related to roles of auxiliary proteins in the cascade, CaV1.2 isoforms, and will help to develop therapies for catecholamine-induced cardiac pathologies.

physiology↗

bioRxiv2019

A revised mechanism of action of hyperaldosteronism-linked mutations in cytosolic domains of GIRK4 (KCNJ5).

G-protein gated, inwardly rectifying potassium channels (GIRK) mediate inhibitory transmission in brain, heart, and adrenal cortex. GIRK4 (KCNJ5) subunits are abundant in the heart and adrenal cortex. Multiple mutations of KCNJ5 cause primary aldosteronism (PA). According to a leading concept, mutations in the pore region of GIRK4 cause loss of K+ selectivity; the ensuing Na+ influx depolarizes zona glomerulosa cells and activates voltage gated Ca2+ channels, inducing hypersecretion of aldosterone. The concept of selectivity loss has been extended to mutations in cytosolic domains of GIRK4 channels, remote from the pore region. We expressed GIRK4R52H, GIRK4E246K, and GIRK4G247R mutants in Xenopus oocytes and human adrenocortical carcinoma cell line (HAC15). Whole-cell currents of heterotetrameric GIRK1/4R52H and GIRK1/4E246K (but not GIRK1/4G247R) channels were greatly reduced compared to GIRK1/4WT. Nevertheless, all heterotetrameric mutants retained full K+ selectivity and inward rectification. When expressed as homotetramers, only GIRK4WT, but none of the mutants, produced whole-cell currents. Confocal imaging, single channel and Forster Resonance Energy Transfer (FRET) analyses showed: 1) reduction of membrane abundance of all mutated channels, especially as homotetramers, 2) impaired interaction with G{beta}{gamma} subunits, and 3) reduced open probability of GIRK1/4R52H. VU0529331, a GIRK4 opener, activated homotetrameric GIRK4G247R channels, but not GIRK4R52H and GIRK4E246K. Our results suggest impaired gating (GIRK4R52H) and expression in plasma membrane (all mutants). We suggest that, contrary to the previously proposed mechanism, R52H and E246K mutants are loss-of-function rather than gain-of-function/selectivity-loss mutants. Hence, GIRK4 openers may be a potential course of treatment for patients with cytosolic N- and C-terminal mutations. Significance StatementMutations in KCNJ5 gene, which encodes for the GIRK4 subunit of G-protein inwardly rectifying K+ channels, are the main cause of primary aldosteronism, a major contributor to secondary hypertension. We report that three mutations in the cytosolic domain of GIRK4 cause loss-of-function, contrary to the prevailing concept that these mutations cause loss of selectivity and subsequent depolarization, i.e. essentially gain-of-function. Our findings correct the existing misconception regarding the biophysical mechanism that impairs the channel function, and may provide indications for future personalized treatment of the disease.

physiology↗

bioRxiv2019

G protein-coupled potassium channels implicated in mouse and cellular models of GNB1 Encephalopathy

De novo mutations in GNB1, encoding the G{beta}1 subunit of G proteins, cause a neurodevelopmental disorder with global developmental delay and epilepsy. Mice carrying a pathogenic mutation, K78R, recapitulate aspects of the disorder, including developmental delay and frequent spike-wave discharges (SWD). Cultured mutant cortical neurons display aberrant bursting activity on multi-electrode arrays. Strikingly, the antiepileptic drug ethosuximide (ETX) restores normal neuronal network behavior in vitro and suppresses SWD in vivo. In contrast, while valproic acid suppresses SWD, it does not restore normal network behavior, suggesting that ETX has mechanistic specificity for the effects of aberrant G{beta}1 signaling. Consistent with this, we show that K78R is a gain-of-function of G protein-coupled potassium channel (GIRK) activation that is potently inhibited by ETX. This work suggests that altered G{beta}1 signaling causes disease in part through effects on GIRK channels, illustrates the utility of cultured neuronal networks in pharmacological screening, and establishes effective pre-clinical models for GNB1 Encephalopathy.

neuroscience↗