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 neurotransmitters in the brain and atrium via seven-helix, G protein-coupled receptors (GPCRs) linked to pertussis toxin-sensitive G proteins of the G i/o family. Opening of the channels is the result of a direct binding of G␤␥ subunits released from the G␣ i/o ␤␥ heterotrimers (1-4). The channel can also be activated by cytosolic Na ϩ and membranal phosphatidylinositol 4,5-bisphosphate (PIP 2 ); the latter is essential for proper GIRK gating by both Na ϩ and G␤␥ (3, 5, 6). Whereas the physiological role of neurotransmitter-induced GIRK activity is well established, the basal activity of these channels (A GIRK,basal ) is often regarded as negligible and physiologically unimportant. This feature distinguishes GIRK from many other K ϩ channels of the Kir family, such as Kir1 and Kir2, which show high intrinsic activity under physiological conditions and are often referred to as "constitutively active." Low A GIRK,basal is supposed to ensure high signal-to-noise ratio for GIRK-related neurotransmitter signaling and to minimize participation of GIRK in resting membrane K ϩ conductance (see Ref. 2). However, some classical and many recent studies challenge this concept. In sinoa...

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, Iso facilitated ACh currents in a way, similar to atrial cells. Cytosolic injection of 30–60 pmol cAMP, but not of Rp-cAMPS (a cAMP analogue that is inhibitory to PKA) mimicked the β2-adrenergic effect. The possibility that the potentiation of GIRK currents was a result of the phosphorylation of the β-adrenergic receptor (β2AR) by PKA was excluded by using a mutant β2AR in which the residues for PKA-mediated modulation were mutated. Overexpression of the α subunit of G proteins (Gαs) led to an increase in basal as well as agonist-induced GIRK1/GIRK4 currents (inhibited by H89). At higher levels of expressed Gαs, GIRK currents were inhibited, presumably due to sequestration of the β/γ subunit dimer of G protein. GIRK1/GIRK5, GIRK1/GIRK2, and homomeric GIRK2 channels were also regulated by cAMP injections. Mutant GIRK1/GIRK4 channels in which the 40 COOH-terminal amino acids (which contain a strong PKA phosphorylation consensus site) were deleted were also modulated by cAMP injections. Hence, the structural determinant responsible is not located within this region. We conclude that, both in atrial myocytes and in Xenopus oocytes, β-adrenergic stimulation potentiates the ACh-evoked GIRK channels via a pathway that involves PKA-catalyzed phosphorylation downstream from β2AR.

Objective: Dravet 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. Methods: Dravet 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. Results: Behavioral tests of WT and DS demonstrated that some deficits manifest already at the pre-epileptic stage, prior to the onset of convulsive seizures. These include motor impairment and hyperactivity in the open field. Deficits in cognitive functions were detected at the onset of severe spontaneous seizures. Power spectral analysis of background EEG activity, measured through development, showed that DS exhibit normal background oscillations at the pre-epileptic stage, a marked reduction in total power during the onset of severe epilepsy, and a subsequent smaller reduction later in life. Importantly, low EEG power at the stage of severe frequent convulsive seizures correlated with increased risk for premature death. Significance: Our data provide a comprehensive developmental trajectory of Dravet epilepsy and Dravet-associated comorbidities in mice, under controlled settings, demonstrating that the convulsive seizures and some nonepileptic comorbidities may be uncoupled. Moreover, we report the existence of an inverse correlation, on average, between the power of background EEG and the severity of epileptic phenotypes, suggesting that such measurements may potentially serve as a biomarker for Dravet severity. K E Y W O R D S background EEG, Dravet syndrome, hyperactivity, motor impairment, power spectral density 2290 | FADILA et AL. SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section. How to cite this article: Fadila S, Quinn S, Turchetti Maia A, et al. Convulsive seizures and some behavioral comorbidities are uncoupled in the Scn1a A1783V Dravet syndrome mouse model.

G protein-gated K+ channels (GIRK; Kir3), activated by Gβγ subunits derived from Gi/o proteins, regulate heartbeat and neuronal excitability and plasticity. Both neurotransmitter-evoked (Ievoked) and neurotransmitter-independent basal (Ibasal) GIRK activities are physiologically important, but mechanisms of Ibasal and its relation to Ievoked are unclear. We have previously shown for heterologously expressed neuronal GIRK1/2, and now show for native GIRK in hippocampal neurons, that Ibasal and Ievoked are interrelated: the extent of activation by neurotransmitter (activation index, Ra) is inversely related to Ibasal. 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 Ibasal and Ievoked 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 Ibasal-Ra 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 Gαi/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 Gαi/o per one GIRK1/2 channel. The unique, unequal association of GIRK1/2 with G protein subunits, and the cooperative nature of GIRK gating by Gβγ, underlie the complex pattern of basal and agonist-evoked activities and allow GIRK1/2 to act as a sensitive bidirectional detector of both Gβγ and Gα.