Cellular & molecular Ca2+ microdomains in olfactory cilia support low signaling amplification of odor transduction

Signal transduction depends critically on the spatial localization of protein constituents. A key question in odor transduction is whether chemotransduction proteins organize into discrete molecular complexes throughout olfactory cilia or distribute homogeneously along the ciliary membrane. Our recordings of Ca2+ changes in individual cilia with unprecedented spatial and temporal resolution, by the use of two-photon microscopy, provide solid evidence for Ca2+ microdomains (transducisomes). Dissociated frog olfactory neurons were preloaded with caged-cAMP and fluo-4 acetoxymethyl ester probe Ca2+ indicator. Ca2+ influx through cyclic nucleotide-gated (CNG) channels was evoked by uniformly photoreleasing cAMP, while ciliary Ca2+ was measured. Discrete fluorescence events were clearly resolved. Events were missing in the absence of external Ca2+, consistent with the absence of internal Ca2+ sources. Fluorescence events at individual microdomains resembled single-CNG channel fluctuations in shape, mean duration and kinetics, indicating that transducisomes typically contain one to three CNG channels. Inhibiting the Na+ ⁄ Ca2+ exchanger or the Ca2+-ATPase prolonged the decay of evoked intraciliary Ca2+ transients, supporting the participation of both transporters in ciliary Ca2+ clearance, and suggesting that both molecules localize close to the CNG channel. Chemosensory transducisomes provide a physical basis for the low amplification and for the linearity of odor responses at low odor concentrations.This work was supported by grant TW007920 from the Fogarty International Center of the NIH (J. Bacigalupo and D. Restrepo), NIDCD grants DC006070 and DC04657 (D. Restrepo), MIDEPLAN ICMP05- 001-F (J. Bacigalupo), FONDECYT 1080653 (J. Bacigalupo), a MECESUP UCH0409 training research fellowship (K. Castillo), the Graduate Department and Academic Affairs, University of Chile (K, Castillo) and a CONICYT doctoral fellowship (K. Castillo)