Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate chemical communication between neurons at synapses. A variant iGluR subfamily, the Ionotropic Receptors (IRs), was recently proposed to detect environmental volatile chemicals in olfactory cilia. Here we elucidate how these peripheral chemosensors have evolved mechanistically from their iGluR ancestors. Using a Drosophila model, we demonstrate that IRs act in combinations of up to three subunits, comprising individual odor-specific receptors and one or two broadly expressed co-receptors. Heteromeric IR complex formation is necessary and sufficient for trafficking to cilia and mediating odor-evoked electrophysiological responses in vivo and in vitro. IRs display heterogeneous ion conduction specificities related to their variable pore sequences, and divergent ligand-binding domains function in odor recognition and cilia localization. Our results provide insights into the conserved and distinct architecture of these olfactory and synaptic ion channels and offer perspectives into use of IRs as genetically encoded chemical sensors.
Animals adapt their behaviors to specific ecological niches, but the genetic and cellular basis of nervous system evolution is poorly understood. We have compared the olfactory circuits of the specialist Drosophila sechellia-which feeds exclusively on Morinda citrifolia fruit-with its generalist cousins D. melanogaster and D. simulans. We show that D. sechellia exhibits derived odor-evoked attraction and physiological sensitivity to the abundant Morinda volatile hexanoic acid and characterize how the responsible sensory receptor (the variant ionotropic glutamate receptor IR75b) and attraction-mediating circuit have evolved. A single amino acid change in IR75b is sufficient to recode it as a hexanoic acid detector. Expanded representation of this sensory pathway in the brain relies on additional changes in the IR75b promoter and trans-acting loci. By contrast, higher-order circuit adaptations are not apparent, suggesting conserved central processing. Our work links olfactory ecology to structural and regulatory genetic changes influencing nervous system anatomy and function.
Pseudogenes are generally considered to be non-functional DNA sequences that arise from protein-coding genes through nonsense or frame-shift mutations1. Although certain pseudogene-derived RNAs have regulatory roles2, and some pseudogene fragments are translated3, no clear functions for pseudogene-derived proteins are known. Olfactory receptor families contain many pseudogenes, reflecting low selection pressures to maintain receptors that are either functionally redundant or detect odours no longer relevant for a species’ fitness4. Here we have characterised a pseudogene in the chemosensory variant ionotropic glutamate receptor (IR) repertoire5,6 of Drosophila sechellia, an insect endemic to the Seychelles that feeds only on ripe fruit of Morinda citrifolia7. This locus, DsecIr75a, bears a premature termination codon (PTC) that appears to be fixed in the population. Unexpectedly, DsecIr75a encodes a functional receptor, due to efficient translational readthrough of the PTC. Readthrough occurs only in neurons, and is independent of the type of termination codon but dependent upon the sequence downstream of the PTC. Furthermore, while the intact D. melanogaster IR75a orthologue detects acetic acid – a chemical cue important for this species to locate fermenting food8,9 but at trace levels in Morinda fruit10 – DsecIR75a has evolved distinct odour-tuning properties, through amino acid changes in its ligand-binding domain. We identify functional PTC-containing loci within different olfactory receptor repertoires and species, suggesting that such “pseudo-pseudogenes” represent a widespread phenomenon.
CD36 transmembrane proteins have diverse roles in lipid uptake, cell adhesion and pathogen sensing. Despite numerous in vitro studies, how they act in native cellular contexts is poorly understood. A Drosophila CD36 homologue, sensory neuron membrane protein 1 (SNMP1), was previously shown to facilitate detection of lipid-derived pheromones by their cognate receptors in olfactory cilia. Here we investigate how SNMP1 functions in vivo. Structure–activity dissection demonstrates that SNMP1's ectodomain is essential, but intracellular and transmembrane domains dispensable, for cilia localization and pheromone-evoked responses. SNMP1 can be substituted by mammalian CD36, whose ectodomain can interact with insect pheromones. Homology modelling, using the mammalian LIMP-2 structure as template, reveals a putative tunnel in the SNMP1 ectodomain that is sufficiently large to accommodate pheromone molecules. Amino-acid substitutions predicted to block this tunnel diminish pheromone sensitivity. We propose a model in which SNMP1 funnels hydrophobic pheromones from the extracellular fluid to integral membrane receptors.
Acid-sensing ion channels (ASICs) are key receptors for extracellular protons. These neuronal nonvoltage-gated Na ؉ channels are involved in learning, the expression of fear, neurodegeneration after ischemia, and pain sensation. We have applied a systematic approach to identify potential pH sensors in ASIC1a and to elucidate the mechanisms by which pH variations govern ASIC gating. We first calculated the pK a value of all extracellular His, Glu, and Asp residues using a Poisson-Boltzmann continuum approach, based on the ASIC three-dimensional structure, to identify candidate pH-sensing residues. The role of these residues was then assessed by site-directed mutagenesis and chemical modification, combined with functional analysis. The localization of putative pH-sensing residues suggests that pH changes control ASIC gating by protonation/ deprotonation of many residues per subunit in different channel domains. Analysis of the function of residues in the palm domain close to the central vertical axis of the channel allowed for prediction of conformational changes of this region during gating. Our study provides a basis for the intrinsic ASIC pH dependence and describes an approach that can also be applied to the investigation of the mechanisms of the pH dependence of other proteins.Acid-sensing ion channels (ASICs) 3 are neuronal nonvoltage-gated Na ϩ channels that are activated by a rapid drop in extracellular pH (1, 2). They are members of the epithelial Na ϩ channel/Degenerin family of ion channel proteins (3). Expression in nociceptive neurons and activation by protons suggest that ASICs may act as pain receptors (4), and evidence for such a role has been provided in several animal pain models (5-7). ASIC1a in the central nervous system plays a role in memory formation and the expression of fear (8, 9). ASIC1a is also an important mediator of cell injury induced by conditions associated with acidosis in the mammalian nervous system (10). Functional ASICs are formed by homo-or heterotrimeric assembly of ASIC subunits 1a, 1b, 2a, 2b, and 3. Each subunit has short intracellular N and C termini and two transmembrane domains that are separated by a large extracellular domain.Extracellular acidification opens ASICs. The ASIC activity is terminated in the continued presence of the acidic stimulus within hundreds of milliseconds to seconds by open channel inactivation, which is also called desensitization (11). Only in some ASIC isoforms and under certain pH conditions can acidification induce a sustained current, which has in most cases a much smaller amplitude than the peak current (12-14). At pH values slightly below the physiological pH, ASICs inactivate without apparent channel opening in a process that is called steady-state inactivation (SSIN) (15). By these two ways ASICs enter the inactivated state, which is a nonconducting, absorbing state. Experimental protocols have been applied to determine the kinetics of the recovery from the inactivated state, showing that channels need exposure for a certain duration ...
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