Domain-specific mutations in Na⁺/K⁺-ATPase α subunit differentially regulate sleep and circadian rhythms in...

The Na⁺/K⁺-ATPase (NKA) is a crucial membrane transporter that maintains cellular membrane potential and ion homeostasis through ATP-dependent exchange of Na⁺ and K⁺ ions. Its functional core is determined by the α subunit (e.g., ATP1A1), which contains key domains including ten transmembrane segments, three well-defined intracellular regulatory regions and a simple extracellular domain. While mutations in the NKA α subunit have been linked to sleep and circadian rhythm disorders, whether different structural domains differentially regulate these processes remains unclear. In this study, we employed CRISPR/Cas9 to generate Drosophila models carrying clinically relevant human ATP1A1 homolog (ATPα) mutations. We found that heterozygous mutations in transmembrane or intracellular actuator domains-viable only in heterozygotes-increased total sleep duration by elevating pressure to fall asleep. In contrast, homozygous intracellular domain mutations (modeling ATPα-CMT2, associated with Charcot-Marie-Tooth disease type 2) concurrently disrupted both sleep architecture and circadian locomotor rhythms, whereas heterozygous mutants showed no significant phenotypes. This reveals distinct domain-specific regulatory mechanisms for sleep and circadian rhythms by NKA α subunit. Further, we demonstrated that NKA regulates sleep primarily through non-LNv circadian neurons, with developmental-stage-dependent effects. Conversely, its maintenance of endogenous circadian rhythms requires adult-stage functionality. These findings elucidate how domain-specific mutations differentially modulate sleep and circadian outputs, while reveling the potential neurons for NKA's regulatory roles. Our work provides new insights into the molecular genetic networks governing sleep-wake and circadian regulation.