Moran Rubinstein

Gαi and Gβγ Jointly Regulate the Conformations of a Gβγ Effector, the Neuronal G Protein-activated K+ Channel (GIRK)

Stable complexes among G proteins and effectors are an emerging concept in cell signaling. The prototypical Gβγ effector G protein-activated K+ channel (GIRK; Kir3) physically interacts with Gβγ but also with Gαi/o. Whether and how Gαi/o subunits regulate GIRK in vivo is unclear. We studied triple interactions among GIRK subunits 1 and 2, Gαi3 and Gβγ. We used in vitro protein interaction assays and in vivo intramolecular Förster resonance energy transfer (i-FRET) between fluorophores attached to N and C termini of either GIRK1 or GIRK2 subunit. We demonstrate, for the first time, that Gβγ and Gαi3 distinctly and interdependently alter the conformational states of the heterotetrameric GIRK1/2 channel. Biochemical experiments show that Gβγ greatly enhances the binding of GIRK1 subunit to Gαi3GDP and, unexpectedly, to Gαi3GTP. i-FRET showed that both Gαi3 and Gβγ induced distinct conformational changes in GIRK1 and GIRK2. Moreover, GIRK1 and GIRK2 subunits assumed unique, distinct conformations when coexpressed with a “constitutively active” Gαi3 mutant and Gβγ together. These conformations differ from those assumed by GIRK1 or GIRK2 after separate coexpression of either Gαi3 or Gβγ. Both biochemical and i-FRET data suggest that GIRK acts as the nucleator of the GIRK-Gα-Gβγ signaling complex and mediates allosteric interactions between GαiGTP and Gβγ. Our findings imply that Gαi/o and the Gαiβγ heterotrimer can regulate a Gβγ effector both before and after activation by neurotransmitters.

Gαi3 primes the G protein‐activated K+ channels for activation by coexpressed Gβγ in intact Xenopus oocytes

G protein‐activated K+ channels (GIRK) mediate postsynaptic inhibitory effects of neurotransmitters in the atrium and in the brain by coupling to G protein‐coupled receptors (GPCRs). In neurotransmitter‐dependent GIRK signalling, Gβγ is released from the heterotrimeric Gαβγ complex upon GPCR activation, activating the channel and attenuating its rectification. Now it becomes clear that Gα is more than a mere Gβγ donor. We have proposed that Gαi3–GDP regulates GIRK gating, keeping its basal activity low but priming (predisposing) the channel for activation by agonist in intact cells, and by Gβγ in excised patches. Here we have further investigated GIRK priming by Gαi3 using a model in which the channel was activated by coexpression of Gβγ, and the currents were measured in intact Xenopus oocytes using the two‐electrode voltage clamp technique. This method enables the bypass of GPCR activation during examination of the regulation of the channel in intact cells. Using this method, we further characterize the priming phenomenon. We tested and excluded the possibility that our estimates of priming are affected by artifacts caused by series resistance or large K+ fluxes. We demonstrate that both Gαi3 and membrane‐attached Gβγ scavenger protein, m‐phosducin, reduce the basal channel activity. However, Gαi3 allows robust channel activation by coexpressed Gβγ, in sharp contrast to m‐phosducin, which causes a substantial reduction in the total Gβγ‐induced current. Furthermore, Gαi3 also does not impair the Gβγ‐dependent attenuation of the channel rectification, in contrast to m‐phosducin, which prevents this Gβγ‐induced modulation. The Gαi3‐induced enhancement of direct activation of GIRK by Gβγ, demonstrated here for the first time in intact cells, strongly supports the hypothesis that Gαi regulates GIRK gating under physiological conditions.

Reciprocal Changes in Phosphorylation and Methylation of Mammalian Brain Sodium Channels in Response to Seizures

Background: Sodium channels underlie neuronal excitability and are regulated by seizures. Results: Mass spectrometric analysis of brain sodium channels revealed novel phosphorylation and methylation sites that decreased and increased, respectively, after seizures. Inducing methylation increased sodium channel activity. Conclusion: Reciprocal phosphorylation and methylation after seizures will alter sodium channel function. Significance: Such regulation would impact neuronal excitability. Voltage-gated sodium (Nav) channels initiate action potentials in brain neurons and are primary therapeutic targets for anti-epileptic drugs controlling neuronal hyperexcitability in epilepsy. The molecular mechanisms underlying abnormal Nav channel expression, localization, and function during development of epilepsy are poorly understood but can potentially result from altered posttranslational modifications (PTMs). For example, phosphorylation regulates Nav channel gating, and has been proposed to contribute to acquired insensitivity to anti-epileptic drugs exhibited by Nav channels in epileptic neurons. However, whether changes in specific brain Nav channel PTMs occur acutely in response to seizures has not been established. Here, we show changes in PTMs of the major brain Nav channel, Nav1.2, after acute kainate-induced seizures. Mass spectrometry-based proteomic analyses of Nav1.2 purified from the brains of control and seizure animals revealed a significant down-regulation of phosphorylation at nine sites, primarily located in the interdomain I-II linker, the region of Nav1.2 crucial for phosphorylation-dependent regulation of activity. Interestingly, Nav1.2 in the seizure samples contained methylated arginine (MeArg) at three sites. These MeArgs were adjacent to down-regulated sites of phosphorylation, and Nav1.2 methylation increased after seizure. Phosphorylation and MeArg were not found together on the same tryptic peptide, suggesting reciprocal regulation of these two PTMs. Coexpression of Nav1.2 with the primary brain arginine methyltransferase PRMT8 led to a surprising 3-fold increase in Nav1.2 current. Reciprocal regulation of phosphorylation and MeArg of Nav1.2 may underlie changes in neuronal Nav channel function in response to seizures and also contribute to physiological modulation of neuronal excitability.

Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by GαiGDP and Gβγ

G protein activated K+ channels (GIRK, Kir3) are switched on by direct binding of Gβγ following activation of Gi/o proteins via G protein‐coupled receptors (GPCRs). Although Gαi subunits do not activate GIRKs, they interact with the channels and regulate the gating pattern of the neuronal heterotetrameric GIRK1/2 channel (composed of GIRK1 and GIRK2 subunits) expressed in Xenopus oocytes. Coexpressed Gαi3 decreases the basal activity (Ibasal) and increases the extent of activation by purified or coexpressed Gβγ. Here we show that this regulation is exerted by the ‘inactive’ GDP‐bound Gαi3GDP and involves the formation of Gαi3βγ heterotrimers, by a mechanism distinct from mere sequestration of Gβγ‘away’ from the channel. The regulation of basal and Gβγ‐evoked current was produced by the ‘constitutively inactive’ mutant of Gαi3, Gαi3G203A, which strongly binds Gβγ, but not by the ‘constitutively active’ mutant, Gαi3Q204L, or by Gβγ‐scavenging proteins. Furthermore, regulation by Gαi3G203A was unique to the GIRK1 subunit; it was not observed in homomeric GIRK2 channels. In vitro protein interaction experiments showed that purified Gβγ enhanced the binding of Gαi3GDP to the cytosolic domain of GIRK1, but not GIRK2. Homomeric GIRK2 channels behaved as a ‘classical’ Gβγ effector, showing low Ibasal and strong Gβγ‐dependent activation. Expression of Gαi3G203A did not affect either Ibasal or Gβγ‐induced activation. In contrast, homomeric GIRK1* (a pore mutant able to form functional homomeric channels) exhibited large Ibasal and was poorly activated by Gβγ. Expression of Gαi3GDP reduced Ibasal and restored the ability of Gβγ to activate GIRK1*, like in GIRK1/2. Transferring the unique distal segment of the C terminus of GIRK1 to GIRK2 rendered the latter functionally similar to GIRK1*. These results demonstrate that GIRK1 containing channels are regulated by both Gαi3GDP and Gβγ, while GIRK2 is a Gβγ‐effector insensitive to Gαi3GDP.

Genetic background modulates impaired excitability of inhibitory neurons in a mouse model of Dravet syndrome

Dominant loss-of-function mutations in voltage-gated sodium channel NaV1.1 cause Dravet Syndrome, an intractable childhood-onset epilepsy. NaV1.1(+/-) Dravet Syndrome mice in C57BL/6 genetic background exhibit severe seizures, cognitive and social impairments, and premature death. Here we show that Dravet Syndrome mice in pure 129/SvJ genetic background have many fewer seizures and much less premature death than in pure C57BL/6 background. These mice also have a higher threshold for thermally induced seizures, fewer myoclonic seizures, and no cognitive impairment, similar to patients with Genetic Epilepsy with Febrile Seizures Plus. Consistent with this mild phenotype, mutation of NaV1.1 channels has much less physiological effect on neuronal excitability in 129/SvJ mice. In hippocampal slices, the excitability of CA1 Stratum Oriens interneurons is selectively impaired, while the excitability of CA1 pyramidal cells is unaffected. NaV1.1 haploinsufficiency results in increased rheobase and threshold for action potential firing and impaired ability to sustain high-frequency firing. Moreover, deletion of NaV1.1 markedly reduces the amplification and integration of synaptic events, further contributing to reduced excitability of interneurons. Excitability is less impaired in inhibitory neurons of Dravet Syndrome mice in 129/SvJ genetic background. Because specific deletion of NaV1.1 in forebrain GABAergic interneuons is sufficient to cause the symptoms of Dravet Syndrome in mice, our results support the conclusion that the milder phenotype in 129/SvJ mice is caused by lesser impairment of sodium channel function and electrical excitability in their forebrain interneurons. This mild impairment of excitability of interneurons leads to a milder disease phenotype in 129/SvJ mice, similar to Genetic Epilepsy with Febrile Seizures Plus in humans.

Dissecting the phenotypes of Dravet syndrome by gene deletion

Neurological and psychiatric syndromes often have multiple disease traits, yet it is unknown how such multi-faceted deficits arise from single mutations. Haploinsufficiency of the voltage-gated sodium channel Nav1.1 causes Dravet syndrome, an intractable childhood-onset epilepsy with hyperactivity, cognitive deficit, autistic-like behaviours, and premature death. Deletion of Nav1.1 channels selectively impairs excitability of GABAergic interneurons. We studied mice having selective deletion of Nav1.1 in parvalbumin- or somatostatin-expressing interneurons. In brain slices, these deletions cause increased threshold for action potential generation, impaired action potential firing in trains, and reduced amplification of postsynaptic potentials in those interneurons. Selective deletion of Nav1.1 in parvalbumin- or somatostatin-expressing interneurons increases susceptibility to thermally-induced seizures, which are strikingly prolonged when Nav1.1 is deleted in both interneuron types. Mice with global haploinsufficiency of Nav1.1 display autistic-like behaviours, hyperactivity and cognitive impairment. Haploinsufficiency of Nav1.1 in parvalbumin-expressing interneurons causes autistic-like behaviours, but not hyperactivity, whereas haploinsufficiency in somatostatin-expressing interneurons causes hyperactivity without autistic-like behaviours. Heterozygous deletion in both interneuron types is required to impair long-term spatial memory in context-dependent fear conditioning, without affecting short-term spatial learning or memory. Thus, the multi-faceted phenotypes of Dravet syndrome can be genetically dissected, revealing synergy in causing epilepsy, premature death and deficits in long-term spatial memory, but interneuron-specific effects on hyperactivity and autistic-like behaviours. These results show that multiple disease traits can arise from similar functional deficits in specific interneuron types.

Recruitment of Gβγ controls the basal activity of G‐protein coupled inwardly rectifying potassium (GIRK) channels: crucial role of distal C terminus of GIRK1

The G‐protein coupled inwardly rectifying potassium (GIRK) channel is an important mediator of neurotransmission via Gβγ subunit of the heterotrimeric Gi/o protein released by G‐protein coupled receptor (GPCR) activation. Channels containing the GIRK1 subunit exhibit high basal currents, whereas channels that are formed by the GIRK2 subunit have very low basal currents. GIRK1‐containing channels, but not channels consisting of GIRK2 only, recruit Gβγ to the plasma membrane. The Gα subunit of the G protein is not recruited by either GIRK1/2 or GIRK2. The unique distal C terminus of GIRK1 (G1‐dCT) endows the channel with strong interaction with Gβγ, and deletion of G1‐dCT abolishes the Gβγ recruitment and reduces the basal currents. These findings suggest that the basal activity of GIRK channels depends on channel‐induced recruitment of Gβγ. The unique C terminus of GIRK1 subunit plays an important role in Gβγ recruitment.

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

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 GIRK1/2s 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.

Preferential Association with Gβγ Over Gα Governs the Activity of a G Protein-Activated K+ Channel

G protein-activated K+ channels (GIRK; Kir3) channels are prototypical effectors of Gβγ. Inhibitory neurotransmitters evoke K+ currents via GIRK, but GIRKs also have basal Gβγ-dependent activity. We proposed that basal and evoked activities are interdependently regulated, but the mechanism remains unknown. We show that heterologous expression of neuronal GIRK1/2 channels is accompanied by a large increase in surface expression of Gβγ but not Gαi. Calibrated protein expression within a range of less than one to several tens of molecules/μm2 and mathematical modeling suggest that each GIRK1/2 “drags” to plasma membrane 3 to 4 molecules of Gβγ but less than 2 molecules of Gαi. The effector-dependent, non-stoichiometric changes in Gβγ and Gαi levels quantitatively and qualitatively account for the reciprocal changes in basal and evoked activities upon incremental expression of GIRK1/2. A similar reciprocal relation between basal and evoked currents is found in hippocampal neurons, suggesting that preferential association of GIRK with Gβγ over Gαi plays a role in controlling excitability.

Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+channel by GαiGDPand Gβγ: Divergent regulation of GIRK1 and GIRK2 subunits by Gαiand Gβγ

G protein activated K+ channels (GIRK, Kir3) are switched on by direct binding of Gβγ following activation of Gi/o proteins via G protein‐coupled receptors (GPCRs). Although Gαi subunits do not activate GIRKs, they interact with the channels and regulate the gating pattern of the neuronal heterotetrameric GIRK1/2 channel (composed of GIRK1 and GIRK2 subunits) expressed in Xenopus oocytes. Coexpressed Gαi3 decreases the basal activity (Ibasal) and increases the extent of activation by purified or coexpressed Gβγ. Here we show that this regulation is exerted by the ‘inactive’ GDP‐bound Gαi3GDP and involves the formation of Gαi3βγ heterotrimers, by a mechanism distinct from mere sequestration of Gβγ‘away’ from the channel. The regulation of basal and Gβγ‐evoked current was produced by the ‘constitutively inactive’ mutant of Gαi3, Gαi3G203A, which strongly binds Gβγ, but not by the ‘constitutively active’ mutant, Gαi3Q204L, or by Gβγ‐scavenging proteins. Furthermore, regulation by Gαi3G203A was unique to the GIRK1 subunit; it was not observed in homomeric GIRK2 channels. In vitro protein interaction experiments showed that purified Gβγ enhanced the binding of Gαi3GDP to the cytosolic domain of GIRK1, but not GIRK2. Homomeric GIRK2 channels behaved as a ‘classical’ Gβγ effector, showing low Ibasal and strong Gβγ‐dependent activation. Expression of Gαi3G203A did not affect either Ibasal or Gβγ‐induced activation. In contrast, homomeric GIRK1* (a pore mutant able to form functional homomeric channels) exhibited large Ibasal and was poorly activated by Gβγ. Expression of Gαi3GDP reduced Ibasal and restored the ability of Gβγ to activate GIRK1*, like in GIRK1/2. Transferring the unique distal segment of the C terminus of GIRK1 to GIRK2 rendered the latter functionally similar to GIRK1*. These results demonstrate that GIRK1 containing channels are regulated by both Gαi3GDP and Gβγ, while GIRK2 is a Gβγ‐effector insensitive to Gαi3GDP.