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.

Upregulated GIRK2 counteracts ethanol-induced changes in excitability & respiration in human neurons

“…In support of our finding that the upregulation of GIRK2 plays an important role in controlling neuronal excitability as well as modulating the response to ethanol, a recent study found that iPSC-derived human neurons derived from AUD-diagnosed participants containing specific KNJ6 SNPs had lower levels of GIRK2 expression, greater neurite area, and heightened excitability than neurons derived from unaffected individuals 6 . Overexpression of GIRK2 in neurons from AUD-diagnosed individuals mimicked the effects of the 7-day IEE protocol, eliminating differences in neuronal morphology induced excitability 6 . Thus, in the context of AUD, increased GIRK2 expression and subsequent neuronal inhibition appears to exert a normalizing influence.…”

“…By contrast, 21-day IEE ↑GIRK2 neurons increased their latency to first spike with glutamate (+ 2.1 s, p = 0.0312 exposure effect; p < 0.00001 interaction effect) ( Figure 5C ). Similar to the measurements of spontaneous activity, ( Figure 4E-F ), calcium peak heights were significantly larger in IEE iso-CTL neurons (+ 0.36 ΔF/F 0 , p = 3×10 - 6 ), and this increase was not observed in ↑GIRK2 neurons ( Figure 5D ). Taken together, these data indicate that 21-day IEE heightens glutamate receptor activity and/or excitability in iso-CTL neurons, whereas ↑GIRK2 neurons exhibit diminished glutamate responses.…”

“…To determine whether differences in glutamate-responsive neurons were due to changes in glutamate sensitivity or in intrinsic excitability, we bath-applied 15 mM KCl in ACSF to directly depolarize the neurons (15 mM KCl is predicted to produce a ∼32 mV positive shift in the resting membrane potential) and measured Ca 2+ spikes in the same population of glutamate-responsive neurons. As before, Ca 2+ flux was elevated in 21-day IEE iso-CTL neurons (+ 0.23 ΔF/F 0 , p < 1×10 - 6 ), while ↑GIRK2 neurons were unaffected ( Figure 6D ). However, 21-day IEE in ↑GIRK2 neurons increased proportion of KCl-responsive ROIs (+8.3%, p = 0.009 IEE effect; p < 1×10 - 6 interaction effect) ( Figure 6A ) and number KCl-evoked Ca 2+ spikes (+ 0.54 spikes, p < 1×10 - 6 ) ( Figure 6B ).…”

“…As before, Ca 2+ flux was elevated in 21-day IEE iso-CTL neurons (+ 0.23 ΔF/F 0 , p < 1×10 - 6 ), while ↑GIRK2 neurons were unaffected ( Figure 6D ). However, 21-day IEE in ↑GIRK2 neurons increased proportion of KCl-responsive ROIs (+8.3%, p = 0.009 IEE effect; p < 1×10 - 6 interaction effect) ( Figure 6A ) and number KCl-evoked Ca 2+ spikes (+ 0.54 spikes, p < 1×10 - 6 ) ( Figure 6B ). Iso-CTL neurons, conversely, had a lower proportion of KCl-responsive ROIs (−16.5%, p < 1×10 6 ) ( Figure 6A ) and no difference in number of evoked spikes (p = 0.4) ( Figure 6B ).…”

“…Most of the associated KCNJ6 SNPs are non-coding, with the exception of a single imputed synonymous SNP, and therefore are expected to primarily impact gene expression, which was recently shown with human neurons containing different KCNJ6 SNPs 1,6 .…”