Circadian clocks regulate much of behavior and physiology, but the mechanisms by which they do so remain poorly understood. While cyclic gene expression is thought to underlie metabolic rhythms, little is known about cycles in cellular physiology. We found that Drosophila insulin-producing cells (IPCs), which are located in the pars intercerebralis and lack an autonomous circadian clock, are functionally connected to the central circadian clock circuit via DN1 neurons. Insulin mediates circadian output by regulating the rhythmic expression of a metabolic gene (sxe2) in the fat body. Patch clamp electrophysiology reveals that IPCs display circadian clock-regulated daily rhythms in firing event frequency and bursting proportion under light:dark conditions. The activity of IPCs and the rhythmic expression of sxe2 are additionally regulated by feeding, as demonstrated by night feeding-induced changes in IPC firing characteristics and sxe2 levels in the fat body. These findings indicate circuit-level regulation of metabolism by clock cells in Drosophila and support a role for the pars intercerebralis in integrating circadian control of behavior and physiology.

SUMMARY The mechanisms by which clock neurons in the Drosophila brain confer an ~24-hr rhythm onto locomotor activity are unclear, but involve the neuropeptide diuretic hormone 44 (DH44), an ortholog of corticotropin-releasing factor. Here we identified DH44 receptor 1 as the relevant receptor for rest:activity rhythms and mapped its site of action to hugin-expressing neurons in the subesophageal zone (SEZ). We traced a circuit that extends from Dh44-expressing neurons in the pars intercerebralis (PI) through hugin+ SEZ neurons to the ventral nerve cord. Hugin neuropeptide, a neuromedin U ortholog, also regulates behavioral rhythms. The DH44 PI-Hugin SEZ circuit controls circadian locomotor activity in a daily cycle but has minimal effect on feeding rhythms, suggesting that the circadian drive to feed can be separated from circadian locomotion. These findings define a linear peptidergic circuit that links the clock to motor outputs to modulate circadian control of locomotor activity.

Despite the clinical ubiquity of anesthesia, the molecular basis of anesthetic action is poorly understood. Amongst the many molecular targets proposed to contribute to anesthetic effects, the voltage gated sodium channels (VGSCs) should also be considered relevant, as they have been shown to be sensitive to all general anesthetics tested thus far. However, binding sites for VGSCs have not been identified. Moreover, the mechanism of inhibition is still largely unknown. The recently reported atomic structures of several members of the bacterial VGSC family offer the opportunity to shed light on the mechanism of action of anesthetics on these important ion channels. To this end, we have performed a molecular dynamics “flooding” simulation on a membrane-bound structural model of the archetypal bacterial VGSC, NaChBac in a closed pore conformation. This computation allowed us to identify binding sites and access pathways for the commonly used volatile general anesthetic, isoflurane. Three sites have been characterized with binding affinities in a physiologically relevant range. Interestingly, one of the most favorable sites is in the pore of the channel, suggesting that the binding sites of local and general anesthetics may overlap. Surprisingly, even though the activation gate of the channel is closed, and therefore the pore and the aqueous compartment at the intracellular side are disconnected, we observe binding of isoflurane in the central cavity. Several sampled association and dissociation events in the central cavity provide consistent support to the hypothesis that the “fenestrations” present in the membrane-embedded region of the channel act as the long-hypothesized hydrophobic drug access pathway.

Halogenated inhaled general anesthetic agents modulate voltagegated ion channels, but the underlying molecular mechanisms are not understood. Many general anesthetic agents regulate voltagegated Na + (Na V ) channels, including the commonly used drug sevoflurane. Here, we investigated the putative binding sites and molecular mechanisms of sevoflurane action on the bacterial Na V channel NaChBac by using a combination of molecular dynamics simulation, electrophysiology, and kinetic analysis. Structural modeling revealed multiple sevoflurane interaction sites possibly associated with NaChBac modulation. Electrophysiologically, sevoflurane favors activation and inactivation at low concentrations (0.2 mM), and additionally accelerates current decay at high concentrations (2 mM). Explaining these observations, kinetic modeling suggests concurrent destabilization of closed states and low-affinity open channel block. We propose that the multiple effects of sevoflurane on NaChBac result from simultaneous interactions at multiple sites with distinct affinities. This multiple-site, multiple-mode hypothesis offers a framework to study the structural basis of general anesthetic action.anesthesia | MD simulations | anesthetics | membrane proteins G eneral anesthetic agents have been in use for more than 160 y.However, we still understand relatively little about their mechanisms of action, which greatly limits our ability to design safer and more effective general anesthetic agents. Ion channels of the central nervous system are known to be key targets of general anesthetic agents, as their modulation can account for the endpoints and side effects of general anesthesia (1-4). Many families of ion channels are modulated by general anesthetic agents, including ligand-gated, voltage-gated, and nongated ion channels (2, 5-7). Mammalian voltage-gated Na + (Na V ) channels, which mediate the upstroke of the action potential, are regulated by numerous inhaled general anesthetic agents (8-14), which generally cause inhibition. Previous work showed that inhaled general anesthetic agents, including sevoflurane, isoflurane, desflurane, and halothane, mediate inhibition by increasing the rate of Na + channel inactivation, hyperpolarizing steady-state inactivation, and slowing recovery from inactivation (11,(15)(16)(17)(18). Inhibition of presynaptic Na V channels in the spinal cord is proposed to lead to inhibition of neurotransmitter release, facilitating immobilization-one of the endpoints of general anesthesia (14,19,20). Despite the importance of Na V channels as general anesthetic targets, little is known about interaction sites or the mechanisms of action.What is known about anesthetic sites in Na V channels comes primarily from the local anesthetic field. Local anesthetic agent binding to Na V channels is well characterized. These amphiphilic drugs enter the channel pore from the intracellular side, causing open-channel block (21). Investigating molecular mechanisms of mammalian Na V channel modulation by general anesthetic agents...

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Background: Halogenated inhaled anesthetics modulate voltage-gated ion channels by unknown mechanisms. Results: Biophysical analyses revealed novel activation of K v channels by the inhaled anesthetic sevoflurane. Conclusion: K v channel activation by sevoflurane results from the positive allosteric modulation of activation gating. Significance: The unique activation of K v channels by sevoflurane demonstrates novel anesthetic specificity and offers new insights into allosteric modulation of channel gating.

Several voltage-gated ion channels are modulated by clinically relevant doses of general anesthetics. However, the structural basis of this modulation is not well understood. Previous work suggested that n-alcohols and inhaled anesthetics stabilize the closed state of the Shaw2 voltage-gated (Kv) channel (K-Shaw2) by directly interacting with a discrete channel site. We hypothesize that the inhibition of K-Shaw2 channels by general anesthetics is governed by interactions between binding and effector sites involving components of the channel's activation gate. To investigate this hypothesis, we applied Ala/Val scanning mutagenesis to the S4-S5 linker and the post-PVP S6 segment, and conducted electrophysiological analysis to evaluate the energetic impact of the mutations on the inhibition of the K-Shaw2 channel by 1-butanol and halothane. These analyses identified residues that determine an apparent binding cooperativity and residue pairs that act in concert to modulate gating upon anesthetic binding. In some instances, due to their critical location, key residues also influence channel gating. Complementing these results, molecular dynamics simulations and in silico docking experiments helped us visualize possible anesthetic sites and interactions. We conclude that the inhibition of K-Shaw2 by general anesthetics results from allosteric interactions between distinct but contiguous binding and effector sites involving inter- and intrasubunit interfaces.

The ability to control transgene expression, both spatially and temporally, is essential for studying model organisms. In Drosophila, spatial control is primarily provided by the GAL4/UAS system, whilst temporal control relies on a temperature-sensitive GAL80 (which inhibits GAL4) and drug-inducible systems. However, these are not ideal. Shifting temperature can impact on many physiological and behavioural traits, and the current drug-inducible systems are either leaky, toxic, incompatible with existing GAL4-driver lines, or do not generate effective levels of expression. Here, we describe the auxin-inducible gene expression system (AGES). AGES relies on the auxin-dependent degradation of a ubiquitously expressed GAL80, and therefore, is compatible with existing GAL4-driver lines. Water-soluble auxin is added to fly food at a low, non-lethal, concentration, which induces expression comparable to uninhibited GAL4 expression. The system works in both larvae and adults, providing a stringent, non-lethal, cost-effective, and convenient method for temporally controlling GAL4 activity in Drosophila.