F654A and K558Q Mutations in NMDA Receptor 1 Affect Ethanol-Induced Behaviors in Drosophila.

N-methyl-D-aspartate (NMDA) receptors regulate synaptic plasticity and modulate a wide variety of behaviors. Mammalian NMDA receptors are inhibited by ethanol (EtOH) even at low concentrations. In mice, the F639A mutation in transmembrane domain (TMD) 3 of the NR1 subunit reduces EtOH sensitivity of the receptor and, in some paradigms, reduces behavioral EtOH sensitivity and increases EtOH consumption. We tested the fly equivalent of the F639A and K544Q mutations for effects on EtOH sensitivity. Drosophila shows a high degree of behavioral and mechanistic conservation in its responses to EtOH. Homologous recombination and CRISPR/Cas9 genome editing were used to generate amino acid changes in the Drosophila NMDAR1 gene, yielding F654A and K558Q alleles. Animals were tested for the degree of EtOH sensitivity, the ability to acquire tolerance to EtOH, EtOH drinking preference, circadian rhythmicity, learning, and locomotor defects. We observed that mutating the NMDAR1 channel also reduces EtOH sensitivity in adult flies. However, in flies, it was the K558Q mutation (orthologous to K544Q in mice) that reduces EtOH sensitivity in a recovery-from-sedation assay. The effects of the F654A mutation (orthologous to F639A in mice) were substantially different in flies than in mammals. In flies, F654A mutation produces phenotypes opposite those in mammals. In flies, the mutant allele is homozygous viable, does not seem to affect health, and increases EtOH sensitivity. Both mutations increased feeding but did not alter the animal's preference for 5% EtOH food. F654A depressed circadian rhythmicity and the capacity of males to court, but it did not depress the capacity for associative learning. K554Q, on the other hand, has little effect on circadian rhythmicity, only slightly suppresses male courtship, and is a strong learning mutant. Mutations in TMD 3 and in the extracellular-vestibule calcium-binding site of the NR1 NMDA subunit affect EtOH sensitivity in Drosophila.