Detection of volatile odorants by olfactory neurons is thought to result from direct activation of seven-transmembrane odorant receptors by odor molecules. Here, we show that detection of the Drosophila pheromone, 11-cis vaccenyl acetate (cVA), is instead mediated by pheromone-induced conformational shifts in the extracellular pheromone-binding protein, LUSH. We show that LUSH undergoes a pheromone-specific conformational change that triggers the firing of pheromone-sensitive neurons. Amino acid substitutions in LUSH that are predicted to reduce or enhance the conformational shift alter sensitivity to cVA as predicted in vivo. One substitution, LUSH(D118A), produces a dominant-active LUSH protein that stimulates T1 neurons through the neuronal receptor components Or67d and SNMP in the complete absence of pheromone. Structural analysis of LUSH(D118A) reveals that it closely resembles cVA-bound LUSH. Therefore, the pheromone-binding protein is an inactive, extracellular ligand converted by pheromone molecules into an activator of pheromone-sensitive neurons and reveals a distinct paradigm for detection of odorants.

The only known volatile pheromone in Drosophila, 11-cis-vaccenyl acetate (cVA), mediates a variety of behaviors including aggregation, mate recognition, and sexual behavior. cVA is detected by a small set of olfactory neurons located in T1 trichoid sensilla on the antennae of males and females. Two components known to be required for cVA reception are the odorant receptor Or67d and the extracellular pheromone-binding protein LUSH. Using a genetic screen for cVA-insensitive mutants, we have identified a third component required for cVA reception: sensory neuron membrane protein (SNMP). SNMP is a homolog of CD36, a scavenger receptor important for lipoprotein binding and uptake of cholesterol and lipids in vertebrates. In humans, loss of CD36 is linked to a wide range of disorders including insulin resistance, dyslipidemia, and atherosclerosis, but how CD36 functions in lipid transport and signal transduction is poorly understood. We show that SNMP is required in pheromone-sensitive neurons for cVA sensitivity but is not required for sensitivity to general odorants. Using antiserum to SNMP infused directly into the sensillum lymph, we show that SNMP function is required on the dendrites of cVA-sensitive neurons; this finding is consistent with a direct role in cVA signal transduction. Therefore, pheromone perception in Drosophila should serve as an excellent model to elucidate the role of CD36 members in transmembrane signaling.CD36 ͉ olfaction ͉ olfactory ͉ sexual behavior ͉ signal transduction C VA (11-cis-vaccenyl acetate) mediates social behaviors in Drosophila, and its reception requires the odorant receptor Or67d and the extracellular pheromone-binding protein LUSH (1-4). Misexpression of Or67d receptors in trichoid neurons that are normally insensitive to pheromone confers cVA sensitivity but only if LUSH is present (3). However, Or67d and LUSH are not sufficient to confer cVA sensitivity to basiconic neurons (T.S.H. and D.P.S., unpublished work). This finding reveals that there are additional factors required for cVA sensitivity present in trichoid sensilla that are lacking in basiconic sensilla. Using a genetic screen, we set out to identify additional components important for cVA sensitivity. We screened Ϸ3,000 mutagenized third-chromosome lines selected for homozygous viability (5). We screened each mutant line for T1 electrophysiological responses to cVA using single sensillum electrophysiological recordings (2, 3, 6). We identified five complementation groups that were cVA-insensitive yet retained spontaneous activity in the pheromone-sensing neurons (the vains phenotype) ( Fig. 1 and Table 1). The presence of spontaneous activity indicates that the neurons are present, are viable, and can sustain action potentials, thereby eliminating nonspecific mutants affecting development or general neuronal function. Of the five complementation groups recovered, two, Or67d and Or83b, affect genes previously implicated in cVA or general odorant detection, two remain unmapped, and the fifth encodes SNMP, a new cVA...

Insect pheromones elicit stereotypic behaviors that are critical for survival and reproduction. Defining the relevant molecular mechanisms mediating pheromone signaling is an important step to manipulate pheromone-induced behaviors in pathogenic or agriculturally important pests. The only volatile pheromone identified in Drosophila is 11-cis-vaccenyl acetate (VA), a male-specific lipid that mediates aggregation behavior. VA activates a few dozen olfactory neurons located in T1 sensilla on the antenna of both male and female flies. Here, we identify a neuronal receptor required for VA sensitivity. We identified two mutants lacking functional T1 sensilla and show that the expression of the VA receptor is dramatically reduced or eliminated. Importantly, we show misexpression of this receptor in non-T1 neurons, normally insensitive to VA, confers pheromone sensitivity at physiologic concentrations. Sensitivity of T1 neurons to VA requires LUSH, an extracellular odorant-binding protein (OBP76a) present in the sensillum lymph bathing trichoid olfactory neuron dendrites. Here, we show LUSH are also required in non-T1 neurons misexpressing the receptor to respond to VA. These data provide new insight into the molecular components and neuronal basis of volatile pheromone perception.

Summary Background Sensory neuron diversity ensures optimal detection of the external world and is a hallmark of sensory systems. An extreme example is the olfactory system, as individual olfactory receptor neurons (ORNs) adopt unique sensory identities by typically expressing a single receptor gene from a large genomic repertoire. In Drosophila, about 50 different ORN classes are generated from a field of precursor cells, giving rise to spatially restricted and distinct clusters of ORNs on the olfactory appendages. Developmental strategies spawning ORN diversity from an initially homogeneous population of precursors are largely unknown. Results Here we unravel the nested and binary logic of the combinatorial code that patterns the decision landscape of precursor states underlying ORN diversity in the Drosophila olfactory system. The transcription factor Rotund (Rn) is a critical component of this code that is expressed in a subset of ORN precursors. Addition of Rn to preexisting transcription factors that assign zonal identities to precursors on the antenna subdivides each zone and almost exponentially increases ORN diversity by branching off novel precursor fates from default ones within each zone. In rn mutants, rn-positive ORN classes are converted to rn-negative ones in a zone-specific manner. Conclusions We provide a model describing how nested and binary changes in combinations of transcription factors could coordinate and pattern a large number of distinct precursor identities within a population to modulate the level of ORN diversity during development and evolution.

cDNAs encoding large-conductance Ca 2+ -activated K + channel a-subunit (rSlo) were obtained from rat brain. From the DNA sequence of multiple rslo clones, we identified a specific sequence variation of 81 nucleotides, which is either absent from or present at the N-terminal region of a putative Ca 2+ -sensing domain of the channel. Transcripts containing such variations were detected in different ratios from several brain regions, and their functional significance was further examined. When heterologously expressed in Xenopus oocytes, both rSlo variants, named rSlo 0 and rSlo 27 , generated Ca 2+ -activated and voltage-activated K + currents characteristic of neuronal large-conductance Ca 2+ -activated K + (BK Ca ) channels. Single-channel recordings of the two channels showed almost identical permeation characteristics and steady-state gating behavior. Noticeable differences between rSlo 0 and rSlo 27 were revealed when the macroscopic currents were measured at various voltages and intracellular Ca 2+ concentrations. rSlo 27 activated was more rapidly than rSlo 0 in the presence of the same voltage stimulus, and the differences in these activation kinetics were dependent on the concentration of intracellular Ca 2+ . Despite their similar apparent affinities for Ca 2+ , rSlo 0 and rSlo 27 showed significant differences in their co-operative gating behavior. The Hill coefficient for intracellular Ca 2 was estimated to be about 3.7 for rSlo 27 regardless of the membrane voltage, and that for rSlo 0 was reduced from about 5 to 2 as the membrane voltage changed from 40 to 140 mV. As activation of BK Ca channels is involved in rapid hyperpolarization of action potentials, the differential processing of rslo transcripts, and the generation of channels with different activation kinetics and Ca 2+ cooperativity may be a mechanism for tuning the excitability of neurons in different brain regions.