A potassium channel β-subunit couples mitochondrial electron transport to sleep

Abstract

The essential but enigmatic functions of sleep^1,2 must be reflected in molecular changes sensed by the brain's sleep-control systems. In the fruitfly Drosophila, about two dozen sleep-inducing neurons³ with projections to the dorsal fan-shaped body (dFB) adjust their electrical output to sleep need⁴, via the antagonistic regulation of two potassium conductances: the leak channel Sandman imposes silence during waking, whereas increased A-type currents through Shaker support tonic firing during sleep⁵. Here we show that oxidative byproducts of mitochondrial electron transport^6,7 regulate the activity of dFB neurons through a nicotinamide adenine dinucleotide phosphate (NADPH) cofactor bound to the oxidoreductase domain^8,9 of Shaker's K_Vβ subunit, Hyperkinetic^10,11. Sleep loss elevates mitochondrial reactive oxygen species in dFB neurons, which register this rise by converting Hyperkinetic to the NADP⁺-bound form. The oxidation of the cofactor slows the inactivation of the A-type current and boosts the frequency of action potentials, thereby promoting sleep. Energy metabolism, oxidative stress, and sleep-three processes implicated independently in lifespan, ageing, and degenerative disease^6,12-14-are thus mechanistically connected. K_Vβ substrates^8,15,16 or inhibitors that alter the ratio of bound NADPH to NADP⁺ (and hence the record of sleep debt or waking time) represent prototypes of potential sleep-regulatory drugs.

Extended Data Figure 1. Chronic or acute dFB-restricted perturbations of redox chemistry have no impact on waking locomotor activity or arousability.

a, Locomotor counts per waking minute of flies expressing R23E10-GAL4-driven SOD1 or pro-oxidant variant SOD1^A4V, in the Trikinetics Drosophila Activity Monitor system, do not differ from their respective parental controls (genotype effect: P>0.2612, Kruskal-Wallis ANOVA with Dunn's post-hoc test). b, Arousability of flies expressing R23E10-GAL4-driven SOD1 (left) or pro-oxidant SOD1^A4V (right) and parental controls (gray colours as in a) (genotype effects: P>0.2487, vibrational force effects: P<0.0001, vibrational force × genotype interactions: P>0.9857, two-way ANOVA). Data are means ± s.e.m. of 6 trials per genotype (n=16–32 flies each). c, Locomotor counts per waking minute of flies expressing R23E10-GAL4-driven miniSOG, with or without RNAi transgenes targeting K_V channel subunits, and parental controls, in a custom video-tracking system. Activity was monitored for 10 min before the photooxidation of miniSOG and then for a 30-min interval that included a 9-min exposure to blue light (genotype effect: P=0.0827, illumination effect: P=0.8059, illumination × genotype interaction: P=0.3086, two-way repeated-measures ANOVA). Data are means ± s.e.m. n, number of flies (a, c) or trials (b). Statistical detail in Supplementary Table 2.

Extended Data Figure 2. Chronic perturbations of redox chemistry in cryptochrome- or Pdf-expressing clock neurons, Kenyon cells, or olfactory projection neurons have no impact on sleep.

a, Sleep in flies expressing cry-GAL4-driven SOD1 or SOD1^A4V in clock neurons and parental controls. Kruskal-Wallis ANOVA with Dunn's post-hoc test failed to detect significant differences of experimental flies from both of their respective parental controls (P>0.1426). b, Sleep in flies expressing Pdf-GAL4-driven SOD1 or SOD1^A4V in clock neurons and parental controls. Kruskal-Wallis ANOVA with Dunn's post-hoc test failed to detect significant differences of experimental flies from both of their respective parental controls (P>0.1732). c, Sleep in flies expressing OK107-GAL4-driven SOD1 or SOD1^A4V in Kenyon cells and parental controls. One-way ANOVA with Holm-Šídák's post-hoc test failed to detect significant differences of experimental flies from both of their respective parental controls (P>0.0603). d, Sleep in flies expressing GH146-GAL4-driven SOD1 or SOD1^A4V in olfactory projection neurons and parental controls. Kruskal-Wallis ANOVA with Dunn's post-hoc test failed to detect significant differences of experimental flies from both of their respective parental controls (P>0.6901). Data are means ± s.e.m. n, number of flies. Statistical detail in Supplementary Table 2.

Extended Data Figure 3. Chronic dFB-restricted manipulations of cryptochrome have no impact on sleep.

Sleep in flies expressing two different R23E10-GAL4-driven cry^RNAi transgenes and parental controls. One-way ANOVA with Holm-Šídák's post-hoc test failed to detect significant differences of experimental flies from both of their respective parental controls (P>0.1718). Date are means ± s.e.m. n, number of flies. Statistical detail in Supplementary Table 2.

Extended Data Figure 4. Blue illumination of miniSOG-negative dFB neurons has no impact on their electrical activity.

a–e, dFB neurons expressing R23E10-GAL4-driven CD8::GFP, before and after a 9-min exposure to blue light. Example voltage responses to current steps (a, sample sizes in b): illumination increases the input resistance (b, R_m; P=0.0098, paired t-test) but not the membrane time constant (b, τ_m; P=0.0723, paired t-test) and leaves unchanged the current-spike frequency function (c, left; current × genotype interaction: P=0.9982, two-way repeated-measures ANOVA) and interspike interval distribution (c, right; P=0.0947, Kolmogorov-Smirnov test). Example I_A (normalized to peak) evoked by voltage steps to +40 mV (d, sample sizes in e): illumination leaves unchanged the I_A amplitude (e; P=0.8040, Wilcoxon test) and both inactivation time constants (e, τ_fast: P=0.6387, τ_slow: P=0.2958, Wilcoxon tests). Asterisks indicate significant differences (P<0.05). Data are means ± s.e.m. n, number of cells. Statistical detail in Supplementary Table 2.

Figure 1. Hyperkinetic senses redox changes linked to sleep history.

a, R23E10-GAL4-driven expression of Hk (left), but not of Hk^K289M (right), in a homozygous Hk¹ mutant background restores wild-type sleep (shaded bands: 95% confidence intervals) relative to parental controls (gray colours and sample sizes as in b). Two-way repeated-measures ANOVA with Holm-Šídák’s post-hoc test detected significant differences from both parental controls (P<0.0001) but not from wild-type (P=0.8599) in flies expressing Hk, and a significant difference from wild-type (P<0.0001) but not from either parental control (P>0.9741) in flies expressing Hk^K289M. b, Sleep in homozygous Hk¹ mutants expressing R23E10-GAL4-driven Hk rescue transgenes and parental, wild-type, and heterozygous controls. One-way ANOVA with Holm-Šídák’s post-hoc test detected significant differences from both parental controls (P<0.0001) but not from wild-type (P=0.9763) in flies expressing Hk, and a significant difference from wild-type (P<0.0001) but not from either parental control (P>0.9704) in flies expressing Hk^K289M. c, Example maximum intensity projections of the somata and dendritic arbors of dFB neurons expressing MitoTimer under R23E10-GAL4 control, in rested and sleep-deprived (SD) flies (sample sizes in d). The red-to-green emission ratio is pseudocoloured according to the key on the right. Scale bar, 10 µm. d, Sleep deprivation during the night (SD effect: P<0.0001, Kruskal-Wallis ANOVA), but not during the day (P>0.6416, Mann-Whitney test), increases MitoTimer's red-to-green ratio in somata and dendrites of dFB neurons but not in Kenyon cells (KCs) (P=0.1328, t-test). Fluorescence ratios are normalized to those of unperturbed controls at the end of sleep deprivation (n=22 and 62 dFB controls for daytime and night-time deprivation; n=20 KC controls). Asterisks indicate significant differences (P<0.05) from both parental controls (b) or rested conditions (d) in pairwise post-hoc comparisons. Data are means ± s.e.m. n, number of flies. Statistical detail in Supplementary Table 1.

Figure 2. dFB-restricted perturbations of redox chemistry alter sleep.

a, Ubiquinone (Q) and cytochrome c (c) ferry electrons (white dots) between the proton-pumping complexes I, III, and IV. When more electrons enter the transport chain than can be used to fuel ATP synthesis, a backlog accumulates in the Q pool. These electrons react directly with O₂, releasing O₂^- into the matrix and the space between the inner and outer mitochondrial membranes (IMM and OMM). Superoxide dismutases (SOD2 in the matrix, SOD1 in the intermembrane space and cytoplasm) convert O₂^- to membrane-permeant H₂O₂; catalase decomposes H₂O₂. AOX uses surplus Q electrons to reduce O₂ to water. Coloured components were manipulated in b–e. b, Sleep in flies expressing R23E10-GAL4-driven MitoTimer and parental controls (genotype effect: P=0.0007, one-way ANOVA). c, Sleep in flies expressing R23E10-GAL4-driven AOX and parental controls (genotype effect: P<0.0001, one-way ANOVA). d, Sleep in flies expressing R23E10-GAL4-driven SOD1 or pro-oxidant SOD1^A4V, with or without RNAi transgenes targeting K_V channel subunits, and parental controls (genotype effect: P<0.0001, one-way ANOVA). e, Sleep in flies expressing R23E10-GAL4-driven catalase and parental controls (genotype effect: P<0.0001, one-way ANOVA). Asterisks indicate significant differences (P<0.05) from both parental controls or in relevant pairwise post-hoc comparisons (brackets). Data are means ± s.e.m. n, number of flies. Statistical detail in Supplementary Table 1.

Figure 3. Optogenetically controlled ROS production in dFB neurons induces sleep.

a, N-myristoylated miniSOG near the Hyperkinetic (Hk) gondola underneath Shaker (Sh). b, Periods of wake (gray) and sleep (black) during and after an initial 9-min exposure to blue light, in flies expressing R23E10-GAL4-driven miniSOG, with or without RNAi transgenes targeting K_V channel subunits, and parental controls. Each row depicts one individual; all individuals were awake at the onset of illumination. The fraction of experimental flies falling asleep differed from both parental controls (P<0.0001) and from flies coexpressing Hk^RNAi (P=0.0030) but not Shal^RNAi (P=0.3622, Fisher’s exact test throughout, sample sizes in c). c, Sleep in flies expressing R23E10-GAL4-driven miniSOG, with or without RNAi transgenes targeting K_V channel subunits, and parental controls (genotype effect: P<0.0001, Kruskal-Wallis ANOVA). d, Cumulative sleep percentages after a 9-min exposure to blue light at zeitgeber time 9.5 h, in flies expressing R23E10-GAL4-driven miniSOG (n=19) and parental controls (n=25 each, gray colours as in c) (time × genotype interaction: P<0.0001, two-way repeated-measures ANOVA). Asterisks indicate significant differences (P<0.05) from both parental controls or in relevant pairwise post-hoc comparisons. Data are means ± s.e.m. n, number of flies. Statistical detail in Supplementary Table 1.

Figure 4. Changes in redox chemistry alter the spiking activity of dFB neurons via I_A.

a–e, dFB neurons expressing R23E10-GAL4-driven miniSOG and CD8::GFP, before and after a 9-min exposure to blue light. Example voltage responses to current steps (a, sample sizes in b): illumination increases the input resistance (b, R_m; P<0.0001, paired t-test) and membrane time constant (b, τ_m; P=0.0041, paired t-test), steepens the current-spike frequency function (c, left; current × genotype interaction: P=0.0014, two-way repeated-measures ANOVA), and shifts the interspike interval distribution toward shorter values (c, right; P<0.0001, Kolmogorov-Smirnov test). Example I_A (normalized to peak) evoked by voltage steps to +40 mV (d, sample sizes in e): illumination leaves the I_A amplitude unchanged (e; P=0.7295, paired t-test) and increases the fast (e, τ_fast; P=0.0245, Wilcoxon test) but not the slow inactivation time constant (e, τ_slow; P=0.3804, Wilcoxon test). f–j, dFB neurons expressing R23E10-GAL4-driven Hk^K289M or Hk rescue transgenes in a homozygous Hk¹ mutant background. Example voltage responses to current steps (f, sample sizes in g): catalytically competent Hk increases the input resistance (g, R_m; P=0.0467, t-test) but not the membrane time constant (g, τ_m; P=0.4962, t-test), steepens the current-spike frequency function (h, left; current × genotype interaction: P<0.0001, two-way repeated-measures ANOVA), and shifts the interspike interval distribution toward shorter values (h, right; P<0.0001, Kolmogorov-Smirnov test). Example I_A (normalized to peak) evoked by voltage steps to +40 mV (i, sample sizes in j): catalytically competent Hk leaves the I_A amplitude unchanged (j; P=0.9827, t-test) and increases the fast (j, τ_fast; P=0.0061, t-test) but not the slow inactivation time constant (j, τ_slow; P=0.1257, Mann-Whitney test). k–o, dFB neurons expressing R23E10-GAL4-driven AOX or SOD1^A4V. Example voltage responses to current steps (k, sample sizes in l): pro-oxidant SOD1^A4V increases the input resistance (l, R_m; P=0.0023, Mann-Whitney test) and membrane time constant (l, τ_m; P=0.0166, Mann-Whitney test), steepens the current-spike frequency function (m, left; current × genotype interaction: P<0.0001, two-way repeated-measures ANOVA), and shifts the interspike interval distribution toward shorter values (m, right; P<0.0001, Kolmogorov-Smirnov test). Example I_A (normalized to peak) evoked by voltage steps to +40 mV (n, sample sizes in o): pro-oxidant SOD1^A4V leaves the I_A amplitude unchanged (o; P=0.4892, t-test) and increases the fast (o, τ_fast; P=0.0027, t-test) but not the slow inactivation time constant (o, τ_slow; P=0.3401, Mann-Whitney test). Asterisks indicate significant differences (P<0.05). Data are means ± s.e.m. n, number of cells. Statistical detail in Supplementary Table 1.