A large family of divergent ionotropic glutamate receptor-like genes in Drosophila
From a bioinformatic screen for novel olfactory molecules (Benton et al., 2007
), we identified 6 antennal-expressed genes encoding proteins annotated as ionotropic glutamate receptors (iGluRs) (data not shown; Littleton and Ganetzky, 2000
). Using these novel receptor sequences as queries, exhaustive BLAST searches of the Drosophila
genome identified a family of 61 predicted genes and 1 pseudogene. These genes are distributed throughout the genome, both as individual sequences and in tandem arrays of up to four genes (, data not shown). We named this family the Ionotropic Receptors (IRs) and assigned individual gene names to the IR
s using nomenclature conventions of Drosophila OR
s (Drosophila Odorant Receptor Nomenclature Committee, 2000
A novel family of divergent ionotropic glutamate receptors in Drosophila
Phylogenetic analysis of predicted IR protein sequences revealed that they are not closely related to members of the canonical families of iGluRs (AMPA, kainate, NMDA, or delta) (). However, they appear to have a similar modular organization to iGluRs, comprising an extracellular N-terminus, a bipartite ligand-binding domain, whose two lobes (S1 and S2) are separated by an ion channel domain, and a short cytoplasmic C-terminus (- and data not shown) (Mayer, 2006
). We note that the gene structure and protein sequence of most receptors are presently only computational predictions. Nevertheless, the family is extremely divergent, exhibiting overall amino acid sequence identity of 10-70%. The most conserved region between IRs and iGluRs spans the ion channel pore (), suggesting that IRs retain ion-conducting properties.
Ligand binding domains in most IRs lack glutamate-interacting residues
The ligand-binding domains are considerably more variable, although alignment of small regions of the S1 and S2 lobes of IRs and iGluRs allowed examination of conservation in amino acid positions that make direct contact with glutamate or artificial agonists in iGluRs (Armstrong et al., 1998
; Armstrong and Gouaux, 2000
; Jin et al., 2003
; Mayer, 2005
) (). While all iGluRs have an arginine (R) residue in S1 that binds the glutamate α-carboxyl group, only 19/61 (31%) IRs retain this residue (). In the first half of the S2 domain, 9/61 (15%) of IRs retain a threonine (T), which contacts the glutamate γ-carboxyl group in all AMPA and kainate receptors (). Interestingly, the iGluRs that lack this T residue (NR1, NR3A, delta) have glycine or serine and not glutamate as a preferred ligand (Mayer et al., 2006
; Naur et al., 2007
). Finally, in the second half of the S2 domain, 100% of the iGluRs have a conserved aspartate (D) or glutamate (E) that interacts with the α-amino group of the glutamate ligand, compared with 10/61 (16%) IRs (). Of 61 IRs, only three (IR8a, IR75a, IR75c) retain the R, D/E, and T residues characteristic of iGluRs, although these residues lie within a divergent structural backbone. Other IRs have a diversity of different amino acids at one or more of these positions. Thus, the ligand-binding specificity of most or all IRs is likely to be both distinct from that of iGluRs and varied within the IR family.
IRs are expressed in chemosensory neurons that do not express ORs or OR83b
We determined the expression of the IR family by both tissue-specific RT-PCR and RNA in situ hybridization. Fifteen IR genes are expressed in the antenna ( and ). Transcripts of these genes were not detected elsewhere in the adult head, body or appendages, except for IR25a and IR76b, which are also expressed in the proboscis (data not shown). Expression of the remaining 46 IR genes was not reproducibly detected in any adult tissue. It is unclear whether these genes are not expressed, expressed at different life stages, or expressed in at levels below the detection threshold of our assays.
A topological map of IR expression in the antenna
We analyzed where in the antenna IR
genes are expressed compared to OR
s by double RNA in situ
hybridization with probes for the OR co-receptor OR83b
and one of several IR
genes, including IR64a
, and IR40a
( and data not shown). IRs
are not expressed in basiconic and trichoid sensilla, as they are not co-expressed with OR83b
, and IR
expression persists in mutants for the proneural gene absent md neurons and olfactory sensilla
), which completely lack these sensilla types (Goulding et al., 2000
; zur Lage et al., 2003
) (, top and middle panels; data not shown). However, expression of these IR
s is dependent upon the proneural gene atonal
, which specifies the coeloconic sensilla as well as a feather-like projection called the arista, and a three-chambered pocket called the sacculus (, bottom panel; data not shown) (Gupta and Rodrigues, 1997
; Jhaveri et al., 2000
). Thus, OR
s and IR
s are expressed in developmentally distinct sensory lineages in the antenna. One exception is the subpopulation of coeloconic OSNs that expresses both IR76b
(Couto et al., 2005
; Yao et al., 2005
) (). We confirmed that IR
-expressing cells in the antenna are neurons by demonstrating that they co-express the neuronal marker elav
( and data not shown).
We next generated a comprehensive map of IR expression (). Each IR was observed to have a topologically-defined expression pattern that is conserved across individuals of both sexes (data not shown). IR8a and IR25a, which encode closely related receptors (), are broadly expressed, detected in overlapping populations of neurons around the sacculus and in the main portion of the antenna (; data not shown). IR25a but not IR8a is also detected in the arista (data not shown; see ). IR21a is expressed in approximately 6 neurons in the arista (), as well as 5-10 neurons near the third chamber of the sacculus (, bottom panel). Three IRs display specific expression in neurons surrounding the sacculus: IR40a and IR93a are co-expressed in 10-15 neurons adjacent to the first and second sacculus chambers (, top panel), while IR64a is found in 10-15 neurons surrounding the third chamber (, middle panel).
Glomerular convergence of IR axons and ciliary localization of IR proteins
The remaining 9 IRs are expressed in coeloconic OSNs distributed across the antenna (). Double and triple RNA in situ hybridization revealed that individual neurons express between 1 and 3 different IR genes and are organized into specific clusters of two or three neurons. Four distinct clusters (cluster A-cluster D), containing two (cluster C) or three (cluster A, B, and D) neurons, could be defined by their expression of stereotyped combinations of IR genes (). Cluster C includes a coeloconic neuron that expresses OR35a and OR83b in addition to IR76b. Although each cluster is distinct, there is overlap between the IRs they express. IR76b is expressed in one neuron in all four clusters, IR75d in three clusters and IR75a in two clusters (). In additional to these selectively-expressed receptors, individual neurons are likely to express one or both of the broadly-expressed IR8a and IR25a (). The combinatorial expression patterns of the IRs raise the possibility that these genes define specific functional properties of these neurons.
Integration of molecular and functional maps of the coeloconic sensilla
Our definition of four distinct clusters of IR
-expressing neurons in the antenna () is consistent with the identification of four types of coeloconic sensilla, named ac1-ac4, which have distinct yet partially overlapping sensory specificities (Yao et al., 2005
). To examine whether IR
expression correlates with the chemosensory properties of these OSNs, we compared the spatial organization of IR
-expressing neurons using probes for unique IR
markers for each cluster type to these functionally distinct sensilla types (). As we lack a unique molecular marker for Cluster B, this cluster was defined as those containing IR75a
-expressing OSNs (present in Cluster B and Cluster C) that are not paired with OR35a
-expressing cluster C neurons (). We found that each cluster has a different, though overlapping, spatial distribution in the antenna (). For example, Cluster A neurons (marked by IR31a
) are restricted to a zone at the anterior of the antenna, just below the arista, while cluster C neurons (marked by IR75b
) are found exclusively in the posterior of the antenna. These stereotyped IR
neuron distributions were observed in antennae from over 20 animals.
Integration of molecular and functional maps in the coeloconic sensilla
The initial description of the coeloconic sensilla classes did not describe their spatial distribution (Yao et al., 2005
). We therefore recorded odor-evoked responses in >100 coeloconic sensilla in several dozen animals across most of the accessible antennal surface, using a panel of odorants that allowed us to identify unambiguously each sensilla type (ammonia for ac1, 1,4-diaminobutane for ac2, propanal and hexanol for ac3, and phenylacetaldehyde for ac4) (Yao et al., 2005
) (, left). After electrophysiological identification, we noted the location of the sensilla on the antennal surface (, right).
This mapping process allowed a correlation of the electrophysiological and molecular properties of the coeloconic sensilla (). For example, ac1 sensilla were only detected in a region on the anterior antennal surface just ventral to the arista, and therefore are most likely correspond to cluster A, containing IR31a-IR75d-IR76b/IR92a
-expressing neurons. Our data fit well with the previous assignment of the OR35a
-expressing neuron to the ac3 sensillum (Yao et al., 2005
), which is found on the posterior of the antenna and is the only coeloconic sensillum class that unambiguously houses two neurons (Yao et al., 2005
) ( and data not shown). While these results allow initial assignment of IRs
to different coeloconic sensilla classes, we note that assignment of specific odor responses to individual IR
-expressing OSNs is not possible from these data alone.
Glomerular convergence of IR OSNs in the antennal lobe
All neurons expressing a given OR extend axons that converge upon a single antennal lobe glomerulus (Couto et al., 2005
; Fishilevich and Vosshall, 2005
), resulting in the representation of a cognate odor ligand as a spatially-defined pattern of neural activity within the brain. To ask whether IR
-expressing neurons have the same wiring logic, we investigated the targeting of OSNs expressing IR76a
by constructing an IR76a
-promoter GAL4 driver that recapitulates the endogenous expression pattern (Brand and Perrimon, 1993
) (). Labeling of these neurons with mCD8:GFP revealed convergence of their axons on to a single glomerulus, ventral medial 4 (VM4), in the antennal lobe (). This glomerulus is one of approximately eight that was previously unaccounted for by maps of axonal projections of OR-expressing OSNs (Couto et al., 2005
; Vosshall and Stocker, 2007
IR proteins localize to sensory cilia
To determine where IRs localize in sensory neurons, we generated antibodies against IR25a. We detected broad expression of IR25a protein in sensory neurons of the arista, sacculus, and coeloconic sensilla (, left). All anti-IR25a immunoreactivity was abolished in an IR25a null mutant (, right). Low levels of IR25a could be detected in the axon segment adjacent to the cell body in some neurons but no staining was observed along the axons as they entered the brain, or at synapses within antennal lobe glomeruli (). In coeloconic neurons, prominent anti-IR25a staining was detected both in the cell body and in the distal tip of the dendrite, which corresponds to the ciliated outer dendritic segment innervating the sensory hair (). Relatively low levels were detected in the inner dendrites, suggesting the existence of a transport mechanism to concentrate receptor protein in cilia. A similar subcellular localization was observed in sacculus and aristal sensory neurons (). The specific targeting of an IR to sensory cilia suggests a role for these proteins in sensory detection.
Inducing novel olfactory sensitivity by ectopic expression of IRs
To test the hypothesis that IR genes encode chemosensory receptors, we investigated whether ectopic IR expression could induce novel olfactory specificities. Three IRs expressed in ac4 sensilla (IR84a, IR76a and IR75d) were individually mis-expressed in ac3 sensilla using the OR35a-GAL4
driver (Fishilevich and Vosshall, 2005
). We used single sensillum recordings to examine which, if any, of these three IRs, could confer sensitivity to phenylacetaldehyde, the only known robust ligand for ac4 but not ac3 sensilla (Yao et al., 2005
). Mis-expression of IR84a conferred a strong response to phenylacetaldehyde () that was not observed in control strains or in animals mis-expressing either IR76a or IR75d (). Ectopically-expressed IR84a did not confer sensitivity to the structurally related odor, phenylacetonitrile, which does not activate either ac3 or ac4 neurons (Yao et al., 2005
). This indicates that mis-expressed IR84a does not simply generate non-specific ligand sensitivity in these neurons.
IR84a mis-expression confers novel olfactory sensitivity to phenylacetaldehyde
We next compared the novel odor responses conferred by IR84a mis-expression to the endogenous phenylacetaldehyde responses of ac4 sensilla by generating dose-response curves (). Stimulus evoked spike frequencies of ac3 sensilla ectopically expressing IR84a are quantitatively very similar to those in ac4 sensilla, even exceeding the endogenous ac4 responses at higher odor concentrations (). These elevated responses are likely to be due to the contribution of weak endogenous phenylacetaldehyde responses that we observed in ac3 sensilla at high stimulus concentrations (), as subtraction of these values produces an IR84a-dependent phenylacetaldehyde dose-response curve that is statistically the same as that of ac4 sensilla (). Thus, ectopic expression of a single IR in ac3 is sufficient to confer a novel ligand- and receptor-specific odor sensitivity that is physiologically indistinguishable from endogenous responses.
To extend this analysis to a second IR, we examined whether mis-expression of one of the IR
genes uniquely expressed in ammonia-sensitive ac1 neurons (IR31a
) was sufficient to confer ectopic responsiveness to this odor. Because ac3 sensilla neurons display endogenous ammonia-evoked responses at modest stimulus concentrations, we used in these experiments the IR76a
-promoter GAL4 transgene to mis-express these receptors in ammonia-insensitive ac4 sensilla () (Yao et al., 2005
). ac4 sensilla mis-expressing IR92a
, but not IR31a
, displayed responses to ammonia (). 1,4-diaminobutane, a control stimulus that does not activate either ac1 or ac4 neurons (Yao et al., 2005
), did not stimulate ac4 sensilla mis-expressing IR92a. We note that the magnitude of the ectopic IR92a ammonia response is lower than native ammonia-evoked responses of ac1 sensilla (Yao et al., 2005
). This may be due to the lack of co-factors present in ac1 sensilla but not in ac4 sensilla. Nevertheless, these results suggest that IR92a comprises at least part of an ammonia-specific chemosensory receptor.
IR92a mis-expression confers novel olfactory sensitivity to ammonia