The GABA A receptor ␥2 subunit mutation, Q351X, associated with generalized epilepsy with febrile seizures plus (GEFSϩ), created a loss of function with homozygous expression. However, heterozygous ␥2(ϩ/Ϫ) gene deletion mice are seizure free, suggesting that the loss of one GABRG2 allele alone in heterozygous patients may not be sufficient to produce epilepsy. Here we show that the mutant ␥2 subunit was immature and retained in the endoplasmic reticulum (ER). With heterozygous coexpression of ␥2S/␥2S(Q351X) subunits and ␣1 and ␤2 subunits, the trafficking deficient mutant ␥2 subunit reduced trafficking of wild-type partnering subunits, which was not seen in the hemizygous gene deletion control. Consequently, the function of the heterozygous receptor channel was reduced to less than the hemizygous control and to less than half of the wild-type receptors with a full gene dose. Pulse-chase experiments demonstrated that in the presence of the mutant ␥2S(Q351X) subunit, wild-type ␣1 subunits degraded more substantially within 1 h of translation. We showed that the basis for this dominant-negative effect on wild-type receptors was due to an interaction between mutant and wild-type subunits. The mutant subunit oligomerized with wild-type subunits and trapped them in the ER, subjecting them to glycosylation arrest and ER-associated degradation (ERAD) through the ubiquitin proteosome system. Thus, we hypothesize that a likely explanation for the GEFSϩ phenotype is a dominant-negative suppression of wild-type receptors by the mutant ␥2S subunit in combination with loss of mutant ␥2S subunit protein function.

Genetic epilepsy and neurodegenerative diseases are two common neurological disorders conventionally viewed as being unrelated. A subset of patients with severe genetic epilepsies with impaired development and often death respond poorly to anticonvulsant drug therapy, suggesting a need for new therapeutic targets. Previously, we reported that multiple GABAA receptor epilepsy mutations caused protein misfolding and abnormal receptor trafficking. Here we establish in a novel model of a severe human genetic epileptic encephalopathy, the Gabrg2+/Q390X knock-in mouse, that in addition to impairing inhibitory neurotransmission, mutant GABAA receptor γ2(Q390X) subunits accumulated and aggregated intracellularly, activated caspase 3 and caused widespread, age-dependent neurodegeneration. These novel findings suggest that the fundamental protein metabolism and cellular consequences of the epilepsy-associated mutant γ2(Q390X) ion channel subunit are not fundamentally different from those associated with neurodegeneration. The study has far-reaching significance for identification of conserved pathological cascades and mechanism-based therapies that overlap genetic epilepsies and neurodegenerative diseases.

Epileptic encephalopathies are a devastating group of severe childhood onset epilepsies with medication-resistant seizures and poor developmental outcomes. Many epileptic encephalopathies have a genetic aetiology and are often associated with de novo mutations in genes mediating synaptic transmission, including GABA receptor subunit genes. Recently, we performed next generation sequencing on patients with a spectrum of epileptic encephalopathy phenotypes, and we identified five novel (A106T, I107T, P282S, R323W and F343L) and one known (R323Q) de novo GABRG2 pathogenic variants (mutations) in eight patients. To gain insight into the molecular basis for how these mutations contribute to epileptic encephalopathies, we compared the effects of the mutations on the properties of recombinant α1β2γ2L GABA receptors transiently expressed in HEK293T cells. Using a combination of patch clamp recording, immunoblotting, confocal imaging and structural modelling, we characterized the effects of these GABRG2 mutations on GABA receptor biogenesis and channel function. Compared with wild-type α1β2γ2L receptors, GABA receptors containing a mutant γ2 subunit had reduced cell surface expression with altered subunit stoichiometry or decreased GABA-evoked whole-cell current amplitudes, but with different levels of reduction. While a causal role of these mutations cannot be established directly from these results, the functional analysis together with the genetic information suggests that these GABRG2 variants may be major contributors to the epileptic encephalopathy phenotypes. Our study further expands the GABRG2 phenotypic spectrum and supports growing evidence that defects in GABAergic neurotransmission participate in the pathogenesis of genetic epilepsies including epileptic encephalopathies.

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CO2 chemoreception may be related to modulation of inward rectifier K+ channels (Kir channels) in brainstem neurons. Kir4.1 is expressed predominantly in the brainstem and inhibited during hypercapnia. Although the homomeric Kir4.1 only responds to severe intracellular acidification, coexpression of Kir4.1 with Kir5.1 greatly enhances channel sensitivities to CO2 and pH. To understand the biophysical and molecular mechanisms underlying the modulation of these currents by CO2 and pH, heteromeric Kir4.1–Kir5.1 were studied in inside-out patches. These Kir4.1–Kir5.1 currents showed a single channel conductance of 59 pS with open-state probability (P open) ∼ 0.4 at pH 7.4. Channel activity reached the maximum at pH 8.5 and was completely suppressed at pH 6.5 with pKa 7.45. The effect of low pH on these currents was due to selective suppression of P open without evident effects on single channel conductance, leading to a decrease in the channel mean open time and an increase in the mean closed time. At pH 8.5, single-channel currents showed two sublevels of conductance at ∼1/4 and 3/4 of the maximal openings. None of them was affected by lowering pH. The Kir4.1–Kir5.1 currents were modulated by phosphatidylinositol-4,5-bisphosphate (PIP2) that enhanced baseline P open and reduced channel sensitivity to intracellular protons. In the presence of 10 μM PIP2, the Kir4.1–Kir5.1 showed a pKa value of 7.22. The effect of PIP2, however, was not seen in homomeric Kir4.1 currents. The CO2/pH sensitivities were related to a lysine residue in the NH2 terminus of Kir4.1. Mutation of this residue (K67M, K67Q) completely eliminated the CO2 sensitivity of both homomeric Kir4.1 and heteromeric Kir4.1–Kir5.1. In excised patches, interestingly, the Kir4.1–Kir5.1 carrying K67M mutation remained sensitive to low pHi. Such pH sensitivity, however, disappeared in the presence of PIP2. The effect of PIP2 on shifting the titration curve of wild-type and mutant channels was totally abolished when Arg178 in Kir5.1 was mutated. Thus, these studies demonstrate a heteromeric Kir channel that can be modulated by both acidic and alkaline pH, show the modulation of pH sensitivity of Kir channels by PIP2, and provide information of the biophysical and molecular mechanisms underlying the Kir modulation by intracellular protons.

Background The widely-used macrolide antibiotic azithromycin increases risk of cardiovascular and sudden cardiac death, although the underlying mechanisms are unclear. Case reports, including the one we document here, demonstrate that azithromycin can cause rapid, polymorphic ventricular tachycardia in the absence of QT prolongation, indicating a novel proarrhythmic syndrome. We investigated the electrophysiologic effects of azithromycin in vivo and in vitro using mice, cardiomyocytes, and human ion channels heterologously expressed in human embryonic kidney (HEK 293) and Chinese hamster ovary (CHO) cells. Methods and Results In conscious telemetered mice, acute intraperitoneal and oral administration of azithromycin caused effects consistent with multi-ion channel block, with significant sinus slowing and increased PR, QRS, QT, and QTc intervals, as seen with azithromycin overdose. Similarly, in HL-1 cardiomyocytes, the drug slowed sinus automaticity, reduced phase 0 upstroke slope, and prolonged action potential duration. Acute exposure to azithromycin reduced peak SCN5A currents in HEK cells (IC50=110±3μM) and Na+ current in mouse ventricular myocytes. However, with chronic (24hour) exposure, azithromycin caused a ~2-fold increase in both peak and late SCN5A currents, with findings confirmed for INa in cardiomyocytes. Mild block occurred for K+ currents representing IKr (CHO cells expressing hERG; IC50=219±21μM) and IKs (CHO cells expressing KCNQ1+KCNE1; IC50=184±12μM), while azithromycin suppressed L-type Ca++ currents (rabbit ventricular myocytes; IC50=66.5±4μM) and IK1 (HEK cells expressing Kir2.1; IC50=44±3μM). Conclusions Chronic exposure to azithromycin increases cardiac Na+ current to promote intracellular Na+ loading, providing a potential mechanistic basis for the novel form of proarrhythmia seen with this macrolide antibiotic.

With a worldwide incidence as high as 6.7% of children, febrile seizures are one of the most common reasons for seeking pediatric care, but the mechanisms underlying generation of febrile seizures are poorly understood. Febrile seizures have been suspected to have a genetic basis, and recently, mutations in GABA A receptor and sodium channel genes have been identified that are associated with febrile seizures and generalized seizures with febrile seizures plus pedigrees. Pentameric GABA A receptors mediate the majority of fast synaptic inhibition in the brain and are composed of combinations of ␣(1-6), ␤(1-3), and ␥(1-3) subunits. In ␣␤␥2 GABA A receptors, the ␥2 subunit is critical for receptor trafficking, clustering, and synaptic maintenance, and mutations in the ␥2 subunit have been monogenically associated with autosomal dominant transmission of febrile seizures. Here, we report that whereas trafficking of wild-type ␣1␤2␥2 receptors was slightly temperature dependent, trafficking of mutant ␣1␤2␥2 receptors containing ␥2 subunit mutations [␥2(R43Q), ␥2(K289M), and ␥2(Q351X)] associated with febrile seizures was highly temperature dependent. In contrast, trafficking of mutant ␣1␤2␥2 receptors containing an ␣1 subunit mutation [␣1(A322D)] not associated with febrile seizures was not highly temperature dependent. Brief increases in temperature from 37 to 40°C rapidly (Ͻ10 min) impaired trafficking and/or accelerated endocytosis of heterozygous mutant ␣1␤2␥2 receptors containing ␥2 subunit mutations associated with febrile seizures but not of wild-type ␣1␤2␥2 receptors or heterozygous mutant ␣1(A322D)␤2␥2 receptors, suggesting that febrile seizures may be produced by a temperatureinduced dynamic reduction of susceptible mutant surface GABA A receptors in response to fever.

Abstract-Activation of protein kinase A (PKA) increases Na ϩ current derived from the human cardiac Na ϩ channel, hH1, in a slow, nonsaturable manner. This effect is prevented by compounds that disrupt plasma membrane recycling, implying enhanced trafficking of channels to the cell membrane as the mechanism responsible for Na ϩ current potentiation. To investigate the molecular basis of this effect, preferred consensus sites (serines 483, 571, and 593) and alternative sites phosphorylated by PKA in the rat heart isoform (serines 525 and 528) were removed in the I-II interdomain linker, a region in the channel previously implicated in the PKA response. Our results demonstrate that the presence of either serine 525 or 528 is required for Na ϩ current potentiation. The role of amino acid sequences that can mediate channel-protein interactions was also examined. Removal of a PDZ domain-binding motif at the carboxy terminus of hH1 did not alter the PKA response. The I-II interdomain linker of the channel contains 3 sites (479RKR481, 533RRR535, and 659RQR661) with the sequence RXR, a motif known to mediate retention of proteins in the endoplasmic reticulum (ER). The PKA-mediated increase in Na ϩ current was abolished when all 3 sites were eliminated, with RRR at position 533 to 535 primarily responsible for this effect. These results demonstrate that both ␣-subunit phosphorylation and the presence of putative ER retention signals are required for the PKA-mediated increase in cardiac Na