Olfactory systems must detect and discriminate amongst an enormous variety of odorants
. To contend with this challenge, diverse species have converged on a common strategy in which odorant identity ...is encoded through the combinatorial activation of large families of olfactory receptors
, thus allowing a finite number of receptors to detect a vast chemical world. Here we offer structural and mechanistic insight into how an individual olfactory receptor can flexibly recognize diverse odorants. We show that the olfactory receptor MhOR5 from the jumping bristletail
Machilis hrabei assembles as a homotetrameric odorant-gated ion channel with broad chemical tuning. Using cryo-electron microscopy, we elucidated the structure of MhOR5 in multiple gating states, alone and in complex with two of its agonists-the odorant eugenol and the insect repellent DEET. Both ligands are recognized through distributed hydrophobic interactions within the same geometrically simple binding pocket located in the transmembrane region of each subunit, suggesting a structural logic for the promiscuous chemical sensitivity of this receptor. Mutation of individual residues lining the binding pocket predictably altered the sensitivity of MhOR5 to eugenol and DEET and broadly reconfigured the receptor's tuning. Together, our data support a model in which diverse odorants share the same structural determinants for binding, shedding light on the molecular recognition mechanisms that ultimately endow the olfactory system with its immense discriminatory capacity.
Living cells respond to mechanical forces applied to their outer membrane through processes referred to as “mechanosensation”. Faced with hypotonic shock, to circumvent cell lysis, bacteria open ...large solute-passing channels to reduce the osmotic pressure gradient. In the vascular beds of vertebrate animals blood flow is regulated directly through mechanical distention-induced opening of stretch-activated channels in smooth muscle cells. Touch sensation is thought to originate in mechanically sensitive ion channels in nerve endings, and hearing in mechanically sensitive ion channels located in specialized cells of the ear. While the ubiquity of mechanosensation in living cells is evident, the ion channels underlying the transduction events in vertebrate animals have remained elusive. Here we demonstrate through electrophysiological recordings that voltage-dependent K ⁺ (Kv) channels exhibit exquisite sensitivity to small (physiologically relevant in magnitude) mechanical perturbations of the cell membrane. The demonstrated mechanosensitivity is quantitatively consistent with membrane tension acting on a late-opening transition through stabilization of a dilated pore. This effect causes a shift in the voltage range over which Kv channels open as well as an increase in the maximum open probability. This mechanically induced shift could allow Kv channels and perhaps other voltage-dependent ion channels to play a role in mechanosensation.
TRAAK channels, members of the two-pore domain K⁺ (potassium ion) channel family K2P, are expressed almost exclusively in the nervous system and control the resting membrane potential. Their gating ...is sensitive to polyunsaturated fatty adds, mechanical deformation of the membrane, and temperature changes. Physiologically, these channels appear to control the noxious input threshold for temperature and pressure sensitivity in dorsal root ganglia neurons. We present the crystal structure of human TRAAK at a resolution of 3.8 angstroms. The channel comprises two protomers, each containing two distinct pore domains, which create a two-fold symmetric K⁺ channel. The extracellular surface features a helical cap, 35 angstroms tall, that creates a bifurcated pore entryway and accounts for the insensitivity of two-pore domain K⁺ channels to inhibitory toxins. Two diagonally opposed gate-forming inner helices form membrane-interacting structures that may underlie this channel's sensitivity to chemical and mechanical properties of the cell membrane.
Connections between the gut and brain monitor the intestinal tissue and its microbial and dietary content
, regulating both physiological intestinal functions such as nutrient absorption and motility
..., and brain-wired feeding behaviour
. It is therefore plausible that circuits exist to detect gut microorganisms and relay this information to areas of the central nervous system that, in turn, regulate gut physiology
. Here we characterize the influence of the microbiota on enteric-associated neurons by combining gnotobiotic mouse models with transcriptomics, circuit-tracing methods and functional manipulations. We find that the gut microbiome modulates gut-extrinsic sympathetic neurons: microbiota depletion leads to increased expression of the neuronal transcription factor cFos, and colonization of germ-free mice with bacteria that produce short-chain fatty acids suppresses cFos expression in the gut sympathetic ganglia. Chemogenetic manipulations, translational profiling and anterograde tracing identify a subset of distal intestine-projecting vagal neurons that are positioned to have an afferent role in microbiota-mediated modulation of gut sympathetic neurons. Retrograde polysynaptic neuronal tracing from the intestinal wall identifies brainstem sensory nuclei that are activated during microbial depletion, as well as efferent sympathetic premotor glutamatergic neurons that regulate gastrointestinal transit. These results reveal microbiota-dependent control of gut-extrinsic sympathetic activation through a gut-brain circuit.
Mosquitoes rely on their sense of smell to find humans to secure a blood meal, transmitting deadly diseases with their bite. In this issue of Cell, De Obaldía and colleagues examine why mosquitoes ...bite some people more than others and report an association with the level of carboxylic acids in the human skin odor.
Piezo1 is a mechanosensitive (MS) ion channel with characteristic fast-inactivation kinetics. We found a slowly-inactivating MS current in mouse embryonic stem (mES) cells and characterized it ...throughout their differentiation into motor-neurons to investigate its components. MS currents were large and slowly-inactivating in the stem-cell stage, and became smaller and faster-inactivating throughout the differentiation. We found that Piezo1 is expressed in mES cells, and its knockout abolishes MS currents, indicating that the slowly-inactivating current in mES cells is carried by Piezo1. To further investigate its slow inactivation in these cells, we cloned Piezo1 cDNA from mES cells and found that it displays fast-inactivation kinetics in heterologous expression, indicating that sources of modulation other than the aminoacid sequence determine its slow kinetics in mES cells. Finally, we report that Piezo1 knockout ES cells showed a reduced rate of proliferation but no significant differences in other markers of pluripotency and differentiation.
We describe the synthesis and characterization of a ruthenium−bipyridyl complex bearing a rhodamine-based fluorescent ligand. The complex is weakly fluorescent due to the quenching of rhodamine. Upon ...irradiation of the MLCT band it releases rhodamine in a fast and clean heterolytic reaction, increasing its fluorescence nearly 6-fold and making it the first visible-light activatable fluorophore based in transition metal chemistry. These properties and its lack of toxicity make it a good candidate for its use as a biologically friendly caged fluorescent probe. The use of this probe as a neuronal marker, and as a flow profiler in a thin, planar cavity and in a model flow injection analysis (FIA) is demonstrated.
Most insects, including human-targeting mosquitoes, detect odors through odorant-activated ion channel complexes consisting of a divergent odorant-binding subunit (OR) and a conserved co-receptor ...subunit (Orco). As a basis for understanding how odorants activate these heteromeric receptors, we report here cryo–electron microscopy structures of two different heteromeric odorant receptor complexes containing ORs from disease-vector mosquitos Aedes aegypti or Anopheles gambiae . These structures reveal an unexpected stoichiometry of one OR to three Orco subunits. Comparison of structures in odorant-bound and unbound states indicates that odorant binding to the sole OR subunit is sufficient to open the channel pore, suggesting a mechanism of OR activation and a conceptual framework for understanding evolution of insect odorant receptor sensitivity.
Editor’s summary There are many molecular strategies used by animals to sense small molecules that are indicative of a meal. Two independent groups of researchers report structural studies on insect-specific tetrameric ligand-gated ion channels. These channels are composed of one odorant receptor (OR) subunit, which binds a ligand, and three Orco subunits, which do not bind ligands. Wang et al . studied an OR-Orco complex from an agriculturally relevant aphid, and Zhao et al . determined structures of the ORs from two mosquito species bound to Orco subunits from another insect. Both teams propose a similar mechanism of gating in which the presence of a single activated OR can lead to opening of the tetrameric channel. Although questions remain about the receptors’ physiological composition and function, these structures are a starting place for developing molecules that target insect ORs for a variety of applications. —Michael A. Funk
The olfactory system must recognize and discriminate amongst an enormous variety of chemicals in the environment. To contend with such diversity, insects have evolved a family of odorant-gated ion ...channels comprised of a highly conserved co-receptor (Orco) and a divergent odorant receptor (OR) that confers chemical specificity. Here, we present the single-particle cryo-electron microscopy structure of an Orco homomer from the parasitic fig wasp Apocrypta bakeri at 3.5 Å resolution, providing structural insight into this receptor family. Orco possesses a novel channel architecture, with four subunits symmetrically arranged around a central pore that diverges into four lateral conduits that open to the cytosol. The Orco tetramer has few inter-subunit interactions within the membrane and is bound together by a small cytoplasmic anchor domain. The minimal sequence conservation among ORs maps largely to the pore and anchor domain, shedding light on how the architecture of this receptor family accommodates its remarkable sequence diversity and facilitates the evolution of odour tuning.
Mechanical force sensation is fundamental to a wide breadth of biology from the classic senses of touch, pain, hearing, and balance to less conspicuous sensations of proprioception, blood pressure, ...and osmolarity and basic aspects of cell growth, differentiation, and development. These diverse and essential systems use force-gated (or mechanosensitive) ion channels that convert mechanical stimuli into cellular electrical signals. TRAAK, TREK1, and TREK2 are K
-selective ion channels of the two-pore domain K
(K2P) family that are mechanosensitive: they are gated open by increasing membrane tension. TRAAK and TREK channels are thought to play roles in somatosensory and other mechanosensory processes in neuronal and non-neuronal tissues. Here, we present protocols for three assays to study mechanical activation of these channels in cell membranes: (1) cell swelling, (2) cell poking, and (3) patched membrane stretching. Patched membrane stretching is also applicable to the study of mechanosensitive K2P channel activity in a cell-free system and a procedure for proteoliposome reconstitution and patching is also presented. These approaches are also readily applicable to the study of other mechanosensitive ion channels.
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