Nontruncating SCN1A mutations associated with severe myoclonic epilepsy of infancy impair cell surface expression.

Mutations in SCN1A, encoding the voltage-gated sodium channel Na(V)1.1, are the most common cause of severe myoclonic epilepsy of infancy (SMEI) or Dravet syndrome. SMEI is most often associated with premature truncations of Na(V)1.1 that cause loss of function, but nontruncating mutations also occur. We hypothesized that some nontruncating mutations might impair trafficking of Na(V)1.1 to the plasma membrane. Here we demonstrated that seven nontruncating missense or in-frame deletion mutations (L986F, delF1289, R1648C, F1661S, G1674R, and G1979E) exhibited reduced cell surface expression relative to wild type (WT) Na(V)1.1 consistent with impaired trafficking. We tested whether two commonly prescribed antiepileptic drugs (phenytoin, lamotrigine), as well as the cystic fibrosis transmembrane conductance regulator (CFTR) trafficking corrector VRT-325, could rescue cell surface and functional expression of two representative Na(V)1.1 mutants (R1648C, G1674R). Treatment of cells with phenytoin increased cell surface expression of WT-Na(V)1.1 and both mutant channels, whereas lamotrigine only increased surface expression of R1648C. VRT-325 did not alter surface expression of WT-Na(V)1.1 or mutant channels. Although phenytoin increased surface expression of G1674R, channel function was not restored, suggesting that this mutation also causes an intrinsic loss of function. Both phenytoin and lamotrigine increased functional expression of R1648C, but lamotrigine also increased persistent sodium current evoked by this mutation. Our findings indicate that certain nontruncating SCN1A mutations associated with SMEI have impaired cell surface expression and that some alleles may be amenable to pharmacological rescue of this defect. However, rescue of dysfunctional Na(V)1.1 channels to the plasma membrane could contribute to exacerbating rather than ameliorating the disease.

Brain activity and desire for Internet video game play.

OBJECTIVE: Recent studies have suggested that the brain circuitry mediating cue-induced desire for video games is similar to that elicited by cues related to drugs and alcohol. We hypothesized that desire for Internet video games during cue presentation would activate similar brain regions to those that have been linked with craving for drugs or pathologic gambling. METHODS: This study involved the acquisition of diagnostic magnetic resonance imaging and functional magnetic resonance imaging data from 19 healthy male adults (age, 18-23 years) following training and a standardized 10-day period of game play with a specified novel Internet video game, "War Rock" (K2 Network, Irvine, CA). Using segments of videotape consisting of 5 contiguous 90-second segments of alternating resting, matched control, and video game-related scenes, desire to play the game was assessed using a 7-point visual analogue scale before and after presentation of the videotape. RESULTS: In responding to Internet video game stimuli, compared with neutral control stimuli, significantly greater activity was identified in left inferior frontal gyrus, left parahippocampal gyrus, right and left parietal lobe, right and left thalamus, and right cerebellum (false discovery rate <0.05, P < .009243). Self-reported desire was positively correlated with the ß values of left inferior frontal gyrus, left parahippocampal gyrus, and right and left thalamus. Compared with the general players, subjects who played more Internet video game showed significantly greater activity in right medial frontal lobe, right and left frontal precentral gyrus, right parietal postcentral gyrus, right parahippocampal gyrus, and left parietal precuneus gyrus. Controlling for total game time, reported desire for the Internet video game in the subjects who played more Internet video game was positively correlated with activation in right medial frontal lobe and right parahippocampal gyrus. DISCUSSION: The present findings suggest that cue-induced activation to Internet video game stimuli may be similar to that observed during cue presentation in persons with substance dependence or pathologic gambling. In particular, cues appear to commonly elicit activity in the dorsolateral prefrontal, orbitofrontal cortex, parahippocampal gyrus, and thalamus.

Multiplexed transposon-mediated stable gene transfer in human cells.

Generation of cultured human cells stably expressing one or more recombinant gene sequences is a widely used approach in biomedical research, biotechnology, and drug development. Conventional methods are not efficient and have severe limitations especially when engineering cells to coexpress multiple transgenes or multiprotein complexes. In this report, we harnessed the highly efficient, nonviral, and plasmid-based piggyBac transposon system to enable concurrent genomic integration of multiple independent transposons harboring distinct protein-coding DNA sequences. Flow cytometry of cell clones derived from a single multiplexed transfection demonstrated approximately 60% (three transposons) or approximately 30% (four transposons) stable coexpression of all delivered transgenes with selection for a single marker transposon. We validated multiplexed piggyBac transposon delivery by coexpressing large transgenes encoding a multisubunit neuronal voltage-gated sodium channel (SCN1A) containing a pore-forming subunit and two accessory subunits while using two additional genes for selection. Previously unobtainable robust sodium current was demonstrated through 38 passages, suitable for use on an automated high-throughput electrophysiology platform. Cotransfection of three large (up to 10.8 kb) piggyBac transposons generated a heterozygous SCN1A stable cell line expressing two separate alleles of the pore-forming subunit and two accessory subunits (total of four sodium channel subunits) with robust functional expression. We conclude that the piggyBac transposon system can be used to perform multiplexed stable gene transfer in cultured human cells, and this technology may be valuable for applications requiring concurrent expression of multiprotein complexes.

Distinct subdomains of the KCNQ1 S6 segment determine channel modulation by different KCNE subunits.

Modulation of voltage-gated potassium (KV) channels by the KCNE family of single transmembrane proteins has physiological and pathophysiological importance. All five KCNE proteins (KCNE1-KCNE5) have been demonstrated to modulate heterologously expressed KCNQ1 (KV7.1) with diverse effects, making this channel a valuable experimental platform for elucidating structure-function relationships and mechanistic differences among members of this intriguing group of accessory subunits. Here, we specifically investigated the determinants of KCNQ1 inhibition by KCNE4, the least well-studied KCNE protein. In CHO-K1 cells, KCNQ1, but not KCNQ4, is strongly inhibited by coexpression with KCNE4. By studying KCNQ1-KCNQ4 chimeras, we identified two adjacent residues (K326 and T327) within the extracellular end of the KCNQ1 S6 segment that determine inhibition of KCNQ1 by KCNE4. This dipeptide motif is distinct from neighboring S6 sequences that enable modulation by KCNE1 and KCNE3. Conversely, S6 mutations (S338C and F340C) that alter KCNE1 and KCNE3 effects on KCNQ1 do not abrogate KCNE4 inhibition. Further, KCNQ1-KCNQ4 chimeras that exhibited resistance to the inhibitory effects of KCNE4 still interact biochemically with this protein, implying that accessory subunit binding alone is not sufficient for channel modulation. These observations indicate that the diverse functional effects observed for KCNE proteins depend, in part, on structures intrinsic to the pore-forming subunit, and that distinct S6 subdomains determine KCNQ1 responses to KCNE1, KCNE3, and KCNE4.

The effect of methylphenidate on Internet video game play in children with attention-deficit/hyperactivity disorder.

OBJECTIVE: A number of studies about attention-deficit/hyperactivity disorder (ADHD) and Internet video game play have examined the prefrontal cortex and dopaminergic system. Stimulants such as methylphenidate (MPH), given to treat ADHD, and video game play have been found to increase synaptic dopamine. We hypothesized that MPH treatment would reduce Internet use in subjects with co-occurring ADHD and Internet video game addictions. METHODS: Sixty-two children (52 males and 10 females), drug-naive, diagnosed with ADHD, and Internet video game players, participated in this study. At the beginning of the study and after 8 weeks of treatment with Concerta (OROS methylphenidate HCl, Seoul, Korea), participants were assessed with Young's Internet Addiction Scale, Korean version (YIAS-K), Korean DuPaul's ADHD Rating Scale, and the Visual Continuous Performance Test. Their Internet usage time was also recorded. RESULTS: After 8 weeks of treatment, the YIAS-K scores and Internet usage times were significantly reduced. The changes in the YIAS-K scores between the baseline and 8-week assessments were positively correlated with the changes in total and inattention scores from the Korean DuPaul's ADHD Rating Scale, as well as omission errors from the Visual Continuous Performance Test. There was also a significant difference in the number of omission errors among non-Internet-addicted, mildly Internet addicted, and severely Internet addicted participants. DISCUSSION: We suggest that Internet video game playing might be a means of self-medication for children with ADHD. In addition, we cautiously suggest that MPH might be evaluated as a potential treatment of Internet addiction.

KCNE4 domains required for inhibition of KCNQ1.

Voltage-gated potassium (Kv) channels are modulated in distinct ways by members of the KCNE family of single transmembrane domain accessory subunits. KCNE4 has a dramatic inhibitory effect on KCNQ1 that differs substantially from the activating effects of KCNE1 and KCNE3. The structural features of KCNE4 that enable this behaviour are unknown. We exploited chimeras of KCNE1, KCNE3 and KCNE4 to identify specific domains responsible for the inhibitory effects on heterologously expressed KCNQ1. Previous structure-function analysis of KCNE1 and KCNE3 identified a critical tripeptide motif within the transmembrane domain that accounts for the differences in KCNQ1 modulation evoked by these two KCNE proteins. Swapping the transmembrane tripeptide motif of KCNE4 with the corresponding amino acid sequence of KCNE1 did not influence the behaviour of either protein. Similarly, exchanging the tripeptide regions of KCNE3 and KCNE4 further demonstrated that this transmembrane motif does not explain the activity of KCNE4. Using a more systematic approach, we demonstrated that the KCNE4 C-terminus was critical for KCNQ1 modulation. Replacement of the KCNE1 or KCNE3 C-termini with that of KCNE4 created chimeric proteins that strongly inhibited KCNQ1. Additional evidence supported a cooperative role of the KCNE4 transmembrane domain. Although the C-terminus was necessary for KCNE4 activity, we demonstrated that a surrogate transmembrane domain derived from the cytokine receptor CD8 did not enable inhibition of KCNQ1, indicating that the KCNE4 C-terminus alone was not sufficient for KCNQ1 modulation. We further demonstrated that the KCNE4 C-terminus interacts with KCNQ1. Our data reveal important structure-function relationships for KCNE4 that help advance our understanding of potassium channel modulation by KCNE proteins.

Craving for alcohol and food during treatment for alcohol dependence: modulation by T allele of 1519T>C GABAAalpha6.

BACKGROUND: Craving for alcohol and food has been studied in association with alcohol dependence and eating disorders, respectively. One subclass of the gamma-aminobutyric acid (GABA) receptor, 1519T>C GABA(A)alpha6 has been reported to be associated with both alcohol dependence and weight gain. In this study, we hypothesized that patients being treated for alcohol dependence would report decreased craving for alcohol, but an increased craving for food during a 4-week treatment period. We further hypothesized that the T allele of the 1519T>C GABA(A)alpha6 gene would modulate the extent of changes in craving for alcohol and food. METHODS: This study included 98 male inpatients being treated for alcohol dependence. A 7-point visual analog scale was applied to evaluate relative levels of alcohol and food craving at baseline and again 4 weeks later. Body weight was also checked at the same periods. Genotyping of the 1519T>C SNP in GABA(A)alpha6 was carried out by restriction fragment length polymorphism. RESULTS: There were significant changes in craving for alcohol and food in all patients with alcohol dependence. During the treatment period, body weight increased in all patients with alcohol dependence. Changes in alcohol and food craving in T-allele carriers (CT + TT) of 1519T>C GABA(A)alpha6 were greater than those observed in CC homozygotes. In T-allele carriers, body weight significantly increased and the changes in weight showed a negative correlation with the change in the craving for alcohol and a positive correlation with the changes in craving for food. DISCUSSION: The current results suggest that in T-allele carriers the change in craving for alcohol during treatment for alcohol dependence is negatively associated with changes in craving for food. The T allele of the 1519T>C GABA(A)alpha6 gene may be one of the modulating factors associated with changes in craving for alcohol and food during treatment of patients with alcohol dependence.

Depression like characteristics of 5HTTLPR polymorphism and temperament in excessive internet users.

INTRODUCTION: Excessive internet use (EIU) has been reported to be comorbid with depression and the manifestation of its symptoms. This study examines the characteristics of excessive internet users that are similar to those of patients with depressive disorders in terms of serotonin transporter gene expression and harm avoidance. METHODS: 91 male adolescents with EIU and 75 healthy comparison subjects were recruited. Between group comparisons were made on genetic polymorphisms of the serotonin transport gene and with respect to novelty seeking and harm avoidance (HA) of Cloninger's Temperament Character Inventory. RESULTS: The homozygous short allelic variant of the serotonin transporter gene (SS-5HTTLPR) is more frequent in the EIU group (chi(2)=4.38, df=1, p<0.05). The HA and Beck Depression Inventory (BDI) scores were significantly higher in the EIU group than in the healthy comparison group (t=7.03, df=164, p<0.01; t=2.12, df=164, p=0.04). EIU subjects expressing SS-5HTTLPR also showed higher HA (HA1, HA2, HA4, and total HA) and Young's internet addiction scale scores than EIU subjects expressing the other serotonin transporter gene allele variants (t=2.47, df=89, p=0.01; t=2.33, df=89, p=0.02; t=2.17, df=89, p=0.03; t=2.25, df=89, p=0.03; t=2.93, df=89, p<0.01 respectively). CONCLUSIONS: The EIU group had higher SS-5HTTLPR frequencies, harm avoidance, and BDI scores. SS-5HTTLPR expression was closely related to harm avoidance in EIU. The results of this study suggest that EIU subjects may have genetic and personality traits similar to depressed patients.

Altered cingulate white matter connectivity in panic disorder patients.

OBJECTIVE: Functional imaging studies of panic disorder subjects suggest an increased activation of the cingulate regions of the brain. Aim of the current study was to explore the white matter connectivity differences between subjects with panic disorder and healthy comparison subjects. METHOD: Structural white matter connectivity, as determined from fractional anisotropy (FA) values obtained by diffusion tensor imaging, was assessed for anterior and posterior cingulate regions in 24 panic disorder patients and 24 age and sex-matched healthy comparison subjects. RESULTS: Subjects with panic disorder exhibited significantly greater FA values in left anterior and right posterior cingulate regions (by 13.3% and 19.6%, respectively) relative to comparison subjects. White matter connectivity for these two cingulate regions was also positively correlated with clinical severity, as determined by Panic Disorder Severity Scale. FA values in left anterior cingulate region negatively correlated with the time of Trail Making Tests and positively with Digit Symbol Substitution Test. CONCLUSIONS: Findings suggest a potential 'enhancement' in white matter connectivity in left anterior and right posterior cingulate regions in panic disorder, and that these changes may play an important role in mediating clinical symptoms of panic disorder.