Gain-of-function KCNJ6 Mutation in a Severe Hyperkinetic Movement Disorder Phenotype.
ABSTRACT: Here, we describe a fourth case of a human with a de novo KCNJ6 (GIRK2) mutation, who presented with clinical findings of severe hyperkinetic movement disorder and developmental delay, similar to the Keppen-Lubinsky syndrome but without lipodystrophy. Whole-exome sequencing of the patient's DNA revealed a heterozygous de novo variant in the KCNJ6 (c.512T>G, p.Leu171Arg). We conducted in vitro functional studies to determine if this Leu-to-Arg mutation alters the function of GIRK2 channels. Heterologous expression of the mutant GIRK2 channel alone produced an aberrant basal inward current that lacked G protein activation, lost K+ selectivity and gained Ca2+ permeability. Notably, the inward current was inhibited by the Na+ channel blocker QX-314, similar to the previously reported weaver mutation in murine GIRK2. Expression of a tandem dimer containing GIRK1 and GIRK2(p.Leu171Arg) did not lead to any currents, suggesting heterotetramers are not functional. In neurons expressing p.Leu171Arg GIRK2 channels, these changes in channel properties would be expected to generate a sustained depolarization, instead of the normal G protein-gated inhibitory response, which could be mitigated by expression of other GIRK subunits. The identification of the p.Leu171Arg GIRK2 mutation potentially expands the Keppen-Lubinsky syndrome phenotype to include severe dystonia and ballismus. Our study suggests screening for dominant KCNJ6 mutations in the evaluation of patients with severe movement disorders, which could provide evidence to support a causal role of KCNJ6 in neurological channelopathies.
Project description:Keppen-Lubinsky syndrome (KPLBS) is a rare disease mainly characterized by severe developmental delay and intellectual disability, microcephaly, large prominent eyes, a narrow nasal bridge, a tented upper lip, a high palate, an open mouth, tightly adherent skin, an aged appearance, and severe generalized lipodystrophy. We sequenced the exomes of three unrelated individuals affected by KPLBS and found de novo heterozygous mutations in KCNJ6 (GIRK2), which encodes an inwardly rectifying potassium channel and maps to the Down syndrome critical region between DIRK1A and DSCR4. In particular, two individuals shared an in-frame heterozygous deletion of three nucleotides (c.455_457del) leading to the loss of one amino acid (p.Thr152del). The third individual was heterozygous for a missense mutation (c.460G>A) which introduces an amino acid change from glycine to serine (p.Gly154Ser). In agreement with animal models, the present data suggest that these mutations severely impair the correct functioning of this potassium channel. Overall, these results establish KPLBS as a channelopathy and suggest that KCNJ6 (GIRK2) could also be a candidate gene for other lipodystrophies. We hope that these results will prompt investigations in this unexplored class of inwardly rectifying K(+) channels.
Project description:Event-related oscillations (EROs) represent highly heritable neuroelectric correlates of cognitive processes that manifest deficits in alcoholics and in offspring at high risk to develop alcoholism. Theta ERO to targets in the visual oddball task has been shown to be an endophenotype for alcoholism. A family-based genome-wide association study was performed for the frontal theta ERO phenotype using 634 583 autosomal single nucleotide polymorphisms (SNPs) genotyped in 1560 family members from 117 families densely affected by alcohol use disorders, recruited in the Collaborative Study on the Genetics of Alcoholism. Genome-wide significant association was found with several SNPs on chromosome 21 in KCNJ6 (a potassium inward rectifier channel; KIR3.2/GIRK2), with the most significant SNP at P = 4.7 × 10(-10)). The same SNPs were also associated with EROs from central and parietal electrodes, but with less significance, suggesting that the association is frontally focused. One imputed synonymous SNP in exon four, highly correlated with our top three SNPs, was significantly associated with the frontal theta ERO phenotype. These results suggest KCNJ6 or its product GIRK2 account for some of the variations in frontal theta band oscillations. GIRK2 receptor activation contributes to slow inhibitory postsynaptic potentials that modulate neuronal excitability, and therefore influence neuronal networks.
Project description:Event related oscillations (EROs) are heritable measures of neurocognitive function that have served as useful phenotype in genetic research. A recent family genome-wide association study (GWAS) by the Collaborative Study on the Genetics of Alcoholism (COGA) found that theta EROs during visual target detection were associated at genome-wide levels with several single nucleotide polymorphisms (SNPs), including a synonymous SNP, rs702859, in the KCNJ6 gene that encodes GIRK2, a G-protein inward rectifying potassium channel that regulates excitability of neuronal networks. The present study examined the effect of the KCNJ6 SNP (rs702859), previously associated with theta ERO to targets in a visual oddball task, on theta EROs during reward processing in a monetary gambling task. The participants were 1601 adolescent and young adult offspring within the age-range of 17-25years (800 males and 801 females) from high-dense alcoholism families as well as control families of the COGA prospective study. Theta ERO power (3.5-7.5Hz, 200-500ms post-stimulus) was compared across genotype groups. ERO theta power at central and parietal regions increased as a function of the minor allele (A) dose in the genotype (AA>AG>GG) in both loss and gain conditions. These findings indicate that variations in the KCNJ6 SNP influence magnitude of theta oscillations at posterior loci during the evaluation of loss and gain, reflecting a genetic influence on neuronal circuits involved in reward-processing. Increased theta power as a function of minor allele dose suggests more efficient cognitive processing in those carrying the minor allele of the KCNJ6 SNPs. Future studies are needed to determine the implications of these genetic effects on posterior theta EROs as possible "protective" factors, or as indices of delays in brain maturation (i.e., lack of frontalization).
Project description:Alcohol abuse and dependence have proven to be complex genetic traits that are influenced by environmental factors. Primate and human studies have shown that early life stress increases the propensity for alcohol abuse in later life. The reinforcing properties of alcohol are mediated by dopaminergic signaling; however, there is little evidence to indicate how stress alters alcohol reinforcement. KCNJ6 (the gene encoding G-protein-coupled inwardly rectifying potassium channel 2 (GIRK2)) is a brain expressed potassium channel with inhibitory effects on dopaminergic tone. The properties of GIRK2 have been shown to be enhanced by the stress peptide corticotrophin-releasing hormone. Therefore, we sought to examine the role of KCNJ6 polymorphisms in adult alcohol dependence and stress-related alcohol abuse in adolescents. We selected 11 SNPs in the promoter region of KCNJ6, which were genotyped in 1152 adult alcohol dependents and 1203 controls. One SNP, rs2836016, was found to be associated with alcohol dependence (p=0.01, false discovery rate). We then assessed rs2836016 in an adolescent sample of 261 subjects, which were characterized for early life stress and adolescent hazardous drinking, defined using the Alcohol Use Disorders Identification Test (AUDIT), to examine gene-environment interactions. In the adolescent sample, the risk genotype of rs2836016 was significantly associated with increased AUDIT scores, but only in those individuals exposed to high levels of psychosocial stress in early life (p=0.01). Our findings show that KCNJ6 is associated with alcohol dependence and may moderate the effect of early psychosocial stress on risky alcohol drinking in adolescents. We have identified a candidate gene for future studies investigating a possible functional link between the response to stress and alcohol reinforcement.
Project description:Opioids are commonly used as effective analgesics for the treatment of acute and chronic pain. However, considerable individual differences have been widely observed in sensitivity to opioid analgesics. We focused on a G-protein-activated inwardly rectifying potassium (GIRK) channel subunit, GIRK2, that is an important molecule in opioid transmission. In our initial polymorphism search, a total of nine single-nucleotide polymorphisms (SNPs) were identified in the whole exon, 5'-flanking, and exon-intron boundary regions of the KCNJ6 gene encoding GIRK2. Among them, G-1250A and A1032G were selected as representative SNPs for further association studies. In an association study of 129 subjects who underwent major open abdominal surgery, the A/A genotype in the A1032G SNP and -1250G/1032A haplotype were significantly associated with increased postoperative analgesic requirements compared with other genotypes and haplotypes. The total dose (mean+/-SEM) of rescue analgesics converted to equivalent oral morphine doses was 20.45+/-9.27 mg, 10.84+/-2.24 mg, and 13.07+/-2.39 mg for the A/A, A/G, and G/G genotypes in the A1032G SNP, respectively. Additionally, KCNJ6 gene expression levels in the 1032A/A subjects were significantly decreased compared with the 1032A/G and 1032G/G subjects in a real-time quantitative PCR analysis using human brain tissues, suggesting that the 1032A/A subjects required more analgesics because of lower KCNJ6 gene expression levels and consequently insufficient analgesic effects. The results indicate that the A1032G SNP and G-1250A/A1032G haplotype could serve as markers that predict increased analgesic requirements. Our findings will provide valuable information for achieving satisfactory pain control and open new avenues for personalized pain treatment.
Project description:G protein-activated inwardly rectifying K+ channels (GIRK) generate slow inhibitory postsynaptic potentials in the brain via G(i/o) protein-coupled receptors. GIRK2, a GIRK subunit, is widely abundant in the brain and has been implicated in various functions and pathologies, such as learning and memory, reward, motor coordination, and Down syndrome. Down syndrome, the most prevalent cause of mental retardation, results from the presence of an extra maternal chromosome 21 (trisomy 21), which comprises the Kcnj6 gene (GIRK2). The present study examined the behaviors and cellular physiology properties in mice harboring a single trisomy of the Kcnj6 gene. Kcnj6 triploid mice exhibit deficits in hippocampal-dependent learning and memory, altered responses to rewards, hampered depotentiation, a form of excitatory synaptic plasticity, and have accentuated long-term synaptic depression. Collectively the findings suggest that triplication of Kcnj6 gene may play an active role in some of the abnormal neurological phenotypes found in Down syndrome.
Project description:Down syndrome (DS), trisomy 21, is caused by increased dose of genes present on human chromosome 21 (HSA21). The gene-dose hypothesis argues that a change in the dose of individual genes or regulatory sequences on HSA21 is necessary for creating DS-related phenotypes, including cognitive impairment. We focused on a possible role for Kcnj6, the gene encoding Kir3.2 (Girk2) subunits of a G-protein-coupled inwardly-rectifying potassium channel. This gene resides on a segment of mouse Chromosome 16 that is present in one extra copy in the genome of the Ts65Dn mouse, a well-studied genetic model of DS. Kir3.2 subunit-containing potassium channels serve as effectors for a number of postsynaptic metabotropic receptors including GABAB receptors. Several studies raise the possibility that increased Kcnj6 dose contributes to synaptic and cognitive abnormalities in DS. To assess directly a role for Kcnj6 gene dose in cognitive deficits in DS, we produced Ts65Dn mice that harbor only 2 copies of Kcnj6 (Ts65Dn:Kcnj6++- mice). The reduction in Kcnj6 gene dose restored to normal the hippocampal level of Kir3.2. Long-term memory, examined in the novel object recognition test with the retention period of 24h, was improved to the level observed in the normosomic littermate control mice (2N:Kcnj6++). Significantly, both short-term and long-term potentiation (STP and LTP) was improved to control levels in the dentate gyrus (DG) of the Ts65Dn:Kcnj6++- mouse. In view of the ability of fluoxetine to suppress Kir3.2 channels, we asked if fluoxetine-treated DG slices of Ts65Dn:Kcnj6+++ mice would rescue synaptic plasticity. Fluoxetine increased STP and LTP to control levels. These results are evidence that increased Kcnj6 gene dose is necessary for synaptic and cognitive dysfunction in the Ts65Dn mouse model of DS. Strategies aimed at pharmacologically reducing channel function should be explored for enhancing cognition in DS.
Project description:G-protein coupled inwardly rectifying potassium (GIRK) channels are effectors determining degree of analgesia experienced upon opioid receptor activation by endogenous and exogenous opioids. The impact of GIRK-related genetic variation on human pain responses has received little research attention. We used a tag single nucleotide polymorphism (SNP) approach to comprehensively examine pain-related effects of KCNJ3 (GIRK1) and KCNJ6 (GIRK2) gene variation. Forty-one KCNJ3 and 69 KCNJ6 tag SNPs were selected, capturing the known variability in each gene. The primary sample included 311 white patients undergoing total knee arthroplasty in whom postsurgical oral opioid analgesic medication order data were available. Primary sample findings were then replicated in an independent white sample of 63 healthy pain-free individuals and 75 individuals with chronic low back pain (CLBP) who provided data regarding laboratory acute pain responsiveness (ischemic task) and chronic pain intensity and unpleasantness (CLBP only). Univariate quantitative trait analyses in the primary sample revealed that 8 KCNJ6 SNPs were significantly associated with the medication order phenotype (P < .05); overall effects of the KCNJ6 gene (gene set-based analysis) just failed to reach significance (P = .054). No significant KCNJ3 effects were observed. A continuous GIRK Related Risk Score (GRRS) was derived in the primary sample to summarize each individual's number of KCNJ6 "pain risk" alleles. This GRRS was applied to the replication sample, which revealed significant associations (P < .05) between higher GRRS values and lower acute pain tolerance and higher CLBP intensity and unpleasantness. Results suggest further exploration of the impact of KCNJ6 genetic variation on pain outcomes is warranted.
Project description:G-protein-gated inward rectifying potassium channels (GIRKs) require G<sub>??</sub> subunits and phosphorylated phosphatidylinositides (PIPs) for gating. Although studies have provided insight into these interactions, the mechanism of how these events are modulated by G<sub>??</sub> and the binding affinity between PIPs and GIRKs remains poorly understood. Here, native ion mobility mass spectrometry is employed to directly monitor small molecule binding events to mouse GIRK2. GIRK2 binds the toxin tertiapin Q and PIPs selectively and with significantly higher affinity than other phospholipids. A mutation in GIRK2 that causes a rotation in the cytoplasmic domain, similarly to G<sub>??</sub>-binding to the wild-type channel, revealed differences in the selectivity towards PIPs. More specifically, PIP isoforms known to weakly activate GIRKs have decreased binding affinity. Taken together, our results reveal selective small molecule binding and uncover a mechanism by which rotation of the cytoplasmic domain can modulate GIRK•PIP interactions.
Project description:G-protein-gated inward rectifying potassium channels (GIRKs) require Gβγ subunits and phosphorylated phosphatidylinositides (PIPs) for gating. Although studies have provided insight into these interactions, the mechanism of how these events are modulated by Gβγ and the binding affinity between PIPs and GIRKs remains poorly understood. Here, native ion mobility mass spectrometry is employed to directly monitor small molecule binding events to mouse GIRK2. GIRK2 binds the toxin tertiapin Q and PIPs selectively and with significantly higher affinity than other phospholipids. A mutation in GIRK2 that causes a rotation in the cytoplasmic domain, similarly to Gβγ-binding to the wild-type channel, revealed differences in the selectivity towards PIPs More specifically, PIP isoforms known to weakly activate GIRKs have decreased binding affinity. Additionally, denaturing mass spectrometry and tryptic digest liquid chromatography mass spectrometry analysis was performed to confirm phosphorylation to GIRK2.