Sleep is highly conserved across animal species. Both wake- and sleep-promoting neurons are implicated in the regulation of wake-sleep transition at dusk inHowever, little is known about how they cooperate and whether they act via different mechanisms. Here, we demonstrated that in female, sleep onset was specifically delayed by blocking the Shaker cognate L channels [Shal; also known as voltage-gated Kchannel 4 (K4)] in wake-promoting cells, including large ventral lateral neurons (l-LNvs) and pars intercerebralis (PI), but not in sleep-promoting dorsal neurons (DN1s). Delayed sleep onset was also observed in males by blocking K4 activity in wake-promoting neurons. Electrophysiological recordings show that K4 channels contribute A-type currents in LNvs and PI cells, but are much less conspicuous in DN1s. Interestingly, blocking K4 in wake-promoting neurons preferentially increased firing rates at dusk ∼ZT13, when the resting membrane potentials and firing rates were at lower levels. Furthermore, pigment-dispersing factor (PDF) is essential for the regulation of sleep onset by K4 in l-LNvs, and downregulation of PDF receptor (PDFR) in PI neurons advanced sleep onset, indicating K4 controls sleep onset via regulating PDF/PDFR signaling in wake-promoting neurons. We propose that K4 acts as a sleep onset controller by suppressing membrane excitability in a clock-dependent manner to balance the wake-sleep transition at dusk. Our results have important implications for the understanding and treatment of sleep disorders such as insomnia.The mechanisms by which our brains reversibly switch from waking to sleep state remain an unanswered and intriguing question in biological research. In this study, we identified that Shal/K4, a well known voltage-gated Kchannel, acts as a controller of wake-sleep transition at dusk incircadian neurons. We find that interference of K4 function with a dominant-negative form (DNK4) in subsets of circadian neurons specifically disrupts sleep onset at dusk, although K4 itself does not exhibit circadian oscillation. K4 preferentially downregulates neuronal firings at ZT9-ZT17, supporting that it plays an essential role in wake-sleep transition at dusk. Our findings may help understand and eventually treat sleep disorders such as insomnia. Drosophila Drosophila Drosophila + + v v v v v v v v v v v v SIGNIFICANCE STATEMENT
