Olfactory marker protein directly buffers cAMP to avoid depolarization-induced silencing of olfactory receptor neurons

Abstract

Olfactory receptor neurons (ORNs) use odour-induced intracellular cAMP surge to gate cyclic nucleotide-gated nonselective cation (CNG) channels in cilia. Prolonged exposure to cAMP causes calmodulin-dependent feedback-adaptation of CNG channels and attenuates neural responses. On the other hand, the odour-source searching behaviour requires ORNs to be sensitive to odours when approaching targets. How ORNs accommodate these conflicting aspects of cAMP responses remains unknown. Here, we discover that olfactory marker protein (OMP) is a major cAMP buffer that maintains the sensitivity of ORNs. Upon the application of sensory stimuli, OMP directly captured and swiftly reduced freely available cAMP, which transiently uncoupled downstream CNG channel activity and prevented persistent depolarization. Under repetitive stimulation, OMP-/-ORNs were immediately silenced after burst firing due to sustained depolarization and inactivated firing machinery. Consequently, OMP-/mice showed serious impairment in odour-source searching tasks. Therefore, cAMP buffering by OMP maintains the resilient firing of ORNs.

Olfactory marker protein interacts with adenosine nucleotide derivatives

“…We consulted the Protein Data Bank (PDB) for a solution structure of OMP determined by nuclear magnetic resonance (Model 1 from a 1ZRI file) [ 17 , 31 ]. The OMP data were further modified by adding polar hydrogen atoms and rendered into an analysis grid using AutoDockTools under a Python platform [ 32 ].…”

“…We used PfuUltra Fusion DNA polymerase (Agilent, CA, USA) and Q5 High-Fidelity DNA polymerase (New England Biolabs, MA, USA) for PCR [ 17 ]. We used the pCI mammalian expression vector (Promega, WI, USA) for exogenous expression in HEK293T cells (ATCC, VA, USA).…”

“…HEK293T cells were cultured in Dulbecco's MEM (DMEM; Wako Pure Chemical, Osaka, Japan) supplemented with 10% foetal bovine serum (FBS: Sigma-Aldrich, MO, USA) without antibiotics at 37 °C in 5% CO 2 . The plasmids containing cDNAs for Rluc-OMP fusion genes or Rluc (5 μg) were transfected into HEK293T cells using Effectene Transfection Reagent (QIAGEN, Hilden, Germany) following the manufacturer's protocol as previously described [ 17 ].…”

“…These investigations in olfaction have been facilitated by the discovery of olfactory marker protein, (OMP), which genetically labels mature ORNs [ [5] , [6] , [7] , [8] ]. Close examinations have revealed that knocking-out of the OMP gene reduces odour discrimination ability in mice [ [9] , [10] , [11] , [12] , [13] , [14] ], prolongs odour response kinetics mediated by the Ca 2+ -permeable cyclic nucleotide-gated A2 (CNGA2) channels [ [15] , [16] , [17] , [18] ], and delays the Ca 2+ extrusion via the Na + -Ca 2+ exchanger (NCX) in ORNs [ 19 ]. Furthermore, OMP-KO delays the maturation of axonal projections from ORNs to the olfactory bulb during development [ 15 , 20 , 21 ], in which basal cAMP levels play a key role [ 17 , 18 , [22] , [23] , [24] , [25] ].…”

“…By searching through the amino acid sequence of OMP, we realized that OMP contains a cyclic nucleotide binding domain (CNBD)-like motif [ 17 ]. OMP was a cAMP binding protein in ORNs and regulated phasic and tonic cAMP signalling kinetics [ [15] , [16] , [17] , [25] , [26] , [27] ]. Previously, we assessed the direct physical interaction between OMP and cAMP in vitro by performing bioluminescence resonance energy transfer (BRET) experiments and determined the apparent affinities to cAMP [ 17 ].…”