Prothrombin Time and International Normalized Ratio as Predictors of Factor VII Coagulation Activity in Pediatric Patients.

BACKGROUND: Factor VII (FVII) deficiency is characterized by normal activated partial thromboplastin time (aPTT) and prolonged prothrombin time (PT) values. It is diagnosed by determining protein level and coagulation activity (FVII:C). FVII:C measurements are expensive and time consuming. OBJECTIVES: To analyze correlations between PT, international normalized ratio (INR), and FVII:C in pediatric patients before otolaryngology surgery and to establish alternative methods for identifying FVII deficiency. METHODS: FVII:C data were collected from 96 patients with normal aPTT and prolonged PT values during preoperative otolaryngology surgery coagulation workup between 2016 and 2020. We compared demographic and clinical parameters using Spearman correlation coefficient and receiver operating characteristic (ROC) curve analysis to determine the accuracy of PT and INR values to predict FVII deficiency. RESULTS: The median values of PT, INR and FVII:C were 13.5 seconds, 1.14, and 67.5%, respectively. In total, 65 participants (67.7%) displayed normal FVII:C compared to 31 (32.3%) with decreased FVII:C. A statistically significant negative correlation was observed between FVII:C and PT values and between FVII:C and INR. Despite statistically significant ROC of 0.653 for PT (P-value = 0.017, 95% confidence interval [95%CI] 0.529-0.776) and 0.669 for INR (P-value = 0.08, 95%CI 0.551-0.788), we were unable to determine an optimal cutoff point to predict FVII:C deficiency with high sensitivity and high specificity. CONCLUSIONS: We could not identify a PT or INR threshold to best predict clinically relevant FVII:C levels. When PT is abnormal, determining FVII:C protein levels is needed for diagnosing FVII deficiency and considering surgical prophylactic treatment.

Convulsive seizures and some behavioral comorbidities are uncoupled in the Scn1aA1783V Dravet syndrome mouse model.

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.

Factors associated with early phosphate levels in preterm infants.

To investigate perinatal factors and early morbidities associated with early serum phosphate (sPhos) levels in a cohort of preterm infants. Retrospective data were obtained from the medical records of a cohort of 454 infants born at < 32 weeks gestational age. Serum phosphate levels were directly associated with gestational age, body weight z-score, and Apgar scores and inversely associated with timing of enteral nutrition initiation and diet consisting of mostly breast milk. Maternal hypertension, lactate levels, early symptomatic hypotension, and total protein supplemented on days 1 to 3 were also inversely associated with sPhos. Morbidities that were found to be associated with sPhos did not persist after adjustment for confounding factors.Conclusions: We report a novel association between early sPhos and timing and content of enteral nutrition, as well as with the early neonatal hemodynamic condition of preterm infants. This information may help identify infants at risk for low sPhos and aid in the nutritional strategy utilized in these patients. This study did not identify early morbidities associated with sPhos. What is Known: • High initial amino acid intake is associated with increased risk of Refeeding like syndrome and hypophosphatemia, among preterm infants. What is New: • Early enteral nutrition, starting within the first 72 h of life, is associated with higher serum phosphate (sPhos) compared to nothing per os (NPO). • sPhos was not associated with early adverse neonatal outcomes.

A Quantitative Model of the GIRK1/2 Channel Reveals That Its Basal and Evoked Activities Are Controlled by Unequal Stoichiometry of Gα and Gßγ.

G protein-gated K+ channels (GIRK; Kir3), activated by Gßγ 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 Gßγ 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 Gßγ 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 Gßγ 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 Gßγ/channel (but not Gα/channel) ratio support a strong association of GIRK1/2 with Gßγ, consistent with recruitment to the cell surface of Gßγ, but not Gα, by GIRK1/2. Our analysis suggests a maximal stoichiometry of 4 Gßγ 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 Gßγ, underlie the complex pattern of basal and agonist-evoked activities and allow GIRK1/2 to act as a sensitive bidirectional detector of both Gßγ and Gα.

Short-term morbidities and neurodevelopmental outcomes in preterm infants exposed to magnesium sulphate treatment.

AIM: The aim of the study is to examine whether baseline serum Mg concentration has an impact on short-term and long-term outcomes in preterm infants exposed antenatally to MgSO4. METHODS: Participants included all infants admitted to the neonatal intensive care unit at <32 weeks of gestational age. Infant serum Mg concentration (iMgC) was examined immediately after birth in those exposed to maternal MgSO4. Data for short-term outcomes were collected from the infants' computerised charts. Neurodevelopmental outcomes at 6-12 months corrected age were assessed using the Griffiths Mental Developmental Scales. RESULTS: Of 197 eligible infants, 145 were exposed to MgSO4. Baseline iMgC was available for 88 infants. Mean iMgC was 3.5 ± 0.88 mg/dL (1.6-5.7 mg/dL). Baseline iMgC was not associated with an increased risk for neither early morbidities nor adverse long-term outcome. However, iMgC above the mean (>3.5 mg/dL) was associated with significantly lower scores on locomotor (P = 0.016) and personal-social (0.041) scales in the first year of life. CONCLUSIONS: In a cohort of preterm infants antenatally exposed to MgSO4, elevated baseline iMgC (>3.5 mg/dL) was associated with lower locomotor scores. Further research is needed in order to study the relationship between supra-physiologic iMgC and its effect on the developing brain.

Occurrence of BNT162b2 Vaccine Adverse Reactions Is Associated with Enhanced SARS-CoV-2 IgG Antibody Response.

Promoting SARS-CoV-2 vaccination has been a global mission since the first vaccines were approved for emergency use. Alongside the excitement following the possibility of eradicating SARS-CoV-2 and ending the COVID-19 pandemic, there has been ample vaccine hesitancy, some due to the abundant reporting of adverse reactions. We report here that the occurrence of BNT162b2 vaccine adverse reactions is associated with enhanced antibody response. We found a statistically significant correlation between having an adverse reaction, whether local or systemic, and higher antibody levels. No sex difference was observed in antibody levels. However, as was recently reported, the antibody response was found to be lower among older vaccinees. The demonstration of a clear correlation between adverse reactions and antibody levels may help reduce vaccination hesitancy by reassuring that the presence of such reactions is an indication of a well-functioning immune system.

Encephalopathy-causing mutations in Gß1 (GNB1) alter regulation of neuronal GIRK channels.

Mutations in the GNB1 gene, encoding the Gß1 subunit of heterotrimeric G proteins, cause GNB1 Encephalopathy. Patients experience seizures, pointing to abnormal activity of ion channels or neurotransmitter receptors. We studied three Gß1 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 Gßγ regulation of the neuronal voltage-gated Ca2+ channel CaV2.2. However, the mutations profoundly affected the Gßγ regulation of the G protein-gated inwardly rectifying potassium channels (GIRK, or Kir3). Changes were observed in Gß1 protein expression levels, Gßγ binding to cytosolic segments of GIRK subunits, and in Gßγ 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 Gßγ, and indicate diverse etiology of GNB1 Encephalopathy cases, bearing a potential for personalized treatment.

A Collision Coupling Model Governs the Activation of Neuronal GIRK1/2 Channels by Muscarinic-2 Receptors.

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 (Gαßγ), opening of the channel is obtained by direct binding of Gßγ subunits. Interestingly, GIRKs are solely activated by Gßγ subunits released from Gαi/o-coupled GPCRs, despite the fact that all receptor types, for instance Gαq-coupled, are also able to provide Gßγ 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.

A unique mechanism of inactivation gating of the Kv channel family member Kv7.1 and its modulation by PIP2 and calmodulin.

Inactivation of voltage-gated K+ (Kv) channels mostly occurs by fast N-type or/and slow C-type mechanisms. Here, we characterized a unique mechanism of inactivation gating comprising two inactivation states in a member of the Kv channel superfamily, Kv7.1. Removal of external Ca2+ in wild-type Kv7.1 channels produced a large, voltage-dependent inactivation, which differed from N- or C-type mechanisms. Glu295 and Asp317 located, respectively, in the turret and pore entrance are involved in Ca2+ coordination, allowing Asp317 to form H-bonding with the pore helix Trp304, which stabilizes the selectivity filter and prevents inactivation. Phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+-calmodulin prevented Kv7.1 inactivation triggered by Ca2+-free external solutions, where Ser182 at the S2-S3 linker relays the calmodulin signal from its inner boundary to the external pore to allow proper channel conduction. Thus, we revealed a unique mechanism of inactivation gating in Kv7.1, exquisitely controlled by external Ca2+ and allosterically coupled by internal PIP2 and Ca2+-calmodulin.

Andersen-Tawil Syndrome Is Associated With Impaired PIP2 Regulation of the Potassium Channel Kir2.1.

Andersen-Tawil syndrome (ATS) type-1 is associated with loss-of-function mutations in KCNJ2 gene. KCNJ2 encodes the tetrameric inward-rectifier potassium channel Kir2.1, important to the resting phase of the cardiac action potential. Kir-channels' activity requires interaction with the agonist phosphatidylinositol-4,5-bisphosphate (PIP2). Two mutations were identified in ATS patients, V77E in the cytosolic N-terminal "slide helix" and M307V in the C-terminal cytoplasmic gate structure "G-loop." Current recordings in Kir2.1-expressing HEK cells showed that each of the two mutations caused Kir2.1 loss-of-function. Biotinylation and immunostaining showed that protein expression and trafficking of Kir2.1 to the plasma membrane were not affected by the mutations. To test the functional effect of the mutants in a heterozygote set, Kir2.1 dimers were prepared. Each dimer was composed of two Kir2.1 subunits joined with a flexible linker (i.e. WT-WT, WT dimer; WT-V77E and WT-M307V, mutant dimer). A tetrameric assembly of Kir2.1 is expected to include two dimers. The protein expression and the current density of WT dimer were equally reduced to ~25% of the WT monomer. Measurements from HEK cells and Xenopus oocytes showed that the expression of either WT-V77E or WT-M307V yielded currents of only about 20% compared to the WT dimer, supporting a dominant-negative effect of the mutants. Kir2.1 sensitivity to PIP2 was examined by activating the PIP2 specific voltage-sensitive phosphatase (VSP) that induced PIP2 depletion during current recordings, in HEK cells and Xenopus oocytes. PIP2 depletion induced a stronger and faster decay in Kir2.1 mutant dimers current compared to the WT dimer. BGP-15, a drug that has been demonstrated to have an anti-arrhythmic effect in mice, stabilized the Kir2.1 current amplitude following VSP-induced PIP2 depletion in cells expressing WT or mutant dimers. This study underlines the implication of mutations in cytoplasmic regions of Kir2.1. A newly developed calibrated VSP activation protocol enabled a quantitative assessment of changes in PIP2 regulation caused by the mutations. The results suggest an impaired function and a dominant-negative effect of the Kir2.1 variants that involve an impaired regulation by PIP2. This study also demonstrates that BGP-15 may be beneficial in restoring impaired Kir2.1 function and possibly in treating ATS symptoms.

Amplitude histogram-based method of analysis of patch clamp recordings that involve extreme changes in channel activity levels.

Many ion channels show low basal activity, which is increased hundreds-fold by the relevant gating factor. A classical example is the activation G-protein-activated K(+) channels (GIRK) by Gbetagamma subunit dimer. The extent of activation (relative to basal current), R(a), is an important physiological parameter, usually readily estimated from whole cell recordings. However, calculation of R(a) often becomes non-trivial in multi-channel patches because of extreme changes in activity upon activation, from a seemingly single-channel pattern to a macroscopic one. In such cases, calculation of the net current flowing through the channels in the patch, I, before and after activation may require different methods of analysis. To address this problem, we utilized neuronal GIRK channels activated by purified Gbetagamma in excised patches of Xenopus oocytes. Channels were expressed at varying densities, from a few to several hundreds per patch. We present a simple and fast method of calculating I using amplitude histogram analysis and establish its accuracy by comparing with I calculated from event lists. This method allows the analysis of extreme changes in I in multichannel patches, which would be impossible using the standard methods of idealization and event list generation.

In vivo, in vitro and in silico correlations of four de novo SCN1A missense mutations.

Mutations in the SCN1A gene, which encodes for the voltage-gated sodium channel NaV1.1, cause Dravet syndrome, a severe developmental and epileptic encephalopathy. Genetic testing of this gene is recommended early in life. However, predicting the outcome of de novo missense SCN1A mutations is difficult, since milder epileptic syndromes may also be associated. In this study, we correlated clinical severity with functional in vitro electrophysiological testing of channel activity and bioinformatics prediction of damaging mutational effects. Three patients, bearing the mutations p.Gly177Ala, p.Ser259Arg and p.Glu1923Arg, showed frequent intractable seizures that had started early in life, with cognitive and behavioral deterioration, consistent with classical Dravet phenotypes. These mutations failed to produce measurable sodium currents in a mammalian expression system, indicating complete loss of channel function. A fourth patient, who harbored the mutation p.Met1267Ile, though presenting with seizures early in life, showed lower seizure burden and higher cognitive function, matching borderland Dravet phenotypes. In correlation with this, functional analysis demonstrated the presence of sodium currents, but with partial loss of function. In contrast, six bioinformatics tools for predicting mutational pathogenicity suggested similar impact for all mutations. Likewise, homology modeling of the secondary and tertiary structures failed to reveal misfolding. In conclusion, functional studies using patch clamp are suggested as a prognostic tool, whereby detectable currents imply milder phenotypes and absence of currents indicate an unfavorable prognosis. Future development of automated patch clamp systems will facilitate the inclusion of such functional testing as part of personalized patient diagnostic schemes.

An inactivation gate in the selectivity filter of KCNQ1 potassium channels.

Inactivation is an inherent property of most voltage-gated K(+) channels. While fast N-type inactivation has been analyzed in biophysical and structural details, the mechanisms underlying slow inactivation are yet poorly understood. Here, we characterized a slow inactivation mechanism in various KCNQ1 pore mutants, including L273F, which hinders entry of external Ba(2+) to its deep site in the pore and traps it by slowing its egress. Kinetic studies, molecular modeling, and dynamics simulations suggest that this slow inactivation involves conformational changes that converge to the outer carbonyl ring of the selectivity filter, where the backbone becomes less flexible. This mechanism involves acceleration of inactivation kinetics and enhancement of Ba(2+) trapping at elevated external K(+) concentrations. Hence, KCNQ1 slow inactivation considerably differs from C-type inactivation where vacation of K(+) from the filter was invoked. We suggest that trapping of K(+) at s(1) due to filter rigidity and hindrance of the dehydration-resolvation transition underlie the slow inactivation of KCNQ1 pore mutants.

External barium affects the gating of KCNQ1 potassium channels and produces a pore block via two discrete sites.

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 Ba(2+) binding kinetics and the concentration and voltage dependence of Ba(2+) steady-state block. Our results indicate that extracellular Ba(2+) 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 Ba(2+) site that occludes the channel pore and could be simulated by a model of voltage-dependent block. (b) A fast superficial Ba(2+) 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 of voltage-dependent block cannot predict the complex impact of Ba(2+) on channel gating in low external K(+) solutions. Ba(2+) binding to this superficial site likely modifies the gating transitions states of KCNQ1. Both sites appear to reside in the permeation pathway as high external K(+) attenuates Ba(2+) inhibition of channel conductance and abolishes its impact on channel gating. Our data suggest that despite the high degree of homology of the pore region among the various K(+) channels, KCNQ1 channels display significant structural and functional uniqueness.

Kinetic modeling of Na(+)-induced, Gbetagamma-dependent activation of G protein-gated K(+) channels.

G protein-activated K(+)(GIRK) channels are activated by numerous neurotransmitters that act on Gi/o proteins, via a direct interaction with the Gbetagamma subunit of G proteins. In addition, GIRK channels are positively regulated by intracellular Na(+) via a direct interaction (fast pathway) and via a GGbetagamma-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 GalphaGDP and Gbetagamma subunits of G proteins. In this scenario, elevated Na(+) enhances basal dissociation of G protein heterotrimers, elevating free cellular Gbetagamma 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 GIRK1F137S channels, which are devoid of direct interaction with Na(+), was measured in excised membrane patches and used as an indicator of free GGbetagamma 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 GalphaGDP and GGbetagamma (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 Galphai3. 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 GGbetagamma effector.

Gbetagamma-dependent and Gbetagamma-independent basal activity of G protein-activated K+ channels.

Cardiac and neuronal G protein-activated K+ channels (GIRK; Kir3) open following the binding of Gbetagamma subunits, released from Gi/o proteins activated by neurotransmitters. GIRKs also possess basal activity contributing to the resting potential in neurons. It appears to depend largely on free Gbetagamma, but a Gbetagamma-independent component has also been envisaged. We investigated Gbetagamma dependence of the basal GIRK activity (A(GIRK,basal)) quantitatively, by titrated expression of Gbetagamma scavengers, in Xenopus oocytes expressing GIRK1/2 channels and muscarinic m2 receptors. The widely used Gbetagamma scavenger, myristoylated C terminus of beta-adrenergic kinase (m-cbetaARK), reduced A(GIRK,basal) by 70-80% and eliminated the acetylcholine-evoked current (I(ACh)). However, we found that m-cbetaARK directly binds to GIRK, complicating the interpretation of physiological data. Among several newly constructed Gbetagamma 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 Gbetagamma and Galpha in the plasma membrane, supporting the existence of preformed complexes of GIRK with G protein subunits. Increased expression of Gbetagamma 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 cbetaARK. These results demonstrate that a major part of Ibasal is Gbetagamma-dependent at all levels of channel expression, and only a small fraction (<10%) may be Gbetagamma-independent.

Single channel analysis of the regulation of GIRK1/GIRK4 channels by protein phosphorylation.

G-Protein activated, inwardly rectifying potassium channels (GIRKs) are important effectors of G-protein beta/gamma-subunits, playing essential roles in the humoral regulation of cardiac activity and also in higher brain functions. G-protein activation of channels of the GIRK1/GIRK4 heterooligomeric composition is controlled via phosphorylation by cyclic AMP dependent protein kinase (PKA) and dephosphorylation by protein phosphatase 2A (PP(2)A). To study the molecular mechanism of this unprecedented example of G-protein effector regulation, single channel recordings were performed on isolated patches of plasma membranes of Xenopus laevis oocytes. Our study shows that: (i) The open probability (P(o)) of GIRK1/GIRK4 channels, stimulated by coexpressed m(2)-receptors, was significantly increased upon addition of the catalytic subunit of PKA to the cytosolic face of an isolated membrane patch. (ii) At moderate concentrations of recombinant G(beta1/gamma2), used to activate the channel, P(o) was significantly reduced in patches treated with PP(2)A, when compared to patches with PKA-cs. (iii) Several single channel gating parameters, including modal gating behavior, were significantly different between phosphorylated and dephosphorylated channels, indicating different gating behavior between the two forms of the protein. Most of these changes were, however, not responsible for the marked difference in P(o) at moderate G-protein concentrations. (iv) An increase of the frequency of openings (f(o)) and a reduction of dwell time duration of the channel in the long-lasting C(5) state was responsible for facilitation of GIRK1/GIRK4 channels by protein phosphorylation. Dephosphorylation by PP(2)A led to an increase of G(beta1/gamma2) concentration required for full activation of the channel and hence to a reduction of the apparent affinity of GIRK1/GIRK4 for G(beta1/gamma2). (v) Although possibly not directly the target of protein phosphorylation/dephosphorylation, the last 20 C-terminal amino acids of the GIRK1 subunit are required for the reduction of apparent affinity for the G-protein by PP(2)A, indicating that they constitute an essential part of the off-switch.

Na+ promotes the dissociation between Galpha GDP and Gbeta gamma, activating G protein-gated K+ channels.

G protein-gated K(+) channels (GIRK, or Kir3) are activated by the direct binding of Gbetagamma or of cytosolic Na(+). Na(+) activation is fast, Gbetagamma-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 Gbetagamma scavenger, hence it is Gbetagamma-dependent; but it does not require GTP. We hypothesized that Na(+) elevates the cellular concentration of free Gbetagamma by promoting the dissociation of the Galphabetagamma heterotrimer into free Galpha(GDP) and Gbetagamma. Direct biochemical measurements showed that Na(+) causes a moderate decrease (approximately 2-fold) in the affinity of interaction between Galpha(GDP) and Gbetagamma. Furthermore, in accord with the predictions of our model, slow Na(+) activation was enhanced by mild coexpression of Galpha(i3). Our findings reveal a previously unknown mechanism of regulation of G proteins and demonstrate a novel Gbetagamma-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 Gbetagamma.