Sequence dependent hypermutation of the immunoglobulin heavy chain in cultured B cells

The variable (V) regions of immunoglobulin heavy and light chains undergo high rates of somatic mutation during the immune response. Although point mutations accumulate throughout the V regions and their immediate flanking sequences, analysis of large numbers of mutations that have arisen in vivo reveal that the triplet AGC appears to be most susceptible to mutation. We have stably transfected B cell lines with γ2a heavy chain constructs containing TAG nonsense codons in their V regions that are part of either a putative (T)AGC hot spot or a (T)AGA non-hot spot motif. Using an ELISA spot assay to detect revertants and fluctuation analysis to determine rates of mutation, the rate of reversion of the TAG nonsense codon has been determined for different motifs in different parts of the V region. In the NSO plasma cell line, the (T)AGC hot spot motif mutates at rates of ≈6 × 10⁻⁴/bp per generation and ≈3 × 10⁻⁵/bp per generation at residues 38 and 94 in the V region. At each of these locations, the (T)AGC hot spot motif is 20–30 times more likely to undergo mutation than the (T)AGA non-hot spot motif. Moreover, the AGA non-hot spot motif mutates at as high a rate as the hot spot motif when it is located adjacent to hot spot motifs, suggesting that more extended sequences influence susceptibility to mutation.     

Vitamin C modulates the metabolic and cytokine profiles,alleviates hepatic endoplasmic reticulum stress,and increases the life span of Gulo?/? mice

Suboptimal intake of dietary vitamin C (ascorbate) increases the risk of several chronic diseases but the exact metabolic pathways affected are still unknown. In this study, we examined the metabolic profile of mice lacking the enzyme gulonolactone oxidase (Gulo) required for the biosynthesis of ascorbate. Gulo^−/− mice were supplemented with 0%, 0.01%, and 0.4% ascorbate (w/v) in drinking water and serum was collected for metabolite measurements by targeted mass spectrometry. We also quantified 42 serum cytokines and examined the levels of different stress markers in liver. The metabolic profiles of Gulo^−/− mice treated with ascorbate were different from untreated Gulo^−/− and normal wild type mice. The cytokine profiles of Gulo^−/− mice, in return, overlapped the profile of wild type animals upon 0.01% or 0.4% vitamin C supplementation. The life span of Gulo^−/− mice increased with the amount of ascorbate in drinking water. It also correlated significantly with the ratios of serum arginine/lysine, tyrosine/phenylalanine, and the ratio of specific species of saturated/unsaturated phosphatidylcholines. Finally, levels of hepatic phosphorylated endoplasmic reticulum associated stress markers IRE1α and eIF2α correlated inversely with serum ascorbate and life span suggesting that vitamin C modulates endoplasmic reticulum stress response and longevity in Gulo^−/− mice.     

E3 ubiquitin ligase GRAIL controls primary T cell activation and oral tolerance

T cell unresponsiveness or anergy is one of the mechanisms that maintain inactivity of self-reactive lymphocytes. E3 ubiquitin ligases are important mediators of the anergic state. The RING finger E3 ligase GRAIL is thought to selectively function in anergic T cells but its mechanism of action and its role in vivo are largely unknown. We show here that genetic deletion of Grail in mice leads not only to loss of an anergic phenotype in various models but also to hyperactivation of primary CD4⁺ T cells. Grail^−/− CD4⁺ T cells hyperproliferate in vitro to TCR stimulation alone or with concomitant anti-CD28 costimulation, with transient increased survival. In vitro differentiated T helper 1 cells show slight but significant hypersecretion of IFN-γ in Grail^−/− mice whereas Th2 and Th17 cytokine secretions are unchanged. Consistent with defective in vitro anergy, oral tolerance is abolished in vivo in OT-II TCR transgenic Grail^−/− mice fed with ovalbumin. In experimental allergic encephalitis, a model of organ-specific autoimmunity, oral tolerization with myelin basic protein was abrogated as well in Grail^−/− mice. On the protein level, Grail^−/− naïve T cells show no significant differences of total and phosphorylated levels of ZAP70, phospholipase Cγ1, and MAP kinases p38 and JNK but elevated baseline levels of MAP kinase ERK1/2. In summary, we define a role for GRAIL in primary T cell activation, survival, and differentiation. In addition, we formally prove an indispensable role for GRAIL in T cell anergy and oral tolerance—a promising, antigen-specific strategy to treat autoimmune diseases.     

Lack of lacto/neolacto-glycolipids enhances the formation of glycolipid-enriched microdomains,facilitating B cell activation

In a previous study, we demonstrated that β1,3-N-acetylglucosaminyltransferase 5 (B3gnt5) is a lactotriaosylceramide (Lc₃Cer) synthase that synthesizes a precursor structure for lacto/neolacto-series glycosphingolipids (GSLs) in in vitro experiments. Here, we generated B3gnt5-deficient (B3gnt5^−/−) mice to investigate the in vivo biological functions of lacto/neolacto-series GSLs. In biochemical analyses, lacto/neolacto-series GSLs were confirmed to be absent and no Lc₃Cer synthase activity was detected in the tissues of these mice. These results demonstrate that β3GnT5 is the sole enzyme synthesizing Lc₃Cer in vivo. Ganglioside GM1, known as a glycosphingolipid-enriched microdomain (GEM) marker, was found to be up-regulated in B3gnt5^−/− B cells by flow cytometry and fluorescence microscopy. However, no difference in the amount of GM1 was observed by TLC-immunoblotting analysis. The GEM-stained puncta on the surface of B3gnt5^−/− resting B cells were brighter and larger than those of WT cells. These results suggest that structural alteration of GEM occurs in B3gnt5^−/− B cells. We next examined whether BCR signaling-related proteins, such as BCR, CD19, and the signaling molecule Lyn, had moved into or out of the GEM fraction. In B3gnt5^−/− B cells, these molecules were enriched in the GEM fraction or adjacent fraction. Moreover, B3gnt5^−/− B cells were more sensitive to the induction of intracellular phosphorylation signals on BCR stimulation and proliferated more vigorously than WT B cells. Together, these results suggest that lacto/neolacto-series GSLs play an important role in clustering of GEMs and tether-specific proteins, such as BCR, CD19, and related signaling molecules to the GEMs.     

The Impact of a Recently Approved Automated Insulin Delivery System on Glycemic,Sleep, and Psychosocial Outcomes in Older Adults With Type 1 Diabetes: A Pilot Study

Background:Older adults with type 1 diabetes (≥65 years) are often under-represented in clinical trials of automated insulin delivery (AID) systems. We sought to test the efficacy of a recently FDA-approved AID system in this population.Methods:Participants with type 1 diabetes used sensor-augmented pump (SAP) therapy for four weeks and then used an AID system (Control-IQ) for four weeks. In addition to glucose control variables, patient-reported outcomes (PRO) were assessed with questionnaires and sleep parameters were assessed by actigraphy.Results:Fifteen older adults (mean age 68.7 ± 3.3, HbA1c of 7.0 ± 0.8) completed the pilot trial. Glycemic outcomes improved during AID compared to SAP. During AID use, mean glucose was 146.0 mg/dL; mean percent time in range (TIR, 70-180 mg/dL) was 79.6%; median time below 70 mg/dL was 1.1%. The AID system was in use 92.6% ± 7.0% of the time. Compared to SAP, while participants were on AID the TIR increased significantly (+10%, P = .002) accompanied by a reduction in both time above 180 mg/dL (−6.9%, P = .005) and below 70 mg/dl (−0.4%, P = .053). Diabetes-related distress decreased significantly while using AID (P = .028), but sleep parameters remained unchanged.Conclusions:Use of this AID system in older adults improved glycemic control with high scores in ease of use, trust, and usability. Participants reported an improvement in diabetes distress with AID use. There were no significant changes in sleep.     

A knockin mouse model of the Bardet-Biedl syndrome 1 M390R mutation has cilia defects, ventriculomegaly, retinopathy, and obesity

Bardet–Biedl syndrome (BBS) is a genetically heterogeneous disorder that results in retinal degeneration, obesity, cognitive impairment, polydactyly, renal abnormalities, and hypogenitalism. Of the 12 known BBS genes, BBS1 is the most commonly mutated, and a single missense mutation (M390R) accounts for ≈80% of BBS1 cases. To gain insight into the function of BBS1, we generated a Bbs1^M390R/M390R knockin mouse model. Mice homozygous for the M390R mutation recapitulated aspects of the human phenotype, including retinal degeneration, male infertility, and obesity. The obese mutant mice were hyperphagic and hyperleptinemic and exhibited reduced locomotor activity but no elevation in mean arterial blood pressure. Morphological evaluation of Bbs1 mutant brain neuroanatomy revealed ventriculomegaly of the lateral and third ventricles, thinning of the cerebral cortex, and reduced volume of the corpus striatum and hippocampus. Similar abnormalities were also observed in the brains of Bbs2^−/−, Bbs4^−/−, and Bbs6^−/− mice, establishing these neuroanatomical defects as a previously undescribed BBS mouse model phenotype. Ultrastructural examination of the ependymal cell cilia that line the enlarged third ventricle of the Bbs1 mutant brains showed that, whereas the 9 + 2 arrangement of axonemal microtubules was intact, elongated cilia and cilia with abnormally swollen distal ends were present. Together with data from transmission electron microscopy analysis of photoreceptor cell connecting cilia, the Bbs1 M390R mutation does not affect axonemal structure, but it may play a role in the regulation of cilia assembly and/or function.     

PU.1 as an essential activator for the expression of gp91phox gene in human peripheral neutrophils, monocytes, and B lymphocytes

We have reported a deficiency of a 91-kDa glycoprotein component of the phagocyte NADPH oxidase (gp91^phox) in neutrophils, monocytes, and B lymphocytes of a patient with X chromosome-linked chronic granulomatous disease. Sequence analysis of his gp91^phox gene revealed a single-base mutation (C → T) at position −53. Electrophoresis mobility-shift assays showed that both PU.1 and hematopoietic-associated factor 1 (HAF-1) bound to the inverted PU.1 consensus sequence centered at position −53 of the gp91^phox promoter, and the mutation at position −53 strongly inhibited the binding of both factors. It was also indicated that a mutation at position −50 strongly inhibited PU.1 binding but hardly inhibited HAF-1 binding, and a mutation at position −56 had an opposite binding specificity for these factors. In transient expression assay using HEL cells, which express PU.1 and HAF-1, the mutations at positions −53 and −50 significantly reduced the gp91^phox promoter activity; however, the mutation at position −56 did not affect the promoter activity. In transient cotransfection study, PU.1 dramatically activated the gp91^phox promoter in Jurkat T cells, which originally contained HAF-1 but not PU.1. In addition, the single-base mutation (C → T) at position −52 that was identified in a patient with chronic granulomatous disease inhibited the binding of PU.1 to the promoter. We therefore conclude that PU.1 is an essential activator for the expression of gp91^phox gene in human neutrophils, monocytes, and B lymphocytes.     

C-type lectin Mincle is an activating receptor for pathogenic fungus, Malassezia

Mincle (also called as Clec4e and Clecsf9) is a C-type lectin receptor expressed in activated phagocytes. Recently, we have demonstrated that Mincle is an FcRγ-associated activating receptor that senses damaged cells. To search an exogenous ligand(s), we screened pathogenic fungi using cell line expressing Mincle, FcRγ, and NFAT-GFP reporter. We found that Mincle specifically recognizes the Malassezia species among 50 different fungal species tested. Malassezia is a pathogenic fungus that causes skin diseases, such as tinea versicolor and atopic dermatitis, and fatal sepsis. However, the specific receptor on host cells has not been identified. Mutation of the putative mannose-binding motif within C-type lectin domain of Mincle abrogated Malassezia recognition. Analyses of glycoconjugate microarray revealed that Mincle selectively binds to α-mannose but not mannan. Thus, Mincle may recognize specific geometry of α-mannosyl residues on Malassezia species and use this to distinguish them from other fungi. Malassezia activated macrophages to produce inflammatory cytokines/chemokines. To elucidate the physiological function of Mincle, Mincle-deficient mice were established. Malassezia-induced cytokine/chemokine production by macrophages from Mincle^−/− mice was significantly impaired. In vivo inflammatory responses against Malassezia was also impaired in Mincle^−/− mice. These results indicate that Mincle is the first specific receptor for Malassezia species to be reported and plays a crucial role in immune responses to this fungus.     

Separation of killing and tumorigenic effects of an alkylating agent in mice defective in two of the DNA repair genes

Alkylation of DNA at the O⁶-position of guanine is one of the most critical events leading to mutation, cancer, and cell death. The enzyme O⁶-methylguanine-DNA methyltransferase repairs O⁶-methylguanine as well as a minor methylated base, O⁴-methylthymine, in DNA. Mouse lines deficient in the methyltransferase (MGMT) gene are hypersensitive to both the killing and to the tumorigenic effects of alkylating agents. We now show that these dual effects of an alkylating agent can be dissociated by introduction of an additional defect in mismatch repair. Mice with mutations in both alleles of the MGMT gene and one of the mismatch repair genes, MLH1, are as resistant to methylnitrosourea (MNU) as are wild-type mice, in terms of survival, but do have numerous tumors after receiving MNU. In contrast to MGMT^−/− MLH1^+/+ mice with decrease in size of the thymus and hypocellular bone marrow after MNU administration, no conspicuous change was found in MGMT^−/− MLH1^−/− mice treated in the same manner. Thus, killing and tumorigenic effects of an alkylating agent can be dissociated by preventing mismatch repair pathways.     

Rate and molecular spectrum of spontaneous mutations in the bacterium Escherichia coli as determined by whole-genome sequencing

Knowledge of the rate and nature of spontaneous mutation is fundamental to understanding evolutionary and molecular processes. In this report, we analyze spontaneous mutations accumulated over thousands of generations by wild-type Escherichia coli and a derivative defective in mismatch repair (MMR), the primary pathway for correcting replication errors. The major conclusions are (i) the mutation rate of a wild-type E. coli strain is ∼1 × 10⁻³ per genome per generation; (ii) mutations in the wild-type strain have the expected mutational bias for G:C > A:T mutations, but the bias changes to A:T > G:C mutations in the absence of MMR; (iii) during replication, A:T > G:C transitions preferentially occur with A templating the lagging strand and T templating the leading strand, whereas G:C > A:T transitions preferentially occur with C templating the lagging strand and G templating the leading strand; (iv) there is a strong bias for transition mutations to occur at 5′ApC3′/3′TpG5′ sites (where bases 5′A and 3′T are mutated) and, to a lesser extent, at 5′GpC3′/3′CpG5′ sites (where bases 5′G and 3′C are mutated); (v) although the rate of small (≤4 nt) insertions and deletions is high at repeat sequences, these events occur at only 1/10th the genomic rate of base-pair substitutions. MMR activity is genetically regulated, and bacteria isolated from nature often lack MMR capacity, suggesting that modulation of MMR can be adaptive. Thus, comparing results from the wild-type and MMR-defective strains may lead to a deeper understanding of factors that determine mutation rates and spectra, how these factors may differ among organisms, and how they may be shaped by environmental conditions.     

Specific involvement of postsynaptic GluN2B-containing NMDA receptors in the developmental elimination of corticospinal synapses

The GluN2B (GluRε2/NR2B) and GluN2A (GluRε1/NR2A) NMDA receptor (NMDAR) subtypes have been differentially implicated in activity-dependent synaptic plasticity. However, little is known about the respective contributions made by these two subtypes to developmental plasticity, in part because studies of GluN2B KO [Grin2b^−/− (2b^−/−)] mice are hampered by early neonatal mortality. We previously used in vitro slice cocultures of rodent cerebral cortex (Cx) and spinal cord (SpC) to show that corticospinal (CS) synapses, once present throughout the SpC, are eliminated from the ventral side during development in an NMDAR-dependent manner. To study subtype specificity of NMDAR in this developmental plasticity, we cocultured Cx and SpC slices derived from postnatal day 0 (P0) animals with different genotypes [2b^−/−, Grin2a^−/− (2a^−/−), or WT mice]. The distribution of CS synapses was studied electrophysiologically and with a voltage-sensitive dye. Synapse elimination on the ventral side was blocked in WT(Cx)-2b^−/−(SpC) pairs but not in WT(Cx)-2a^−/−(SpC) or 2b^−/−(Cx)-WT(SpC) pairs. CS axonal regression was also observed through live imaging of CS axons labeled with enhanced yellow fluorescent protein (EYFP) through exo utero electroporation. These findings suggest that postsynaptic GluN2B is selectively involved in CS synapse elimination. In addition, the elimination was not blocked in 2a^−/− SpC slices, where Ca²⁺ entry through GluN2B-mediated CS synaptic currents was reduced to the same level as in 2b^−/− slices, suggesting that the differential effect of GluN2B and GluN2A in CS synapse elimination might not be explained based solely on greater Ca²⁺ entry through GluN2B-containing channels.     

Neural deficits contribute to respiratory insufficiency in Pompe disease

Pompe disease is a severe form of muscular dystrophy due to glycogen accumulation in all tissues, especially striated muscle. Disease severity is directly related to the deficiency of acid α-glucosidase (GAA), which degrades glycogen in the lysosome. Respiratory dysfunction is a hallmark of the disease, muscle weakness has been viewed as the underlying cause, and the possibility of an associated neural contribution has not been evaluated previously. Therefore, we examined behavioral and neurophysiological aspects of breathing in 2 animal models of Pompe disease—the Gaa^−/− mouse and a transgenic line (MTP) expressing GAA only in skeletal muscle, as well as a detailed analysis of the CNS in a Pompe disease patient. Glycogen content was elevated in the Gaa^−/− mouse cervical spinal cord. Retrograde labeling of phrenic motoneurons showed significantly greater soma size in Gaa^−/− mice vs. isogenic controls, and glycogen was observed in Gaa^−/− phrenic motoneurons. Ventilation, assessed via plethysmography, was attenuated during quiet breathing and hypercapnic challenge in Gaa^−/− mice (6 to >21 months of age) vs. controls. We confirmed that MTP mice had normal diaphragmatic contractile properties; however, MTP mice had ventilation similar to the Gaa^−/− mice during quiet breathing. Neurophysiological recordings indicated that efferent phrenic nerve inspiratory burst amplitudes were substantially lower in Gaa^−/− and MTP mice vs. controls. In human samples, we demonstrated similar pathology in the cervical spinal cord and greater accumulation of glycogen in spinal cord compared with brain. We conclude that neural output to the diaphragm is deficient in Gaa^−/− mice, and therapies targeting muscle alone may be ineffective in Pompe disease.     

Interaction analysis of Mycobacterium tuberculosis between the host environment and highly mutated genes from population genetic structure comparison

We aimed to investigate the genetic and demographic differences and interactions between areas where observed genomic variations in Mycobacterium tuberculosis (M. tb) were distributed uniformly in cold and hot spots.The cold and hot spot areas were identified using the reported incidence of TB over the previous 5 years. Whole genome sequencing was performed on 291 M. tb isolates between January and June 2018. Analysis of molecular variance and a multifactor dimensionality reduction (MDR) model was applied to test gene-gene-environment interactions. Adjusted odds ratios (OR) and 95% confidence intervals (CI) were computed to test the extent to which genetic mutation affects the TB epidemic using a multivariate logistic regression model.The percentage of the Beijing family strain in hot spots was significantly higher than that in cold spots (64.63% vs 50.69%, P = .022), among the elderly, people with a low BMI, and those having a history of contact with a TB patient (all P < .05). Individuals from cold spot areas had a higher frequency of out-of-town traveling (P < .05). The mutation of Rv1186c, Rv3900c, Rv1508c, Rv0210, and an Intergenic Region (SNP site: 3847237) showed a significant difference between cold and hot spots. (P < .001). The MDR model displayed a clear negative interaction effect of age groups with BMI (interaction entropy: −3.55%) and mutation of Rv0210 (interaction entropy: −2.39%). Through the mutations of Rv0210 and BMI had a low independent effect (interaction entropy: −1.46%).Our data suggests a statistically significant role of age, BMI and the polymorphisms of Rv0210 genes in the transmission and development of M. tb. The results provide clues for the study of susceptibility genes of M. tb in different populations. The characteristic strains showed a local epidemic. Strengthening genotype monitoring of strains in various regions can be used as an early warning signal of epidemic spillover.     

Tumors of DNA mismatch repair-deficient hosts exhibit dramatic increases in genomic instability

DNA mismatch repair (MMR) deficiency is associated with an increased mutational burden and predisposition to certain malignancies. Relatively little is known, however, about gene-specific mutation frequencies within MMR-deficient primary tumors. Thymic lymphomas from Msh2^−/− mice were thus analyzed by using a lacI-based transgenic shuttle-phage mutation detection system. All tumors exhibited greatly elevated lacI gene mutation frequencies, ranging from 3.2- to 17.4-fold above the ≈15-fold elevations present within normal Msh2^−/− thymi. In addition, lacI genes harboring multiple changes, including clusters of mutations, were found in thymic tumor DNA. The results suggest that an additional mutator activity, such as an error-prone DNA polymerase, leads to increased genomic instability in these MMR-deficient tumors.     

Akt1 is critical for acute inflammation and histamine-mediated vascular leakage

Akt1 is implicated in cell metabolism, survival migration, and gene expression; however, little is known about the role of specific Akt isoforms during inflammation in vivo. Thus, we directly explored the roles of the isoforms Akt1 and Akt2 in acute inflammation models by using mice deficient in either Akt1 or Akt2. Akt1^−/− mice showed a markedly reduced edema versus Akt2^−/− and WT controls, and the reduced inflammation was associated with a dramatic decrease in neutrophil and monocyte infiltration. The loss of Akt1 did not affect leukocyte functions in vitro, and bone marrow transplant experiments suggest that host Akt1 regulates leukocyte emigration into inflamed tissues. Moreover, carrageenan-induced edema and the direct propermeability actions of bradykinin and histamine were reduced dramatically in Akt1^−/− versus WT mice. These findings are supported by in vitro experiments showing that Akt1 deficiency or blockade of nitric oxide synthase markedly reduces histamine-stimulated changes in transendothelial electrical resistance of microvascular endothelial cells. Collectively, these results suggest that Akt1 is necessary for acute inflammation and exerts its actions primarily via regulation of vascular permeability, leading to edema and leukocyte extravasation.     

From the Cover: A human IL10 BAC transgene reveals tissue-specific control of IL-10 expression and alters disease outcome

Interleukin (IL)-10 is an immunoregulatory cytokine that is produced by diverse cell populations. Studies in mice suggest that the cellular source of IL-10 is a key determinant in various disease pathologies, yet little is known regarding the control of tissue-specific human IL-10 expression. To assess cell type-specific human IL-10 regulation, we created a human IL-10 transgenic mouse with a bacterial artificial chromosome (hIL10BAC) in which the IL10 gene is positioned centrally. Since human IL-10 is biologically active in the mouse, we could examine the in vivo capacity of tissue-specific human IL-10 expression to recapitulate IL-10-dependent phenotypes by reconstituting Il10^−/− mice (Il10^−/−/hIL10BAC). In response to LPS, Il10^−/−/hIL10BAC mice proficiently regulate IL-10-target genes and normalize sensitivity to LPS toxicity via faithful human IL-10 expression from macrophages and dendritic cells. However, in the Leishmania donovani model of pathogen persistence, Il10^−/−/hIL10BAC mice did not develop the characteristic IL-10⁺IFN-γ⁺CD4 T cell subset thought to mediate persistence and, like Il10^−/− mice, cleared the parasites. Furthermore, the IL-10-promoting cytokine IL-27 failed to regulate transgenic human IL-10 production in CD4⁺ T cells in vitro which together suggests that the hIL10BAC encodes for weak T cell-specific IL-10 expression. Thus, the hIL10BAC mouse is a model of human gene structure and function revealing tissue-specific regulatory requirements for IL-10 expression which impacts disease outcomes.     

Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice

Receptor tyrosine kinases Flt-1 and Flk-1/KDR, and their ligand, the vascular endothelial growth factor (VEGF), were shown to be essential for angiogenesis in the mouse embryo by gene targeting. Flk-1/KDR null mutant mice exhibited impaired endothelial and hematopoietic cell development. On the other hand, Flt-1 null mutation resulted in early embryonic death at embryonic day 8.5, showing disorganization of blood vessels, such as overgrowth of endothelial cells. Flt-1 differs from Flk-1 in that it displays a higher affinity for VEGF but lower kinase activity, suggesting the importance of its extracellular domain. To examine the biological role of Flt-1 in embryonic development and vascular formation, we deleted the kinase domain without affecting the ligand binding region. Flt-1 tyrosine kinase-deficient homozygous mice (flt-1^TK−/−) developed normal vessels and survived. However, VEGF-induced macrophage migration was strongly suppressed in flt-1^TK−/− mice. These results indicate that Flt-1 without tyrosine kinase domain is sufficient to allow embryonic development with normal angiogenesis, and that a receptor tyrosine kinase plays a main biological role as a ligand-binding molecule.     

Normal cerebellar development but susceptibility to seizures in mice lacking G protein-coupled, inwardly rectifying K+ channel GIRK2

G protein-gated, inwardly rectifying K⁺ channels (GIRK) are effectors of G protein-coupled receptors for neurotransmitters and hormones and may play an important role in the regulation of neuronal excitability. GIRK channels may be important in neurodevelopment, as suggested by the recent finding that a point mutation in the pore region of GIRK2 (G156S) is responsible for the weaver (wv) phenotype. The GIRK2 G156S gene gives rise to channels that exhibit a loss of K⁺ selectivity and may also exert dominant-negative effects on G_βγ-activated K⁺ currents. To investigate the physiological role of GIRK2, we generated mutant mice lacking GIRK2. Unlike wv/wv mutant mice, GIRK2 −/− mice are morphologically indistinguishable from wild-type mice, suggesting that the wv phenotype is likely due to abnormal GIRK2 function. Like wv/wv mice, GIRK2 −/− mice have much reduced GIRK1 expression in the brain. They also develop spontaneous seizures and are more susceptible to pharmacologically induced seizures using a γ-aminobutyric acid antagonist. Moreover, wv/− mice exhibit much milder cerebellar abnormalities than wv/wv mice, indicating a dosage effect of the GIRK2 G156S mutation. Our results indicate that the weaver phenotypes arise from a gain-of-function mutation of GIRK2 and that GIRK1 and GIRK2 are important mediators of neuronal excitability in vivo.