Ankyrin Repeats Convey Force to Gate the NOMPC Mechanotransduction Channel.

How metazoan mechanotransduction channels sense mechanical stimuli is not well understood. The NOMPC channel in the transient receptor potential (TRP) family, a mechanotransduction channel for Drosophila touch sensation and hearing, contains 29 Ankyrin repeats (ARs) that associate with microtubules. These ARs have been postulated to act as a tether that conveys force to the channel. Here, we report that these N-terminal ARs form a cytoplasmic domain essential for NOMPC mechanogating in vitro, mechanosensitivity of touch receptor neurons in vivo, and touch-induced behaviors of Drosophila larvae. Duplicating the ARs elongates the filaments that tether NOMPC to microtubules in mechanosensory neurons. Moreover, microtubule association is required for NOMPC mechanogating. Importantly, transferring the NOMPC ARs to mechanoinsensitive voltage-gated potassium channels confers mechanosensitivity to the chimeric channels. These experiments strongly support a tether mechanism of mechanogating for the NOMPC channel, providing insights into the basis of mechanosensitivity of mechanotransduction channels.

Activation and closed-state inactivation mechanisms of the human voltage-gated KV4 channel complexes.

The voltage-gated ion channel activity depends on both activation (transition from the resting state to the open state) and inactivation. Inactivation is a self-restraint mechanism to limit ion conduction and is as crucial to membrane excitability as activation. Inactivation can occur when the channel is open or closed. Although open-state inactivation is well understood, the molecular basis of closed-state inactivation has remained elusive. We report cryo-EM structures of human KV4.2 channel complexes in inactivated, open, and closed states. Closed-state inactivation of KV4 involves an unprecedented symmetry breakdown for pore closure by only two of the four S4-S5 linkers, distinct from known mechanisms of open-state inactivation. We further capture KV4 in a putative resting state, revealing how voltage sensor movements control the pore. Moreover, our structures provide insights regarding channel modulation by KChIP2 and DPP6 auxiliary subunits. Our findings elucidate mechanisms of closed-state inactivation and voltage-dependent activation of the KV4 channel.

Mechanosensitive Ion Channels: Structural Features Relevant to Mechanotransduction Mechanisms.

Activation of mechanosensitive ion channels underlies a variety of fundamental physiological processes that require sensation of mechanical force. Different mechanosensitive channels adapt distinctive structures and mechanotransduction mechanisms to fit their biological roles. How mechanosensitive channels work, especially in animals, has been extensively studied in the past decade. Here we review key findings in the functional and structural characterizations of these channels and highlight the structural features relevant to the mechanotransduction mechanism of each specific channel.

TMEM16F forms a Ca2+-activated cation channel required for lipid scrambling in platelets during blood coagulation.

Collapse of membrane lipid asymmetry is a hallmark of blood coagulation. TMEM16F of the TMEM16 family that includes TMEM16A/B Ca(2+)-activated Cl(-) channels (CaCCs) is linked to Scott syndrome with deficient Ca(2+)-dependent lipid scrambling. We generated TMEM16F knockout mice that exhibit bleeding defects and protection in an arterial thrombosis model associated with platelet deficiency in Ca(2+)-dependent phosphatidylserine exposure and procoagulant activity and lack a Ca(2+)-activated cation current in the platelet precursor megakaryocytes. Heterologous expression of TMEM16F generates a small-conductance Ca(2+)-activated nonselective cation (SCAN) current with subpicosiemens single-channel conductance rather than a CaCC. TMEM16F-SCAN channels permeate both monovalent and divalent cations, including Ca(2+), and exhibit synergistic gating by Ca(2+) and voltage. We further pinpointed a residue in the putative pore region important for the cation versus anion selectivity of TMEM16F-SCAN and TMEM16A-CaCC channels. This study thus identifies a Ca(2+)-activated channel permeable to Ca(2+) and critical for Ca(2+)-dependent scramblase activity during blood coagulation. PAPERFLICK:

Structure, function and pharmacology of human itch GPCRs.

The MRGPRX family of receptors (MRGPRX1-4) is a family of mas-related G-protein-coupled receptors that have evolved relatively recently1. Of these, MRGPRX2 and MRGPRX4 are key physiological and pathological mediators of itch and related mast cell-mediated hypersensitivity reactions2-5. MRGPRX2 couples to both Gi and Gq in mast cells6. Here we describe agonist-stabilized structures of MRGPRX2 coupled to Gi1 and Gq in ternary complexes with the endogenous peptide cortistatin-14 and with a synthetic agonist probe, respectively, and the development of potent antagonist probes for MRGPRX2. We also describe a specific MRGPRX4 agonist and the structure of this agonist in a complex with MRGPRX4 and Gq. Together, these findings should accelerate the structure-guided discovery of therapeutic agents for pain, itch and mast cell-mediated hypersensitivity.

TMEM16F exacerbates tau pathology and mediates phosphatidylserine exposure in phospho-tau-burdened neurons.

TMEM16F is a calcium-activated phospholipid scramblase and nonselective ion channel, which allows the movement of lipids bidirectionally across the plasma membrane. While the functions of TMEM16F have been extensively characterized in multiple cell types, the role of TMEM16F in the central nervous system remains largely unknown. Here, we sought to study how TMEM16F in the brain may be involved in neurodegeneration. Using a mouse model that expresses the pathological P301S human tau (PS19 mouse), we found reduced tauopathy and microgliosis in 6- to 7-mo-old PS19 mice lacking TMEM16F. Furthermore, this reduction of pathology can be recapitulated in the PS19 mice with TMEM16F removed from neurons, while removal of TMEM16F from microglia of PS19 mice did not significantly impact tauopathy at this time point. Moreover, TMEM16F mediated aberrant phosphatidylserine exposure in neurons with phospho-tau burden. These studies raise the prospect of targeting TMEM16F in neurons as a potential treatment of neurodegeneration.

Paralytic, the Drosophila voltage-gated sodium channel, regulates proliferation of neural progenitors.

Proliferating cells, typically considered "nonexcitable," nevertheless, exhibit regulation by bioelectric signals. Notably, voltage-gated sodium channels (VGSC) that are crucial for neuronal excitability are also found in progenitors and up-regulated in cancer. Here, we identify a role for VGSC in proliferation of Drosophila neuroblast (NB) lineages within the central nervous system. Loss of paralytic (para), the sole gene that encodes Drosophila VGSC, reduces neuroblast progeny cell number. The type II neuroblast lineages, featuring a population of transit-amplifying intermediate neural progenitors (INP) similar to that found in the developing human cortex, are particularly sensitive to para manipulation. Following a series of asymmetric divisions, INPs normally exit the cell cycle through a final symmetric division. Our data suggests that loss of Para induces apoptosis in this population, whereas overexpression leads to an increase in INPs and overall neuroblast progeny cell numbers. These effects are cell autonomous and depend on Para channel activity. Reduction of Para expression not only affects normal NB development, but also strongly suppresses brain tumor mass, implicating a role for Para in cancer progression. To our knowledge, our studies are the first to identify a role for VGSC in neural progenitor proliferation. Elucidating the contribution of VGSC in proliferation will advance our understanding of bioelectric signaling within development and disease states.

Dendrite regeneration of adult Drosophila sensory neurons diminishes with aging and is inhibited by epidermal-derived matrix metalloproteinase 2.

Dendrites possess distinct structural and functional properties that enable neurons to receive information from the environment as well as other neurons. Despite their key role in neuronal function, current understanding of the ability of neurons to regenerate dendrites is lacking. This study characterizes the structural and functional capacity for dendrite regeneration in vivo in adult animals and examines the effect of neuronal maturation on dendrite regeneration. We focused on the class IV dendritic arborization (c4da) neuron of the Drosophila sensory system, which has a dendritic arbor that undergoes dramatic remodeling during the first 3 d of adult life and then maintains a relatively stable morphology thereafter. Using a laser severing paradigm, we monitored regeneration after acute and spatially restricted injury. We found that the capacity for regeneration was present in adult neurons but diminished as the animal aged. Regenerated dendrites recovered receptive function. Furthermore, we found that the regenerated dendrites show preferential alignment with the extracellular matrix (ECM). Finally, inhibition of ECM degradation by inhibition of matrix metalloproteinase 2 (Mmp2) to preserve the extracellular environment characteristics of young adults led to increased dendrite regeneration. These results demonstrate that dendrites retain regenerative potential throughout adulthood and that regenerative capacity decreases with aging.

A twist on potassium channel gating.

The selectivity filter of potassium channels, the narrowest segment of the pore, permits only potassium ions to diffuse through the pore. Clarke et al. (2010) now present 11 structures of an inwardly rectifying potassium channel, providing evidence that the selectivity filter functions in channel gating and conformational changes in the cytoplasmic domains correlate with pore opening.

A photo-switchable assay system for dendrite degeneration and repair in Drosophila melanogaster.

Neurodegeneration arising from aging, injury, or diseases has devastating health consequences. Whereas neuronal survival and axon degeneration have been studied extensively, much less is known about how neurodegeneration affects dendrites, in part due to the limited assay systems available. To develop an assay for dendrite degeneration and repair, we used photo-switchable caspase-3 (caspase-Light-Oxygen-Voltage-sensing [caspase-LOV]) in peripheral class 4 dendrite arborization (c4da) neurons to induce graded neurodegeneration by adjusting illumination duration during development and adulthood in Drosophila melanogaster. We found that both developing and mature c4da neurons were able to survive while sustaining mild neurodegeneration induced by moderate caspase-LOV activation. Further, we observed active dendrite addition and dendrite regeneration in developing and mature c4da neurons, respectively. Using this assay, we found that the mouse Wallerian degeneration slow (WldS) protein can protect c4da neurons from caspase-LOV-induced dendrite degeneration and cell death. Furthermore, our data show that WldS can reduce dendrite elimination without affecting dendrite addition. In summary, we successfully established a photo-switchable assay system in both developing and mature neurons and used WldS as a test case to study the mechanisms underlying dendrite regeneration and repair.

Kv1.1 preserves the neural stem cell pool and facilitates neuron maturation during adult hippocampal neurogenesis.

Adult hippocampal neurogenesis is critical for learning and memory, and aberrant adult neurogenesis has been implicated in cognitive decline associated with aging and neurological diseases [J. T. Gonçalves, S. T. Schafer, F. H. Gage, Cell 167, 897­914 (2016)]. In previous studies, we observed that the delayed-rectifier voltage-gated potassium channel Kv1.1 controls the membrane potential of neural stem and progenitor cells and acts as a brake on neurogenesis during neonatal hippocampal development [S. M. Chou et al., eLife 10, e58779 (2021)]. To assess the role of Kv1.1 in adult hippocampal neurogenesis, we developed an inducible conditional knockout mouse to specifically remove Kv1.1 from adult neural stem cells via tamoxifen administration. We determined that Kv1.1 deletion in adult neural stem cells causes overproliferation and depletion of radial glia-like neural stem cells, prevents proper adult-born granule cell maturation and integration into the dentate gyrus, and moderately impairs hippocampus-dependent contextual fear learning and memory. Taken together, these findings support a critical role for this voltage-gated ion channel in adult neurogenesis.

Rare variants in ANO1, encoding a calcium-activated chloride channel, predispose to moyamoya disease.

Moyamoya disease, a cerebrovascular disease leading to strokes in children and young adults, is characterized by progressive occlusion of the distal internal carotid arteries and the formation of collateral vessels. Altered genes play a prominent role in the aetiology of moyamoya disease, but a causative gene is not identified in the majority of cases. Exome sequencing data from 151 individuals from 84 unsolved families were analysed to identify further genes for moyamoya disease, then candidate genes assessed in additional cases (150 probands). Two families had the same rare variant in ANO1, which encodes a calcium-activated chloride channel, anoctamin-1. Haplotype analyses found the families were related, and ANO1 p.Met658Val segregated with moyamoya disease in the family with an LOD score of 3.3. Six additional ANO1 rare variants were identified in moyamoya disease families. The ANO1 rare variants were assessed using patch-clamp recordings, and the majority of variants, including ANO1 p.Met658Val, displayed increased sensitivity to intracellular Ca2+. Patients harbouring these gain-of-function ANO1 variants had classic features of moyamoya disease, but also had aneurysm, stenosis and/or occlusion in the posterior circulation. Our studies support that ANO1 gain-of-function pathogenic variants predispose to moyamoya disease and are associated with unique involvement of the posterior circulation.

A gate keeper for axonal transport.

The axon and dendritic arbor of neurons require different sets of membrane proteins to carry out their functions. In this issue, Song et al. (2009) describe how a cytoplasmic diffusion barrier in the axon initial segment of rat hippocampal neurons ensures that only axonal (and not dendritic) membrane proteins enter the axon.

TMEM16C is involved in thermoregulation and protects rodent pups from febrile seizures.

Febrile seizures (FSs) are the most common convulsion in infancy and childhood. Considering the limitations of current treatments, it is important to examine the mechanistic cause of FSs. Prompted by a genome-wide association study identifying TMEM16C (also known as ANO3) as a risk factor of FSs, we showed previously that loss of TMEM16C function causes hippocampal neuronal hyperexcitability [Feenstra et al., Nat. Genet. 46, 1274-1282 (2014)]. Our previous study further revealed a reduction in the number of warm-sensitive neurons that increase their action potential firing rate with rising temperature of the brain region harboring these hypothalamic neurons. Whereas central neuronal hyperexcitability has been implicated in FSs, it is unclear whether the maximal temperature reached during fever or the rate of body temperature rise affects FSs. Here we report that mutant rodent pups with TMEM16C eliminated from all or a subset of their central neurons serve as FS models with deficient thermoregulation. Tmem16c knockout (KO) rat pups at postnatal day 10 (P10) are more susceptible to hyperthermia-induced seizures. Moreover, they display a more rapid rise of body temperature upon heat exposure. In addition, conditional knockout (cKO) mouse pups (P11) with TMEM16C deletion from the brain display greater susceptibility of hyperthermia-induced seizures as well as deficiency in thermoregulation. We also found similar phenotypes in P11 cKO mouse pups with TMEM16C deletion from Ptgds-expressing cells, including temperature-sensitive neurons in the preoptic area (POA) of the anterior hypothalamus, the brain region that controls body temperature. These findings suggest that homeostatic thermoregulation plays an important role in FSs.

In vivo dendrite regeneration after injury is different from dendrite development.

Neurons receive information along dendrites and send signals along axons to synaptic contacts. The factors that control axon regeneration have been examined in many systems, but dendrite regeneration has been largely unexplored. Here we report that, in intact Drosophila larvae, a discrete injury that removes all dendrites induces robust dendritic growth that recreates many features of uninjured dendrites, including the number of dendrite branches that regenerate and responsiveness to sensory stimuli. However, the growth and patterning of injury-induced dendrites is significantly different from uninjured dendrites. We found that regenerated arbors cover much less territory than uninjured neurons, fail to avoid crossing over other branches from the same neuron, respond less strongly to mechanical stimuli, and are pruned precociously. Finally, silencing the electrical activity of the neurons specifically blocks injury-induced, but not developmental, dendrite growth. By elucidating the essential features of dendrites grown in response to acute injury, our work builds a framework for exploring dendrite regeneration in physiological and pathological conditions.

Expression cloning of TMEM16A as a calcium-activated chloride channel subunit.

Calcium-activated chloride channels (CaCCs) are major regulators of sensory transduction, epithelial secretion, and smooth muscle contraction. Other crucial roles of CaCCs include action potential generation in Characean algae and prevention of polyspermia in frog egg membrane. None of the known molecular candidates share properties characteristic of most CaCCs in native cells. Using Axolotl oocytes as an expression system, we have identified TMEM16A as the Xenopus oocyte CaCC. The TMEM16 family of "transmembrane proteins with unknown function" is conserved among eukaryotes, with family members linked to tracheomalacia (mouse TMEM16A), gnathodiaphyseal dysplasia (human TMEM16E), aberrant X segregation (a Drosophila TMEM16 family member), and increased sodium tolerance (yeast TMEM16). Moreover, mouse TMEM16A and TMEM16B yield CaCCs in Axolotl oocytes and mammalian HEK293 cells and recapitulate the broad CaCC expression. The identification of this new family of ion channels may help the development of CaCC modulators for treating diseases including hypertension and cystic fibrosis.

Electron cryo-microscopy structure of the mechanotransduction channel NOMPC.

Mechanosensory transduction for senses such as proprioception, touch, balance, acceleration, hearing and pain relies on mechanotransduction channels, which convert mechanical stimuli into electrical signals in specialized sensory cells. How force gates mechanotransduction channels is a central question in the field, for which there are two major models. One is the membrane-tension model: force applied to the membrane generates a change in membrane tension that is sufficient to gate the channel, as in the bacterial MscL channel and certain eukaryotic potassium channels. The other is the tether model: force is transmitted via a tether to gate the channel. The transient receptor potential (TRP) channel NOMPC is important for mechanosensation-related behaviours such as locomotion, touch and sound sensation across different species including Caenorhabditis elegans, Drosophila and zebrafish. NOMPC is the founding member of the TRPN subfamily, and is thought to be gated by tethering of its ankyrin repeat domain to microtubules of the cytoskeleton. Thus, a goal of studying NOMPC is to reveal the underlying mechanism of force-induced gating, which could serve as a paradigm of the tether model. NOMPC fulfils all the criteria that apply to mechanotransduction channels and has 29 ankyrin repeats, the largest number among TRP channels. A key question is how the long ankyrin repeat domain is organized as a tether that can trigger channel gating. Here we present a de novo atomic structure of Drosophila NOMPC determined by single-particle electron cryo-microscopy. Structural analysis suggests that the ankyrin repeat domain of NOMPC resembles a helical spring, suggesting its role of linking mechanical displacement of the cytoskeleton to the opening of the channel. The NOMPC architecture underscores the basis of translating mechanical force into an electrical signal within a cell.

Cryo-EM structures of the TMEM16A calcium-activated chloride channel.

Calcium-activated chloride channels (CaCCs) encoded by TMEM16A control neuronal signalling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the gastrointestinal system. To understand how CaCCs mediate and control anion permeation to fulfil these physiological functions, knowledge of the mammalian TMEM16A structure and identification of its pore-lining residues are essential. TMEM16A forms a dimer with two pores. Previous CaCC structural analyses have relied on homology modelling of a homologue (nhTMEM16) from the fungus Nectria haematococca that functions primarily as a lipid scramblase, as well as subnanometre-resolution electron cryo-microscopy. Here we present de novo atomic structures of the transmembrane domains of mouse TMEM16A in nanodiscs and in lauryl maltose neopentyl glycol as determined by single-particle electron cryo-microscopy. These structures reveal the ion permeation pore and represent different functional states. The structure in lauryl maltose neopentyl glycol has one Ca2+ ion resolved within each monomer with a constricted pore; this is likely to correspond to a closed state, because a CaCC with a single Ca2+ occupancy requires membrane depolarization in order to open (C.J.P. et al., manuscript submitted). The structure in nanodiscs has two Ca2+ ions per monomer and its pore is in a closed conformation; this probably reflects channel rundown, which is the gradual loss of channel activity that follows prolonged CaCC activation in 1 mM Ca2+. Our mutagenesis and electrophysiological studies, prompted by analyses of the structures, identified ten residues distributed along the pore that interact with permeant anions and affect anion selectivity, as well as seven pore-lining residues that cluster near pore constrictions and regulate channel gating. Together, these results clarify the basis of CaCC anion conduction.

Glial ensheathment of the somatodendritic compartment regulates sensory neuron structure and activity.

Sensory neurons perceive environmental cues and are important of organismal survival. Peripheral sensory neurons interact intimately with glial cells. While the function of axonal ensheathment by glia is well studied, less is known about the functional significance of glial interaction with the somatodendritic compartment of neurons. Herein, we show that three distinct glia cell types differentially wrap around the axonal and somatodendritic surface of the polymodal dendritic arborization (da) neuron of the Drosophila peripheral nervous system for detection of thermal, mechanical, and light stimuli. We find that glial cell-specific loss of the chromatin modifier gene dATRX in the subperineurial glial layer leads to selective elimination of somatodendritic glial ensheathment, thus allowing us to investigate the function of such ensheathment. We find that somatodendritic glial ensheathment regulates the morphology of the dendritic arbor, as well as the activity of the sensory neuron, in response to sensory stimuli. Additionally, glial ensheathment of the neuronal soma influences dendritic regeneration after injury.

TMEM16A controls EGF-induced calcium signaling implicated in pancreatic cancer prognosis.

Pancreatic cancer typically spreads rapidly and has poor survival rates. Here, we report that the calcium-activated chloride channel TMEM16A is a biomarker for pancreatic cancer with a poor prognosis. TMEM16A is up-regulated in 75% of cases of pancreatic cancer and high levels of TMEM16A expression are correlated with low patient survival probability. TMEM16A up-regulation is associated with the ligand-dependent EGFR signaling pathway. In vitro, TMEM16A is required for EGF-induced store-operated calcium entry essential for pancreatic cancer cell migration. TMEM16A also has a profound impact on phosphoproteome remodeling upon EGF stimulation. Moreover, molecular actors identified in this TMEM16A-dependent EGFR-induced calcium signaling pathway form a gene set that makes it possible not only to distinguish neuro-endocrine tumors from other forms of pancreatic cancer, but also to subdivide the latter into three clusters with distinct genetic profiles that could reflect their molecular underpinning.