However, results demonstrate that the noncoding haplotype alters expression of GIRK2 protein and KCNJ6 mRNA. iN from subjects with the homozygous variant KCNJ6 haplotype exhibited lower expression of GIRK2 by immunocytochemistry (Fig. 3D), but while the difference in neuronal mRNA levels as detected by scRNAseq analysis matched this trend, it did not reach significance (p = 0.0508; Fig. 1C). FISH analysis, however, confirmed that KCNJ6 mRNA was reduced in the AF group (Fig. 5J). KCNJ6 mRNA was also detected in fewer MAP2+ cells and at lower levels per cell (Fig. 5J.b, c). The results of counting GIRK2 puncta and evaluating KCNJ6 mRNA by scRNAseq and FISH support a diminished expression of GIRK2 in neurons from the affected group. Secondary effects of KCNJ6 mRNA and GIRK2 protein regulation include many predictors of altered neuronal connectivity and signaling. Genes that were reduced in the AF group (GO:BP Down in Fig. 1E; Supplementary Fig. 3B) point to changes in synaptic transmission and projection morphology. Since we focused on KCNJ6 haplotype differences in cells without ethanol, we did not expect to match findings in, for example, human AUD brains [52], which found prominent effects in astrocytes and microglia.
