1 Inhibitory odorant signaling in mammalian olfactory receptor neurons 2 3

18 Odorants inhibit as well as excite olfactory receptor neurons (ORNs) in many 19 species of animals. Cyclic nucleotide-dependent activation of canonical mammalian 20 ORNs is well established but it is still unclear how odorants inhibit these cells. Here we 21 further implicate phosphoinositide-3-kinase (PI3K), an indispensable element of PI 22 signaling in many cellular processes, in olfactory transduction in rodent ORNs. We show 23 that odorants rapidly and transiently activate PI3K in the olfactory cilia and in the 24 olfactory epithelium in vitro. We implicate known G-protein coupled isoforms of PI3K 25 and show that they modulate not only the magnitude but also the onset kinetics of the 26 electrophysiological response of ORNs to complex odorants. Finally, we show that the 27 ability of a single odorant to inhibit another can be PI3K-dependent. Our collective 28 results provide compelling support for the idea that PI3K-dependent signaling mediates 29 inhibitory odorant input to mammalian ORNs and at least in part contributes to the 30 mixture suppression typically seen in the response of ORNs to complex natural 31 odorants. 32 33 INTRODUCTION 34 It is increasingly clear that the mammalian sense of smell is organized into 35 subsystems, including functional subsets of ORNs in the main olfactory epithelium (OE) 36 itself (reviews: (Breer et al. 2006; Ma 2007; Munger et al. 2008). Organizational 37 complexity extends to individual ORNs since it has been long known that odorants can 38 inhibit as well as excite canonical ORNs in the OE, as in most species of animals 39 (review:(Ache and Young 2005). Canonical ORNs are excited by the binding of odor 40 molecules to their cognate odorant receptor (OR), which triggers a cyclic nucleotide 41 signaling cascade that targets an olfactory cyclic nucleotide-gated (CNG) ion channel 42 (Kaupp and Seifert 2002). The resulting Ca-influx into the ORN secondarily targets a 43 Ca-activated chloride channel which further amplifies the output of the cell (Kleene 44 1993). Negative feedback from the elevated Ca concentration causes a 45 Ca/calmodulin-dependent decrease in the sensitivity of the CNG channel to cAMP 46 (Bradley et al. 2004; Bradley et al. 2005). How odorants inhibit these cells, however, is 47 still not understood. 48 A long-standing point of controversy has been whether phosphoinositide (PI)49 signaling plays a role in mammalian olfactory transduction (Gold 1999; Noe and Breer 5