Daniel Yakubovich - Academia.edu (original) (raw)

Papers by Daniel Yakubovich

Biophysical Journal, Feb 1, 2023

Epilepsia, 2020

ObjectiveDravet syndrome (Dravet) is a severe childhood epileptic encephalopathy. The disease beg... more ObjectiveDravet syndrome (Dravet) is a severe childhood epileptic encephalopathy. The disease begins with a febrile stage, characterized by febrile seizures with otherwise normal development. Progression to the worsening stage features recurrent intractable seizures and the presentation of additional nonepileptic comorbidities, including global developmental delay, hyperactivity, and motor deficits. Later in life, at the stabilization stage, seizure burden decreases, whereas Dravet‐associated comorbidities persist. To date, it remains debated whether the nonepileptic comorbidities result from severe epilepsy or represent an independent phenotypic feature.MethodsDravet mice (DS) faithfully recapitulate many clinical aspects of Dravet. Using wild‐type (WT) and DS at different ages, we monitored multiple behavioral features as well as background electroencephalogram (EEG) activity during the different stages of Dravet epilepsy.ResultsBehavioral tests of WT and DS demonstrated that some...

Science Advances, 2020

A unique mechanism of Kv7.1 inactivation is controlled by external Ca2 + and allosterically coupl... more A unique mechanism of Kv7.1 inactivation is controlled by external Ca2 + and allosterically coupled by PIP2 and calmodulin.

iScience, 2021

Summary Mutations in the GNB1 gene, encoding the Gβ1 subunit of heterotrimeric G proteins, cause ... more Summary Mutations in the GNB1 gene, encoding the Gβ1 subunit of heterotrimeric G proteins, cause GNB1 Encephalopathy. Patients experience seizures, pointing to abnormal activity of ion channels or neurotransmitter receptors. We studied three Gβ1 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 Gi/o, or the Gβγ regulation of the neuronal voltage-gated Ca2+ channel CaV2.2. However, the mutations profoundly affected the Gβγ regulation of the G protein-gated inwardly rectifying potassium channels (GIRK, or Kir3). Changes were observed in Gβ1 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.

Frontiers in Pharmacology, 2020

The G protein-activated Inwardly Rectifying K +-channel (GIRK) modulates heart rate and neuronal ... more The G protein-activated Inwardly Rectifying K +-channel (GIRK) modulates heart rate and neuronal excitability. Following G-Protein Coupled Receptor (GPCR)-mediated activation of heterotrimeric G proteins (Gabg), opening of the channel is obtained by direct binding of Gbg subunits. Interestingly, GIRKs are solely activated by Gbg subunits released from Ga i/ocoupled GPCRs, despite the fact that all receptor types, for instance Ga q-coupled, are also able to provide Gbg subunits. It is proposed that this specificity and fast kinetics of activation stem from pre-coupling (or pre-assembly) of proteins within this signaling cascade. However, many studies, including our own, point towards a diffusion-limited mechanism, namely collision coupling. Here, we set out to address this long-standing question by combining electrophysiology, imaging, and mathematical modeling. Muscarinic-2 receptors (M2R) and neuronal GIRK1/2 channels were coexpressed in Xenopus laevis oocytes, where we monitored protein surface expression, current amplitude, and activation kinetics. Densities of expressed M2R were assessed using a fluorescently labeled GIRK channel as a molecular ruler. We then incorporated our results, along with available kinetic data reported for the G-protein cycle and for GIRK1/2 activation, to generate a comprehensive mathematical model for the M2R-G-protein-GIRK1/2 signaling cascade. We find that, without assuming any irreversible interactions, our collision coupling kinetic model faithfully reproduces the rate of channel activation, the changes in agonist-evoked currents and the acceleration of channel activation by increased receptor densities.

Biophysical Journal, 2020

Dravet Syndrome (Dravet) is a severe childhood epileptic encephalopathy. The disease begins aroun... more Dravet Syndrome (Dravet) is a severe childhood epileptic encephalopathy. The disease begins around the age of six months, with a febrile stage, characterized by febrile seizures with otherwise normal development. By the end of the first year of life, the disease progresses to the worsening stage, featuring recurrent intractable seizures and the appearance of additional comorbidities, including global developmental delay, cognitive deficits, hyperactivity and motor problems. Later, in early school years, Dravet reaches the stabilization stage, in which seizure burden decreases, while Dravet-associated comorbidities persist. Dravet syndrome mouse models (DS) faithfully recapitulate the three stages of the human syndrome. Here, we performed power spectral analyses of background EEG activity in DS and their wild-type (WT) littermates, demonstrating disease stage-related alterations. Specifically, while the febrile stage activity resembled that of WT mice, we observed a marked reduction ...

Biophysical Journal, 2019

We recorded the time and dose dependent differential drug response on field potentials (FPs) from... more We recorded the time and dose dependent differential drug response on field potentials (FPs) from 23 independent constructs simultaneously before and after exposure to the Dox concentration range of 0.1 to 10 mM. We observed a time and dose dependent effect on FP amplitude and cell viability. At 1 h post-treatment there was no significant relative FP amplitude change at any concentration when compared to pre-treatment indicating minimally altered electrical properties of the network. However, a time and dose dependent attenuation of FP amplitude ensued at 24 h relative to control (15526% for 0.1 mM and 45512% for 1 mM) and gradually increased by 48 h (33521% for 0.1 mM and 8856% for 1 mM). Viability measured by percentage of active electrodes also decreased in a dose and time dependent fashion. Exposure to the highest concentration of 10 mM abolished electrical activity in all 5 constructs probed by 24 h while the same occurred in the 6 constructs exposed to 1 mM after 72 hours. The lowest concentration of 0.1 mM had the least cardiotoxic effect with minimal effect on viability thus the cumulative effect on the FP properties could be tracked on the same constructs over 72 h. At this concentration the normalized RR interval was significantly prolonged at all time points (8% increase at 1 h, 17% at 24 h, 88% at 48 h and 52% at 72 h) when compared to control. These results strongly suggest that the proposed model has the capability to mimic the Dox induced cardiotoxicity.

PLOS Computational Biology, 2015

G protein-gated K + channels (GIRK; Kir3), activated by Gβγ subunits derived from G i/o proteins,... more G protein-gated K + channels (GIRK; Kir3), activated by Gβγ subunits derived from G i/o proteins, regulate heartbeat and neuronal excitability and plasticity. Both neurotransmitterevoked (I evoked) and neurotransmitter-independent basal (I basal) GIRK activities are physiologically important, but mechanisms of I basal and its relation to I evoked are unclear. We have previously shown for heterologously expressed neuronal GIRK1/2, and now show for native GIRK in hippocampal neurons, that I basal and I evoked are interrelated: the extent of activation by neurotransmitter (activation index, R a) is inversely related to I basal. To unveil the underlying mechanisms, we have developed a quantitative model of GIRK1/2 function. We characterized single-channel and macroscopic GIRK1/2 currents, and surface densities of GIRK1/ 2 and Gβγ expressed in Xenopus oocytes. Based on experimental results, we constructed a mathematical model of GIRK1/2 activity under steady-state conditions before and after activation by neurotransmitter. Our model accurately recapitulates I basal and I evoked in Xenopus oocytes, HEK293 cells and hippocampal neurons; correctly predicts the dose-dependent activation of GIRK1/2 by coexpressed Gβγ and fully accounts for the inverse I basal-R a correlation. Modeling indicates that, under all conditions and at different channel expression levels, between 3 and 4 Gβγ dimers are available for each GIRK1/2 channel. In contrast, available Gα i/o decreases from~2 to less than one Gα per channel as GIRK1/2's density increases. The persistent Gβγ/channel (but not Gα/channel) ratio support a strong association of GIRK1/2 with Gβγ, consistent with recruitment to the cell surface of Gβγ, but not Gα, by GIRK1/2. Our analysis suggests a maximal stoichiometry of 4 Gβγ but only 2 Gα i/o per one GIRK1/2 channel. The unique, unequal association of GIRK1/2 with G protein subunits,

Biophysical Journal, 2014

Voltage-gated K þ (Kv) are tetramers of a-subunits each consisting of 6 transmembrane segments (S... more Voltage-gated K þ (Kv) are tetramers of a-subunits each consisting of 6 transmembrane segments (S1-S6) and a cytoplasmic N-and C-terminus. The S5-S6 segments of each subunit assemble to generate the central pore while the S1-S4 segments form the voltage-sensing domains. The PXP motif in the middle of S6 provides a degree of flexibility to the bottom half of the S6 segment which is necessary for channel gating. This region is also critical for the interaction with channel blockers. Based on sequence homology, eight Shakerrelated Kv subfamilies have been identified: Kv1-Kv6, Kv8-Kv9. The silent (KvS) subunits (Kv5-Kv9) cannot form homotetramers but assemble with Kv2 subunits into Kv2/KvS heterotetramers that display unique biophysical properties. KvS subunits lack the 2 nd proline residue of the PXP motif which may impact on the pharmacological profile of channel blockers. We tested this hypothesis by using the Kv1.5(P511G) mutant in which the 2 nd proline of the PXP motif was replaced by a glycine. Homotetrameric Kv1.5(P511G) channels were insensitive to 4-AP while heterotetrameric Kv1.5-Kv1.5(P511G) channels (stoichiometry controlled by using dimers), still displayed current inhibition. However, Kv1.5-Kv1.5(P511G) channels were significantly less sensitive displaying an IC 50 values of 16 mM instead of 270mM for wild type (WT) Kv1.5. Similarly, heterotetrameric Kv2/KvS channels displayed an altered affinity for 4-AP compared to WT Kv2.1; 18 mM (IC 50 for Kv2.1) inhibited 17%, 60%, 82% and 13% of Kv5.1, Kv6.3, Kv8.1 and Kv9.3-containing currents, respectively. Furthermore, the heterotetrameric Kv2/KvS channels displayed also a subtle change in the affinity for the open channel blockers quinidine and flecainide. These results suggest that the absence of a complete PXP motif in one or two out of four subunits alters the pharmacological profile. (Supported by FWO fellowships to JS and EB & grant FWO-G.0449.11N to DJS).

The Journal of Physiology, 2000

The G protein-activated K¤ channels (GIRK, or Kir3, family) play important roles in the regulatio... more The G protein-activated K¤ channels (GIRK, or Kir3, family) play important roles in the regulation of heartbeat and in inhibitory neurotransmission in the brain (for review, see Yamada et al. 1998). This is the only ion channel family whose members are known to be activated by G protein âã subunits (Gâã) by a direct protein-protein interaction (reviewed by Kurachi, 1995; Wickman & Clapham, 1995; Dascal, 1997; Jan & Jan, 1997). Theoretically, this provides a unique opportunity to study the molecular details of the interaction of Gâã with its effector with millisecond resolution and, from there, to understand better the mechanisms of G protein-effector interactions in general. However, the high density of channels in cardiac and neuronal cells, in which GIRK channels are endogenously expressed, makes it difficult to obtain patches with one channel. This precluded a comprehensive description of the closed states and hampered the study of the slow kinetic properties of GIRK gating. In this study, we utilized the potential of a heterologous expression system, the Xenopus oocyte, in which the channel density in the membrane can be controlled by regulating the level of expression, to overcome this problem. The GIRK family includes GIRK1, which was initially cloned from atrium (Dascal et al. 1993; Kubo et al. 1993), and additional subunits (GIRK2 to GIRK5). In most cases, functional GIRK channels are heterotetramers formed by GIRK1 with the other subunits: GIRK2, GIRK3 and GIRK4 in the brain (Lesage et al. 1994, 1995; Duprat et al.

Journal of Molecular Neuroscience, 2005

G protein-activated K + (GIRK) channels are activated by numerous neurotransmitters that act on G... more G protein-activated K + (GIRK) channels are activated by numerous neurotransmitters that act on G i/o proteins, via a direct interaction with the Gβγ subunit of G proteins. In addition, GIRK channels are positively regulated by intracellular Na + via a direct interaction (fast pathway) and via a Gβγ-dependent mechanism (slow pathway). The slow modulation has been proposed to arise from the recently described phenomenon of Na +induced reduction of affinity of interaction between Gα GDP and Gβγ subunits of G proteins. In this scenario, elevated Na + enhances basal dissociation of G protein heterotrimers, elevating free cellular Gβγ and activating GIRK. However, it is not clear whether this hypothesis can account for the quantitative and kinetic aspects of the observed regulation. Here, we report the development of a quantitative model of slow, Na +-dependent, G protein-mediated activation of GIRK. Activity of GIRK1 F137S channels, which are devoid of direct interaction with Na + , was measured in excised membrane patches and used as an indicator of free Gβγ levels. The change in channel activity was used to calculate the Na +-dependent change in the affinity of G protein subunit interaction. Under a wide range of initial conditions, the model predicted that a relatively small decrease in the affinity of interaction of Gα GDP and Gβγ (about twofold under most conditions) accounts for the twofold activation of GIRK induced by Na + , in agreement with biochemical data published previously. The model also correctly described the slow time course of Na + effect and explained the previously observed enhancement of Na +-induced activation of GIRK by coexpressed Gα i3. This is the first quantitative model that describes the basal equilibrium between free and bound G protein subunits and its consequences on regulation of a Gβγ effector. to be the basal dissociation of G protein subunits, which occurs without any receptor stimulation. Although the interaction between Gα GDP (GDPbound Gα) and Gβγ is a high-affinity one, it is reversible (Sarvazyan et al., 1998, 2002). Simple calculations based on mass-action law show that this reaction alone, in the absence any of GPCR activation, might give rise to high basal levels of free Gβγ. Furthermore, this equilibrium is dynamic: We have demonstrated recently that it is modulated by intracellular Na + , which might therefore lead to changes in cellular levels of free Gα GDP and Gβγ(Rishal et al., 2003). Unfortunately, at present, no indicators are available to follow the changes in free [Gβγ] in intact cells.

The Journal of General Physiology, 2004

The pore properties and the reciprocal interactions between permeant ions and the gating of KCNQ ... more The pore properties and the reciprocal interactions between permeant ions and the gating of KCNQ channels are poorly understood. Here we used external barium to investigate the permeation characteristics of homomeric KCNQ1 channels. We assessed the Ba2+ binding kinetics and the concentration and voltage dependence of Ba2+ steady-state block. Our results indicate that extracellular Ba2+ exerts a series of complex effects, including a voltage-dependent pore blockade as well as unique gating alterations. External barium interacts with the permeation pathway of KCNQ1 at two discrete and nonsequential sites. (a) A slow deep Ba2+ site that occludes the channel pore and could be simulated by a model of voltage-dependent block. (b) A fast superficial Ba2+ site that barely contributes to channel block and mostly affects channel gating by shifting rightward the voltage dependence of activation, slowing activation, speeding up deactivation kinetics, and inhibiting channel inactivation. A model...

The Journal of General Physiology, 2000

To investigate possible effects of adrenergic stimulation on G protein–activated inwardly rectify... more To investigate possible effects of adrenergic stimulation on G protein–activated inwardly rectifying K+ channels (GIRK), acetylcholine (ACh)-evoked K+ current, IKACh, was recorded from adult rat atrial cardiomyocytes using the whole cell patch clamp method and a fast perfusion system. The rise time of IKACh was 0.4 ± 0.1 s. When isoproterenol (Iso) was applied simultaneously with ACh, an additional slow component (11.4 ± 3.0 s) appeared, and the amplitude of the elicited IKACh was increased by 22.9 ± 5.4%. Both the slow component of activation and the current increase caused by Iso were abolished by preincubation in 50 μM H89 {N-[2-((p -bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, a potent inhibitor of PKA}. This heterologous facilitation of GIRK current by β-adrenergic stimulation was further studied in Xenopus laevis oocytes coexpressing β2-adrenergic receptors, m2 -receptors, and GIRK1/GIRK4 subunits. Both Iso and ACh elicited GIRK currents in these oocytes. Furthermore,...

Journal of Biological Chemistry, 2002

G protein-gated K ؉ channels (GIRK, or Kir3) are activated by the direct binding of G␤␥ or of cyt... more G protein-gated K ؉ channels (GIRK, or Kir3) are activated by the direct binding of G␤␥ or of cytosolic Na ؉. Na ؉ activation is fast, G␤␥-independent, and probably via a direct, low affinity (EC 50 , 30-40 mM) binding of Na ؉ to the channel. Here we demonstrate that an increase in intracellular Na ؉ concentration, [Na ؉ ] in , within the physiological range (5-20 mM), activates GIRK within minutes via an additional, slow mechanism. The slow activation is observed in GIRK mutants lacking the direct Na ؉ effect. It is inhibited by a G␤␥ scavenger, hence it is G␤␥-dependent; but it does not require GTP. We hypothesized that Na ؉ elevates the cellular concentration of free G␤␥ by promoting the dissociation of the G␣␤␥ heterotrimer into free G␣ GDP and G␤␥. Direct biochemical measurements showed that Na ؉ causes a moderate decrease (ϳ2-fold) in the affinity of interaction between G␣ GDP and G␤␥. Furthermore, in accord with the predictions of our model, slow Na ؉ activation was enhanced by mild coexpression of G␣ i3. Our findings reveal a previously unknown mechanism of regulation of G proteins and demonstrate a novel G␤␥-dependent regulation of GIRK by Na ؉. We propose that Na ؉ may act as a regulatory factor, or even a second messenger, that regulates effectors via G␤␥.

Journal of Biological Chemistry, 2005

Cardiac and neuronal G protein-activated K ؉ channels (GIRK; Kir3) open following the binding of ... more Cardiac and neuronal G protein-activated K ؉ channels (GIRK; Kir3) open following the binding of G␤␥ subunits, released from G i/o proteins activated by neurotransmitters. GIRKs also possess basal activity contributing to the resting potential in neurons. It appears to depend largely on free G␤␥, but a G␤␥-independent component has also been envisaged. We investigated G␤␥ dependence of the basal GIRK activity (A GIRK,basal) quantitatively, by titrated expression of G␤␥ scavengers, in Xenopus oocytes expressing GIRK1/2 channels and muscarinic m2 receptors. The widely used G␤␥ scavenger, myristoylated C terminus of ␤-adrenergic kinase (m-c␤ARK), reduced A GIRK,basal by 70-80% and eliminated the acetylcholine-evoked current (I ACh). However, we found that m-c␤ARK directly binds to GIRK, complicating the interpretation of physiological data. Among several newly constructed G␤␥ scavengers, phosducin with an added myristoylation signal (m-phosducin) was most efficient in reducing GIRK currents. m-phosducin relocated to the membrane fraction and did not bind GIRK. Titrated expression of m-phosducin caused a reduction of A GIRK,basal by up to 90%. Expression of GIRK was accompanied by an increase in the level of G␤␥ and G␣ in the plasma membrane, supporting the existence of preformed complexes of GIRK with G protein subunits. Increased expression of G␤␥ and its constitutive association with GIRK may underlie the excessively high A GIRK,basal observed at high expression levels of GIRK. Only 10-15% of A GIRK,basal persisted upon expression of both m-phosducin and c␤ARK. These results demonstrate that a major part of I basal is G␤␥-dependent at all levels of channel expression, and only a small fraction (<10%) may be G␤␥-independent. G protein-activated, inwardly rectifying K ϩ channels (GIRK, Kir3) 1 mediate postsynaptic inhibitory effects of various neu

Biophysical Journal, 2012

The gate at the pore-forming domain of potassium channels is allosterically controlled by a stimu... more The gate at the pore-forming domain of potassium channels is allosterically controlled by a stimulus-sensing domain. Using Cd2þ as a probe, we examined the structural elements responsible for gating in an inward-rectifier Kþ channel. We generated a cysteine-free mouse Kir3.2 mutant (C1234) in which the four cysteine residues that are potentially exposed to the cytoplasm were replaced. These residues were as follows: Cys65 in the N-terminal portion of the cytoplasmic domain, Cys190 in the second transmembrane helix, and Cys221 and Cys321 in the bC and bI strands, respectively, of the C-terminal portion of the cytoplasmic domain. One of four endogenous cysteines of Kir3.2 (Cys65) the cysteines of which potentially face the cytoplasm contributes to a high-affinity site for inhibition by internal Cd2þ. Crystal structure of its cytoplasmic domain in complex with Cd2þ reveals that octahedral coordination geometry supports the high-affinity binding at Cys65. This mode of action causes the tethering of Cys65 on the N-terminus to His233 on the CD loop in the stimulus-sensing domain. We previously reported that the interaction of these structural elements via an ionic bond between His69 and Asp228 maintained the channel in a closed state by lowering the sensitivity to phosphatidylinositol-4,5-bisphosphate. Therefore, the mode of binding of Cd2þ reveals that ''inverse agonist''-like action is a mechanism underlying the inhibition of Kir3.2 by Cd2þ and also that the N-terminus and CD loop are the structural elements participate in gating.

Biophysical Journal, 2010