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Alcohol reverses the effects of KCNJ6 (GIRK2) noncoding variants on excitability of human glutamatergic neurons.

ABSTRACT: Synonymous and noncoding single nucleotide polymorphisms (SNPs) in the KCNJ6 gene, encoding G protein-gated inwardly rectifying potassium channel subunit 2 (GIRK2), have been linked with increased electroencephalographic frontal theta event-related oscillations (ERO) in subjects diagnosed with alcohol use disorder (AUD). To identify molecular and cellular mechanisms while retaining the appropriate genetic background, we generated induced excitatory glutamatergic neurons (iN) from iPSCs derived from four AUD-diagnosed subjects with KCNJ6 variants ("Affected: AF") and four control subjects without variants ("Unaffected: UN"). Neurons were analyzed for changes in gene expression, morphology, excitability and physiological properties. Single-cell RNA sequencing suggests that KCNJ6 AF variant neurons have altered patterns of synaptic transmission and cell projection morphogenesis. Results confirm that AF neurons express lower levels of GIRK2, have greater neurite area, and elevated excitability. Interestingly, exposure to intoxicating concentrations of ethanol induces GIRK2 expression and reverses functional effects in AF neurons. Ectopic overexpression of GIRK2 alone mimics the effect of ethanol to normalize induced excitability. We conclude that KCNJ6 variants decrease GIRK2 expression and increase excitability and that this effect can be minimized or reduced with ethanol.

SUBMITTER: Popova D

PROVIDER: S-EPMC9542475 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Alcohol reverses the effects of KCNJ6 (GIRK2) noncoding variants on excitability of human glutamatergic neurons.

Popova Dina D Gameiro-Ros Isabel I Youssef Mark M MM Zalamea Petronio P Morris Ayeshia D AD Prytkova Iya I Jadali Azadeh A Kwan Kelvin Y KY Kamarajan Chella C Salvatore Jessica E JE Xuei Xiaoling X Chorlian David B DB Porjesz Bernice B Kuperman Samuel S Dick Danielle M DM Goate Alison A Edenberg Howard J HJ Tischfield Jay A JA Pang Zhiping P ZP Slesinger Paul A PA Hart Ronald P RP

Molecular psychiatry 20221007 2

Synonymous and noncoding single nucleotide polymorphisms (SNPs) in the KCNJ6 gene, encoding G protein-gated inwardly rectifying potassium channel subunit 2 (GIRK2), have been linked with increased electroencephalographic frontal theta event-related oscillations (ERO) in subjects diagnosed with alcohol use disorder (AUD). To identify molecular and cellular mechanisms while retaining the appropriate genetic background, we generated induced excitatory glutamatergic neurons (iN) from iPSCs derived f ...[more]

PMID: 36207584

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Project description:Increased expression of G-coupled Inwardly Rectifying Potassium Channel Subunit 2 (GIRK2) has been linked to an electrophysiological endophenotype for alcohol use disorder. We have increased GIRK2 expression in human glutamatergic (NGN2-induced) neurons using two distinct strategies (CRISPRa targeting of the endogenous gene and ectopic expression with a lentiviral vector) to investigate the interplay between GIRK2 expression levels and neuronal response to alcohol (ethanol). We used six healthy donor cell lines (2 CRISPRa and 4 lenti) to draw comparisons within the same genetic background. Findings from functional data (calcium imaging, electrophysiology, and mitochondrial stress tests) indicate that increased GIRK2 expression, combined with prolonged (21-day) intermittent ethanol exposure (IEE), counteracts IEE-induced changes in glutamatergic activity and cellular respiration. To understand the transcriptomic underpinnings of these functional data, we conducted bulk RNAseq in the same donor cohort. Differential gene expression analysis indicates that both CRISPRa and lenti-increased GIRK2 interacted with IEE to produce unique differential regulation of synaptic genes, including glutamate receptors and voltage-gated ion channels, distinct from the effects of IEE or increased GIRK2 alone. Increased expression of G-coupled Inwardly Rectifying Potassium Channel Subunit 2 (GIRK2) has been linked to an electrophysiological endophenotype for alcohol use disorder. We have increased GIRK2 expression in human glutamatergic (NGN2-induced) neurons using two distinct strategies (CRISPRa targeting of the endogenous gene and ectopic expression with a lentiviral vector) to investigate the interplay between GIRK2 expression levels and neuronal response to alcohol (ethanol). We used six healthy donor cell lines (2 CRISPRa and 4 lenti) to draw comparisons within the same genetic background. Findings from functional data (calcium imaging, electrophysiology, and mitochondrial stress tests) indicate that increased GIRK2 expression, combined with prolonged (21-day) intermittent ethanol exposure (IEE), counteracts IEE-induced changes in glutamatergic activity and cellular respiration. To understand the transcriptomic underpinnings of these functional data, we conducted bulk RNAseq in the same donor cohort. Differential gene expression analysis indicates that both CRISPRa and lenti-increased GIRK2 interacted with IEE to produce unique differential regulation of synaptic genes, including glutamate receptors and voltage-gated ion channels, distinct from the effects of IEE or increased GIRK2 alone.

Dataset Information

Alcohol reverses the effects of KCNJ6 (GIRK2) noncoding variants on excitability of human glutamatergic neurons.

Publications

Alcohol reverses the effects of KCNJ6 (GIRK2) noncoding variants on excitability of human glutamatergic neurons.

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