Explore the vast range of titles available.

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 neurons 3 with projections to the dorsal fan-shaped body (dFB) adjust their electrical output to sleep need 4 , 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 sleep 5 . 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 V β 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 V β 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. Sleep deprivation in Drosophila elevates reactive oxygen species in sleep-promoting neurons, leading to changes in potassium currents and spiking activity and thereby connecting energy metabolism, oxidative stress, and sleep.

Sleep-promoting neurons in Drosophila are shown to switch between electrical activity and silence as a function of sleep need; the switch is operated by dopamine and involves the antagonistic regulation of two potassium channels. A dopamine switch for sleep regulation The risks and costs associated with animal sleep are obvious but the beneficial trade-offs remain largely unknown, in part because of a lack of mechanistic understanding of sleep homeostasis. Now, Gero Miesenböck and colleagues report that sleep-promoting neurons that innervate the Drosophila fan-shaped body switch between electrical activity and silence as a function of sleep requirement. The switch is operated by dopamine and involves antagonistic modulation of voltage-dependent and voltage-independent potassium channels, thus linking sleep homeostasis to the molecular biophysics of identified neurons. Elsewhere in this issue of Nature , Michael Rosbash and colleagues identify a subset of dorsal clock neurons in Drosophila as sleep-promoting cells, that participate in a feedback loop with pacemaker neurons to drive both midday siesta and night-time sleep. Sleep disconnects animals from the external world, at considerable risks and costs that must be offset by a vital benefit. Insight into this mysterious benefit will come from understanding sleep homeostasis: to monitor sleep need, an internal bookkeeper must track physiological changes that are linked to the core function of sleep 1 . In Drosophila , a crucial component of the machinery for sleep homeostasis is a cluster of neurons innervating the dorsal fan-shaped body (dFB) of the central complex 2 , 3 . Artificial activation of these cells induces sleep 2 , whereas reductions in excitability cause insomnia 3 , 4 . dFB neurons in sleep-deprived flies tend to be electrically active, with high input resistances and long membrane time constants, while neurons in rested flies tend to be electrically silent 3 . Correlative evidence thus supports the simple view that homeostatic sleep control works by switching sleep-promoting neurons between active and quiescent states 3 . Here we demonstrate state switching by dFB neurons, identify dopamine as a neuromodulator that operates the switch, and delineate the switching mechanism. Arousing dopamine 4 , 5 , 6 , 7 , 8 caused transient hyperpolarization of dFB neurons within tens of milliseconds and lasting excitability suppression within minutes. Both effects were transduced by Dop1R2 receptors and mediated by potassium conductances. The switch to electrical silence involved the downregulation of voltage-gated A-type currents carried by Shaker and Shab, and the upregulation of voltage-independent leak currents through a two-pore-domain potassium channel that we term Sandman. Sandman is encoded by the CG8713 gene and translocates to the plasma membrane in response to dopamine. dFB-restricted interference with the expression of Shaker or Sandman decreased or increased sleep, respectively, by slowing the repetitive discharge of dFB neurons in the ON state or blocking their entry into the OFF state. Biophysical changes in a small population of neurons are thus linked to the control of sleep–wake state.

Voltage gated potassium (Kv) channels regulate processes from cellular excitability to immune response and are major pharmaceutical targets. Despite recent structural advances, the closed state structure of the strictly coupled Kv1 family remains elusive. Here, we capture the structure of the Shaker potassium channel with a closed pore by uncoupling its voltage sensor domains from the pore domain. Structural determination of the uncoupled I384R mutant by single particle Cryo-EM reveals a fully closed pore coexisting with activated, non-relaxed voltage sensors. Comparison with the open pore structure suggests a roll-and-turn movement along the length of the pore-forming S6 helices, contrasting with canonical gating models based on limited movements of S6. These rotational-translational motions place two hydrophobic residues, one in the inner cavity and the other at the bundle crossing region, directly at the permeation pathway, limiting the pore radius to less than 1 Å. The selectivity filter is captured in a noncanonical state, partially expanded at G446, unlike previously described dilated or pinched filter conformations. Together, these findings suggest a reinterpretation of the mechanism of activation gating for strictly coupled Kv1 channels, highlighting the strictly sensor-pore coupling that underlies different functional states. Voltage-gated potassium channels control excitability, but their non-conductive structure remained elusive. Here, authors reveal the Shaker channel’s closed-pore structure, showing distinct “roll-and-turn” helix movements that redefine activation gating in the Kv1 family.

Root-to-shoot translocation and shoot homeostasis of potassium(K) determine nutrient balance, growth, and stress tolerance of vascular plants. To maintain the cation-anion balance, xylem loading of K⁺ in the roots relies on the concomitant loading of counteranions, like nitrate (NO₃⁻). However, the coregulation of these loading steps is unclear. Here, we show that the bidirectional, low-affinity Nitrate Transporter1 (NRT1)/Peptide Transporter (PTR) family member NPF7.3/NRT1.5 is important for the NO₃⁻-dependent K⁺ translocation in Arabidopsis (Arabidopsis thaliana). Lack of NPF7.3/NRT1.5 resulted in K deficiency in shoots under low NO₃⁻ nutrition, whereas the root elemental composition was unchanged. Gene expression data corroborated K deficiency in thenrt1.5-5shoot, whereas the root responded with a differential expression of genes involved in cation-anion balance. A grafting experiment confirmed that the presence of NPF7.3/NRT1.5 in the root is a prerequisite for proper root-to-shoot translocation of K⁺ under low NO₃⁻ supply. Because the depolarization-activated Stelar K⁺ Outward Rectifier (SKOR) has previously been described as a major contributor for root-to-shoot translocation of K⁺ in Arabidopsis, we addressed the hypothesis that NPF7.3/NRT1.5-mediated NO₃⁻ translocation might affect xylem loading and root-to-shoot K⁺ translocation through SKOR. Indeed, growth ofnrt1.5-5andskor-2single and double mutants under different K/NO₃⁻ regimes revealed that both proteins contribute to K⁺ translocation from root to shoot. SKOR activity dominates under high NO₃⁻ and low K⁺ supply, whereas NPF7.3/NRT1.5 is required under low NO₃⁻ availability. This study unravels nutritional conditions as a critical factor for the joint activity of SKOR and NPF7.3/NRT1.5 for shoot K homeostasis.

The S4–S5 linker physically links voltage sensor and pore domain in voltage-gated ion channels and is essential for electromechanical coupling between both domains. Little dynamic information is available on the movement of the cytosolic S4–S5 linker due to lack of a direct electrical or optical readout. To understand the movements of the gating machinery during activation and inactivation, we incorporated fluorescent unnatural amino acids at four positions along the linker of the Shaker KV channel. Using two-color voltage-clamp fluorometry, we compared S4–S5 linker movements with charge displacement, S4 movement, and pore opening. We found that the proximal S4–S5 linker moves with the S4 helix throughout the gating process, whereas the distal portion undergoes a separate motion related to late gating transitions. Both pore and S4–S5 linker undergo rearrangements during C-type inactivation. In presence of accelerated C-type inactivation, the energetic coupling between movement of the distal S4–S5 linker and pore opening disappears.

Voltage-dependent potassium channels (Kv) play a crucial role in membrane repolarization during action potentials. They undergo voltage-dependent structural conformational transitions according to their distribution across their energy landscape. Understanding these transitions helps us comprehend their molecular function. Here, we used sudden and sustained temperature changes (Tstep) combined with different voltage protocols and mutations to dissect the energy landscape of the Shaker K + channel. We used two mutations, ILT (V369I, I372L, and S376T) and I384N, that affect the coupling between the voltage sensor (VSD) and the pore domain (PD), to obtain the temperature dependence of VSD last transition and the intrinsic temperature dependence of the pore, respectively. Our findings support a loose or tight conformation of the electromechanical coupling. In the loose conformation, the movement of the VSD is necessary but not sufficient to efficiently propagate the electromechanical energy to open the pore. In contrast, this movement is effectively translated into pore opening in the tight conformation. Our results describe the energy landscape of the Shaker channel and how its temperature dependence can be modulated by affecting its electromechanical coupling. Voltage‐gated potassium channels allow nerve communication by coupling voltage sensing to pore opening. Here, the authors applied rapid temperature steps and mutations to map the channel’s energy landscape, revealing how distinct energy barriers contribute to channel activation.

Voltage-dependent potassium channels (Kvs) gate in response to changes in electrical membrane potential by coupling a voltage-sensing module with a K⁺-selective pore. Animal toxins targeting Kvs are classified as pore blockers, which physically plug the ion conduction pathway, or as gating modifiers, which disrupt voltage sensor movements. A third group of toxins blocks K⁺ conduction by an unknown mechanism via binding to the channel turrets. Here, we show that Conkunitzin-S1 (Cs1), a peptide toxin isolated from cone snail venom, binds at the turrets of Kv1.2 and targets a network of hydrogen bonds that govern water access to the peripheral cavities that surround the central pore. The resulting ectopic water flow triggers an asymmetric collapse of the pore by a process resembling that of inherent slow inactivation. Pore modulation by animal toxins exposes the peripheral cavity of K⁺ channels as a novel pharmacological target and provides a rational framework for drug design.

Potassium channels: the active-to-inactive switch Switching between conductive and non-conductive states is central to the function of ion channels. In potassium channels, inactivation gating occurs by two distinct molecular mechanisms: N-type inactivation (a rapid autoinhibitory process in which an N-terminal particle blocks conduction by binding to the open pore) and C-type inactivation (originating from conformational transitions at the selectivity filter). In the first of two papers, Eduardo Perozo and co-workers solve the X-ray crystal structure of the K + channel KcsA in an 'open-inactivated' conformation together with a series of crystal structures of channels that are 'trapped' in a set of partially open conformations. In the second paper, the authors identify the underlying mechanism by which movements in the inner gate of this channel trigger conformational changes at the selectivity filter, leading to the non-conductive C-type inactivated state. K + channels can convert between conductive and non-conductive forms through mechanisms that range from flicker transitions (which occur in microseconds) to C-type inactivation (which occurs on millisecond to second timescales). Here, the mechanisms are revealed through which movements of the inner gate of the K + channel KcsA trigger conformational changes at the selectivity filter, leading to the non-conductive C-type inactivated state. The coupled interplay between activation and inactivation gating is a functional hallmark of K + channels 1 , 2 . This coupling has been experimentally demonstrated through ion interaction effects 3 , 4 and cysteine accessibility 1 , and is associated with a well defined boundary of energetically coupled residues 2 . The structure of the K + channel KcsA in its fully open conformation, in addition to four other partial channel openings, richly illustrates the structural basis of activation–inactivation gating 5 . Here, we identify the mechanistic principles by which movements on the inner bundle gate trigger conformational changes at the selectivity filter, leading to the non-conductive C-type inactivated state. Analysis of a series of KcsA open structures suggests that, as a consequence of the hinge-bending and rotation of the TM2 helix, the aromatic ring of Phe 103 tilts towards residues Thr 74 and Thr 75 in the pore-helix and towards Ile 100 in the neighbouring subunit. This allows the network of hydrogen bonds among residues Trp 67, Glu 71 and Asp 80 to destabilize the selectivity filter 6 , 7 , allowing entry to its non-conductive conformation. Mutations at position 103 have a size-dependent effect on gating kinetics: small side-chain substitutions F103A and F103C severely impair inactivation kinetics, whereas larger side chains such as F103W have more subtle effects. This suggests that the allosteric coupling between the inner helical bundle and the selectivity filter might rely on straightforward mechanical deformation propagated through a network of steric contacts. Average interactions calculated from molecular dynamics simulations show favourable open-state interaction-energies between Phe 103 and the surrounding residues. We probed similar interactions in the Shaker K + channel where inactivation was impaired in the mutant I470A. We propose that side-chain rearrangements at position 103 mechanically couple activation and inactivation in KcsA and a variety of other K + channels.

Most ion channels are multimeric (typically comprising 3–5 subunits). The subunits are encoded by homologous members of a gene family, generating an enormous set of possible heteromeric combinations. In this study, we provide evidence that the preferred target of conopeptide κM-RIIIJ is a heteromeric K v 1 channel consisting of three K v 1.2 subunits and one K v 1.1 or K v 1.6 subunit. We define the molecular interaction of κM-RIIIJ with these asymmetric K v 1 channels and show that in dorsal root ganglia (DRG) neurons, different κM-RIIIJ concentrations can distinguish discrete subpopulations of neurons. Our results highlight the potential of natural products and venom components, such as conopeptides, to generally elucidate native physiological roles of specific heteromeric ion channel isoforms at the cellular, circuit, and systems level. The vast complexity of native heteromeric K + channels is largely unexplored. Defining the composition and subunit arrangement of individual subunits in native heteromeric K + channels and establishing their physiological roles is experimentally challenging. Here we systematically explored this “zone of ignorance” in molecular neuroscience. Venom components, such as peptide toxins, appear to have evolved to modulate physiologically relevant targets by discriminating among closely related native ion channel complexes. We provide proof-of-principle for this assertion by demonstrating that κM-conotoxin RIIIJ (κM-RIIIJ) from Conus radiatus precisely targets “asymmetric” K v channels composed of three K v 1.2 subunits and one K v 1.1 or K v 1.6 subunit with 100-fold higher apparent affinity compared with homomeric K v 1.2 channels. Our study shows that dorsal root ganglion (DRG) neurons contain at least two major functional K v 1.2 channel complexes: a heteromer, for which κM-RIIIJ has high affinity, and a putative K v 1.2 homomer, toward which κM-RIIIJ is less potent. This conclusion was reached by ( i ) covalent linkage of members of the mammalian Shaker-related K v 1 family to K v 1.2 and systematic assessment of the potency of κM-RIIIJ block of heteromeric K + channel-mediated currents in heterologous expression systems; ( ii ) molecular dynamics simulations of asymmetric K v 1 channels providing insights into the molecular basis of κM-RIIIJ selectivity and potency toward its targets; and ( iii ) evaluation of calcium responses of a defined population of DRG neurons to κM-RIIIJ. Our study demonstrates that bioactive molecules present in venoms provide essential pharmacological tools that systematically target specific heteromeric K v channel complexes that operate in native tissues.

A Shaker Kv-channel V478W mutant shows enhanced C-type inactivation with disruption of the outermost K + site in the selectivity filter (IS1). The crystal structure of Kv1.2-2.1 bearing the equivalent mutation reveals an empty IS1. C-type inactivation underlies important roles played by voltage-gated K + (Kv) channels. Functional studies have provided strong evidence that a common underlying cause of this type of inactivation is an alteration near the extracellular end of the channel's ion-selectivity filter. Unlike N-type inactivation, which is known to reflect occlusion of the channel's intracellular end, the structural mechanism of C-type inactivation remains controversial and may have many detailed variations. Here we report that in voltage-gated Shaker K + channels lacking N-type inactivation, a mutation enhancing inactivation disrupts the outermost K + site in the selectivity filter. Furthermore, in a crystal structure of the Kv1.2-2.1 chimeric channel bearing the same mutation, the outermost K + site, which is formed by eight carbonyl-oxygen atoms, appears to be slightly too small to readily accommodate a K + ion and in fact exhibits little ion density; this structural finding is consistent with the functional hallmark of C-type inactivation.